Changes in stiffness of the optic nerve and involvement of neurofilament light chains in the course of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis.

Multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), are often accompanied by optic neuritis associated with neurofilament disruption. In this study, the stiffness of the optic nerve was investigated by atomic force microscopy (AFM) in mice with induced EAE in the successive phases of the disease: onset, peak, and chronic. AFM results were compared with the intensity of the main pathological processes in the optic nerve: inflammation, demyelination, and axonal loss, as well as with the density of astrocytes, assessed by quantitative histology and immunohistochemistry. Optic nerve tissue and serum levels of neurofilament light chain protein (NEFL) were also examined by immunostaining and ELISA, respectively. The stiffness of the optic nerve in EAE mice was lower than that in control and naïve animals. It increased in the onset and peak phases and sharply decreased in the chronic phase. Serum NEFL level showed similar dynamics, while tissue NEFL level decreased in the onset and peak phases, indicating a leak of NEFL from the optic nerve to body fluids. Inflammation and demyelination gradually increased to reach the maximum in the peak phase of EAE, and inflammation slightly declined in the chronic phase, while demyelination did not. The axonal loss also gradually increased and had the highest level in the chronic phase. Among these processes, demyelination and especially axonal loss most effectively decrease the stiffness of the optic nerve. NEFL level in serum can be regarded as an early indicator of EAE, as it rapidly grows in the onset phase of the disease.

Biomechanical changes in the liver tissue induced by a mouse model of multiple sclerosis (EAE) and the effect of anti-VLA-4 mAb treatment.

Experimental autoimmune encephalomyelitis (EAE) is a mouse model of demyelinating diseases, such as multiple sclerosis (MS). MS can be accompanied by autoimmune hepatitis. In this study, nanomechanical, biorheological and histological examinations were carried out by atomic force microscopy (AFM), rheology, and immunofluorescence microscopy to investigate changes in the liver tissue of EAE mice and the effect of natalizumab, a monoclonal antibody against α4-integrin (VLA-4) cell adhesion molecule, used in MS therapy. Liver samples collected from EAE mice in three successive phases of the disease showed inflammatory changes manifested by leukocyte infiltrations and elevated levels of proinflammatory cytokine IL-1ß. Liver stiffness and viscoelasticity increased in the onset phase of EAE, decreased in the peak phase and increased again in the chronic phase to reach the highest values. These changes were not associated with inflammation parameters which increased in the peak phase and decreased to the lowest values in the chronic phase. Moreover, anti-VLA treatment, which reduced the inflammation parameters, had an ambiguous effect on stiffness and viscoelasticity: it increased them in the peak phase but decreased in the chronic phase. The observed discrepancies can result from a complex network of interactions between inflammation and fibrosis, as well as between liver cells and the extracellular matrix influencing the biomechanical properties of the liver tissue.

Small-molecule inhibitor - tyrphostin AG1296 regulates proliferation, survival and migration of rhabdomyosarcoma cells.

Rhabdomyosarcoma (RMS) is the most commonly occurring malignant soft tissue tumor in children. Despite improving its treatment methods, the current outcome in the advanced stages of this tumor is not satisfactory. RMS cells are characterized by abnormal cellular signaling due to the changes in the activity of the tyrosine kinases. Thus, substances blocking the mitogenic signal transmitted by receptors with tyrosine kinase activity raise hopes for inhibition of the uncontrolled cell growth. In this study, we examined the anticancer activity of tyrphostin AG1296, a tyrosine kinase inhibitor that binds to the intracellular domain of the PDGF (platelet-derived growth factor) receptor in human RMS alveolar and embryonal cell lines. We have discovered that tyrphostin AG1296 completely inhibited cell proliferation and effectively inhibited cell viability. Tyrphostin AG1296 induced apoptosis of the RMS cells and significantly inhibited their migration. Additionally, investigated inhibitor slightly inhibited expression of AKT and phosphorylation of ERK in alveolar RMS cells. Importantly, the inhibitor exerted also potent effects on the nanomechanical properties and cytoskeleton organization of RMS cells. To conclude, tyrphostin AG1296 is a promising compound in the treatment of alveolar RMS. Undoubtedly, a better knowledge of receptor pathomechanism of tyrosine kinases may contribute to developing new, more effective ways of RMS treatment.

Changes in spinal cord stiffness in the course of experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis.

Experimental autoimmune encephalomyelitis (EAE) is a commonly used mouse model of multiple sclerosis, a chronic inflammatory disease of the central nervous system (CNS) characterized by demyelination leading to brain and spinal cord malfunctions. We postulate that not only biological but also biomechanical properties play an important role in impairements of CNS function. Atomic force microscopy (AFM) was applied to investigate mechanical properties of spinal cords collected from EAE mice in preonset, onset, peak, and chronic disease phases. Biomechanical changes were compared with histopathological alterations observed in the successive phases. The deformability of gray matter did not change, while rigidity of white matter increased during the onset phase, remained at the same level in the peak phase and decreased in the chronic phase. Inflammatory infiltration and laminin content accompanied the tissue rigidity increase, whereas demyelination and axonal damage showed an opposite effect. The increase in white matter rigidity can be regarded as an early signature of EAE.

Comparing surface properties of melanoma cells using time of flight secondary ions mass spectrometry.

Various techniques have been already reported to differentiate between normal (non-malignant) and cancerous cells based on their physico-chemical properties. This is relatively simple when studied cancerous cells originate from distant stages of cancer progression. Here, studies on chemical properties of two closely related human melanoma cell lines are presented: WM115 melanoma cells were taken from the vertical growth phase while WM266-4 from the skin metastatic site of the same patient. Their chemical properties were studied by two techniques, namely time-of-flight secondary ion mass spectra (ToF SIMS) and photothermal microspectroscopy (PTMS), used to record mass and photothermal spectra of cells, respectively. In our approach, independently of the spectra type, its full range, i.e. masses and wavenumbers within the range 0-500 kDa and 500-4000 cm-1, underwent a similar methodology for principal component analysis (PCA). PCA outcome shows results groupped depending on the sample type (either WM115 or WM266-4 cells). The results are independent of the method applied to study chemical properties of melanoma cells, indicating that cancer-related changes are large enough to be identified with these techniques and to differentiate between cells originating from vertical growth phase and skin metastatis.

Data on step-by-step atomic force microscopy monitoring of changes occurring in single melanoma cells undergoing ToF SIMS specialized sample preparation protocol.

Data included in this article are associated with the research article entitled 'Protocol of single cells preparation for time-of-flight secondary ion mass spectrometry' (Bobrowska et al., 2016 in press) [1]. This data file contains topography images of single melanoma cells recorded using atomic force microscopy (AFM). Single cells cultured on glass surface were subjected to the proposed sample preparation protocol applied to prepare biological samples for time-of-flight secondary ion mass spectrometry (ToF SIMS) measurements. AFM images were collected step-by-step for the single cell, after each step of the proposed preparation protocol. It consists of four main parts: (i) paraformaldehyde fixation, (ii) salt removal, (iii) dehydrating, and (iv) sample drying. In total 13 steps are required, starting from imaging of a living cell in a culture medium and ending up at images of a dried cell in the air. The protocol was applied to melanoma cells from two cell lines, namely, WM115 melanoma cells originated from primary melanoma site and WM266-4 ones being the metastasis of WM115 cells to skin.

Influence of propofol and volatile anaesthetics on the inflammatory response in the ventilated lung.

BACKGROUND: The immunomodulatory effects of volatile anaesthetics in vitro and the protective effect of propofol in lung injury spurred us to study the effects of volatile anaesthetics and propofol on lung tissue in vivo. METHODS: Twenty-seven pigs were randomized to 4-h general anaesthesia with propofol (8 mg/kg/h, group P, n=9), sevoflurane [minimum alveolar concentration (MAC)=1.0, group S, n=9) or desflurane (MAC=1.0, group D, n=9). Four healthy animals served as the no-ventilation group. Bronchoalveolar lavage fluid (BALF) was obtained to measure the cell counts, platelet-activating factor acetylhydrolase (PAF-AcH), phospholipase A(2) (PLA(2)) and superoxide dismutase (SOD) activity. Lung tissues were evaluated histologically and for caspase-3 expression. RESULTS: Volatile anaesthetics reduced PAF-AcH levels without affecting PLA(2) activity and resulted in decreased alveolar macrophage and increased lymphocyte counts in BALF (sevoflurane: 29 ± 23%; desflurane: 26 ± 6%, both P<0.05 compared with 4 ± 2% in the no-ventilation group). These findings were accompanied by atelectasis and inflammatory cells' infiltration in the inhalational anaesthetics groups. Also, sevoflurane reduced SOD activity and both sevoflurane and desflurane induced significant caspase-3 expression. In contrast, propofol resulted in a minor degree of inflammation and preserved BALF cells' composition without triggering apoptosis. CONCLUSION: Halogenated anaesthetics seem to trigger an immune lymphocytic response in the lung, inducing significant apoptosis and impairment of PAF-AcH. In contrast, propofol preserves anti-inflammatory and anti-oxidant defences during mechanical ventilation, thus preventing the emergence of apoptosis.

HPLC-fluorimetric assay of phospholipase A2. Application to biological samples with high protein content and various reaction conditions.

Phospholipase A(2) (PLA(2)) quantitation in real-time, using (7-nitro-2-1,3-benzoxadiazol-4-yl)amino-derivatives of phosphatidylcholine (NBD-PCs) as substrates, is influenced by high protein content, color or turbidity. The purpose of the study was to overcome such limitations by a complementary reversed-phase HPLC step to separate the substrates from the products of the reaction. Plasma and bronchoalveolar lavage (BAL) fluid were applied as enzymic sources, while standard porcine PLA(2) and human plasma PAF-acetylhydrolase (PAF-AH) were employed as positive controls. The method was validated with a radiometric assay and compared with the real-time fluorimetric assay. Regarding PLA(2) and PAF-AH determination, the isocratic elution systems CH(3)OH-H(2)O (80:20, v/v) and CH(3)OH-H(2)O-CH(3)COOH (60:40:0.2, v/v/v) separated efficiently C(12)-NBD-FA/C(12)-NBD-PC and C(6)-NBD-FA/C(6)-NBD-PC, with 4.4 and 2.2 resolution, respectively. Analysis time was ∼15 min/injection. The reproducibility, expressed as relative standard deviation, was ≤13% for PLA(2) and ≤16% for PAF-AH, respectively. The assay was linear for PLA(2) activities extending from 1 pmol up to at least 250 nmol FA/h/mL sample, similar to the radiometric assay. It was appropriate for samples with high protein content, where the real-time fluorimetric assay was insufficient. The HPLC method was also evaluated under elevated temperatures, various pH values and Ca(2+) concentrations.

Intracellular nanomanipulation by a photonic-force microscope with real-time acquisition of a 3D stiffness matrix.

A traditional photonic-force microscope (PFM) results in huge sets of data, which requires tedious numerical analysis. In this paper, we propose instead an analog signal processor to attain real-time capabilities while retaining the richness of the traditional PFM data. Our system is devoted to intracellular measurements and is fully interactive through the use of a haptic joystick. Using our specialized analog hardware along with a dedicated algorithm, we can extract the full 3D stiffness matrix of the optical trap in real time, including the off-diagonal cross-terms. Our system is also capable of simultaneously recording data for subsequent offline analysis. This allows us to check that a good correlation exists between the classical analysis of stiffness and our real-time measurements. We monitor the PFM beads using an optical microscope. The force-feedback mechanism of the haptic joystick helps us in interactively guiding the bead inside living cells and collecting information from its (possibly anisotropic) environment. The instantaneous stiffness measurements are also displayed in real time on a graphical user interface. The whole system has been built and is operational; here we present early results that confirm the consistency of the real-time measurements with offline computations.

Desferrioxamine attenuates minor lung injury following surgical acute liver failure.

Acute liver failure (ALF) can be complicated by lung dysfunction. The aim of this study was to test the hypothesis that inhibition of oxidative stress through iron chelation with desferrioxamine (DFX) attenuates pulmonary injury caused by ALF. 14 adult female domestic pigs were subjected to surgical devascularisation of the liver and were randomised to a study group (DFX group, n = 7), which received post-operative intravenous infusion of DFX (14.5 mg x kg(-1) x h(-1) for the first 6 h post-operatively and 2.4 mg x kg(-1) x h(-1) until completion of 24 h), and a control group (n = 7). Post-operative lung damage was evaluated by histological and bronchoalveolar lavage fluid (BALF) analysis. DFX resulted in reduced BALF protein levels and tissue phospholipase (PL)A(2) activity. Plasma malondialdehyde and BALF nitrate and nitrite concentrations were lower, while catalase activity in the lung was higher after DFX treatment. PLA(2), platelet-activating factor acetylhydrolase and total cell counts in BALF did not differ between groups. Histological examination revealed reduced alveolar collapse, pneumonocyte necrosis and total lung injury in the DFX-treated animals. DFX reduced systemic and pulmonary oxidative stress during ALF. The limited activity of PLA(2) and the attenuation of pneumonocyte necrosis could represent beneficial mechanisms by which DFX improves alveolar-capillary membrane permeability and prevents alveolar space collapse.

The systemic inflammatory response in coronary artery bypass grafting: what is the role of the very low ejection fraction (EF < or = 30%)?

AIM: Patients with depressed left ventricular function are more susceptible to develop postoperative complications after cardiac surgery. The aim of the present study was to examine the effect of severe left ventricular dysfunction on the activation of systemic inflammatory reaction during and after coronary artery bypass grafting (CABG). METHODS: Clinical prospective study; 32 selected patients underwent CABG; 16 patients had depressed left ventricular function before the operation (low ejection fraction [EF] <30%)--Low EF group (study group). Sixteen patients had normal left ventricular function (normal EF, >50%)--Normal EF group (control group). The levels of inflammatory mediators TNF-alpha, IL-6, IL-8 and IL-10 were measured preoperatively, during and after cardiopulmonary bypass (CPB) and 24 hours postoperatively. RESULTS: Higher levels of almost all of inflammatory mediators were detected in patients with depressed left ventricular function compared with patients of normal EF group. IL-6 levels were found statistically significant higher in Low EF group before the induction of anesthesia (P=0.039) and after the administration of protamine (P=0.02). IL-8 levels were found statistically significant higher in Low EF group before the induction of anesthesia (P=0.05), 30 min after the start of CPB (P=0.02), after the administration of protamine (P=0.015) and 24 hours after the end of the operation (P=0.05). No statistically significant differences were demonstrated between the 2 groups of study relative to TNF-alpha and IL-10. CONCLUSION: A greater activation of systemic inflammatory reaction occurred in patients with depressed left ventricular function than in patients with normal cardiac function when they underwent CABG with extracorporeal circulation.

Rheological properties of erythrocytes in patients with high risk of cardiovascular disease.

Rheological properties of erythrocytes from patients with high risk of cardiovascular disease (CVD) were analyzed in relation to individual patient risk factors as well as to the medication. Additionally, comparative statistical analysis was performed considering plasma concentration of the selected mediators of vascular endothelium: 6-keto-prostaglandin F(1alpha) (PGF(1alpha)), sVCAM-1 and E-selectin adhesion molecules and interleukin-6 (IL-6). It was found that antihypertensive therapy with angiotensin-converting enzyme inhibitor (ACEI) is accompanied by improvement of RBC rheology: the increase of deformability and the decrease of aggregability. This improvement is probably mediated by endothelial prostacyclin and nitric oxide which are generated by ACEI. A correlation was observed between RBC deformability/aggregability and the patient's hematocrit level, what implicates that the hematocrit level should be explicitly taken into consideration when investigating rheological properties of erythrocytes. A strong relationship was also found between the plasma concentration of sVCAM-1 and patient's age.

Acute lung injury probably associated with infusion of propofol emulsion.

We present a case of acute lung injury associated with propofol infusion in a mechanically ventilated patient with intracerebral haemorrhage. Diagnosis was based on the exclusion of other risk factors inducing acute lung injury and on the clinical improvement after discontinuation of the propofol emulsion. Laboratory data such as the increase in total phospholipids, neutral lipids and free fatty acids in the broncho-alveolar lavage fluid, the remarkably high percentage of alveolar macrophages including fat droplets and the similar lipid composition of propofol and broncho-alveolar lavage fluid support the relationship between propofol and acute lung injury.

Anti-phospholipid antibodies in serum from patients with Guillain-Barré syndrome.

OBJECTIVE: The Guillain-Barré syndrome (GBS) is an acute inflammatory polyneuropathy related to autoimmunity. However, no conclusive etiological concept has yet been found. We examined the variation in autoantibodies to lipids in serum of GBS patients in response to the course of the disease but investigated titer modifications during treatment with gamma-globulin. DESIGN AND SETTING: Prospective clinical study in a 14-bed general ICU. PATIENTS: Nine patients with GBS and nine controls were included in the study. MEASUREMENTS AND RESULTS: Four blood samples were obtained before and after treatment. Serum samples, diluted 1:60, were tested by enzyme-linked immunosorbent assay for IgM, IgA, and IgG antibodies to phosphatidylcholine, phosphatidylinositol, cardiolipin, phosphatidic acid, phosphatidylserine, phosphatidylglycerol, phosphatidylethanolamine, sphingomyelin, and gangliosides. Anti-phospholipid antibodies of the IgM, IgA, and IgG families were detected in all GBS patients but in none of the controls. Phosphatidylinositol, cardiolipin, phosphatidylcholine, and phosphatidic acid were the main antigens. All patients developed anti-phosphatidylinositol antibodies of the IgM family and anti-cardiolipin antibodies of the IgA and IgG families. A decrease in the level of anti-phospholipid autoantibodies was observed after 1 day of treatment with gamma-globulin. Two days after ending gamma-globulin administration the IgG antibodies increased again. CONCLUSIONS: Our findings suggest that in GBS there is an extensive immune reaction, which is altered after gamma-globulin treatment. Anti-cardiolipin and anti-phosphatidylinositol antibodies could be useful markers for the response to treatment.

Probing molecular interaction between concanavalin A and mannose ligands by means of SFM.

Recently, the scanning force microscope (SFM) has been widely used for direct monitoring of specific interactions between biologically active molecules. Such studies have employed the SFM liquid-cell setup, which allows measurements to be made in the native environment with force resolution down to a tenth of a picoNewton. In this study, the ligand-receptor strength of monoclonal anti-human prostatic acid phosphatase and prostatic acid phosphatase, representing an antigen-antibody system with a single type of interaction, was determined. Then, the interaction force occurring between concanavalin A and the carbohydrate component of the glycoproteins arylsulfatase A and carboxypeptidase Y was measured. High mannose-type glycans were sought on the human prostate carcinoma cell surface. Application of an analysis based on the Poisson distribution of the number of bonds formed in all these measured systems allowed the strength of the molecular interaction to be calculated. The values of the force acting between two single molecules were 530+/-25, 790+/-32, and 940+/-39 pN between prostatic acid phosphatase and monoclonal anti-human prostatic acid phosphatase, between concanavalin A and arylsulfatase A, and between concanavalin A and carboxypeptidase Y, respectively. The value calculated from data collected for the force between concanavalin A and mannose-containing ligands present on the surface of human prostate carcinoma cells was smaller, 116+/-17 pN. The different values of the binding force between concanavalin A and mannose-containing ligands were attributed to the structural changes of the carbohydrate components.

Bronchoalveolar lavage alterations during prolonged ventilation of patients without acute lung injury.

Mechanical ventilation deteriorates previously injured lung, but little is known about its effect on healthy human lung. This work was designed to assess the effect of prolonged mechanical ventilation on bronchoalveolar lavage (BAL) fluid composition of patients without acute lung injury. Twenty-two ventilated patients (tidal volume 8-10 mL x kg(-1), positive end-expiratory pressure 3-5 cmH2O) without lung injury, who did not develop any complication from the respiratory system during the 2-week study period, were studied. They were subjected to three consecutive BALs, the first during 36 h from intubation, the second at the end of the first week of mechanical ventilation and the third at the end of the second week of mechanical ventilation. Total BAL protein increased during mechanical ventilation (148 +/- 62, 381 +/- 288, 353 +/- 215 microg x mL(-1) BAL for the first, second and third BAL, respectively). In contrast, BAL phospholipids decreased (2.7 +/- 1.1, 1.4 +/- 0.6, 1.2 +/- 0.7 microg x mL(-1) BAL, respectively). Large surfactant aggregates were reduced and inflammatory markers, such as platelet activating factor (PAF), PAF-acetylhydrolase and neutrophils, significantly increased after 1 week, but partially remitted after 2 weeks of mechanical ventilation. In summary, this study demonstrates that prolonged mechanical ventilation even of patients without acute lung injury is associated with the presence of inflammatory markers and surfactant alterations.

Ventilator-associated pneumonia and atelectasis: evaluation through bronchoalveolar lavage fluid analysis.

OBJECTIVE: Surfactant offers protection against alveolar collapse and contributes to the local defense mechanism, but it is unclear if surfactant alterations have a role in the development of atelectasis or ventilator-associated pneumonia (VAP). The present study was undertaken to monitor surfactant, as well as biochemical BAL fluid alterations, during the course of VAP and atelectasis in mechanically ventilated patients without primary cardiopulmonary disease, to elucidate the pathogenesis and to differentiate these two entities. DESIGN. Prospective controlled study. SETTING: 14-bed general ICU of a 750-bed University Hospital. PATIENTS: Sixty-one ventilated patients, without primary cardiopulmonary disease-normal initial chest X-ray, satisfactory oxygenation (PaO(2)/FiO(2)>300 mmHg), and expected time of ventilation exceeding 2 weeks-were initially enrolled. Twelve of them developed VAP and eight lobar or segmental atelectasis during the 2-week study period. INTERVENTIONS: An initial BAL was performed in all patients within 48 h from admission. Patients who developed VAP or atelectasis were subjected to a second and third BAL during and after the resolution of VAP or atelectasis, respectively. MEASUREMENTS AND RESULTS: VAP and atelectasis resulted in a significant increase of total protein and markers of inflammation, such as PAF and neutrophils, which partially remitted after their resolution. Large surfactant aggregates, which contribute to surface tension decrease, were significantly reduced during both entities and remained low even after their resolution. CONCLUSIONS: BAL alterations during VAP and atelectasis suggest increased alveolar-capillary permeability, severe surfactant abnormalities, and signs of local inflammatory reaction. These alterations are associated with the observed deteriorated gas exchange and lung mechanics and could predispose to further lung injury in ventilated patients.