Brownian nanoimaging of interface dynamics and ligand-receptor binding at cell surfaces in 3-D.

We describe a method for nanoimaging interfacial dynamics and ligand-receptor binding at surfaces of live cells in 3-D. The imaging probe is a 1-µm diameter glass bead confined by a soft laser trap to create a "cloud" of fluctuating states. Using a facile on-line method of video image analysis, the probe displacements are reported at ~10 ms intervals with bare precisions (±SD) of 4-6 nm along the optical axis (elevation) and 2 nm in the transverse directions. We demonstrate how the Brownian distributions are analyzed to characterize the free energy potential of each small probe in 3-D taking into account the blur effect of its motions during CCD image capture. Then, using the approach to image interactions of a labeled probe with lamellae of leukocytic cells spreading on cover-glass substrates, we show that deformations of the soft distribution in probe elevations provide both a sensitive long-range sensor for defining the steric topography of a cell lamella and a fast telemetry for reporting rare events of probe binding with its surface receptors. Invoking established principles of Brownian physics and statistical thermodynamics, we describe an off-line method of super resolution that improves precision of probe separations from a non-reactive steric boundary to ~1 nm.

Assessment of the effectiveness and safety of natalizumab for treating relapsing-remitting multiple sclerosis.

OBJECTIVE: Assessing the effectiveness and safety of natalizumab for treating relapsing-remitting multiple sclerosis in a tertiary hospital. METHOD: Observational, prospective study of adult patients treated with natalizumab from May 2007 until February 2009. TREATMENT: 300 mg natalizumab every four weeks. Response criteria: assessment of disease progression, appearance of flare-ups and assessment of magnetic resonance images. Adverse reactions during treatment with natalizumab were recorded. RESULTS: Thirty patients (73% female); average age 34 ± 8.4 years; mean baseline EDSS 3.4 ± 1.3; number of flare-ups in the past year 2.1 ± 1.2. TREATMENT was discontinued in five patients, due to refusal in one case, ineffectiveness in two cases and anaphylaxis in the other two cases. Fourteen patients completed one year of treatment with satisfactory results. A lower EDSS score by 36%, 47%, 31%, 54% and 28% was obtained at 3, 6, 9, 12 and 15 months of treatment respectively. The prevalence of relapse-free patients was 94%, 76% and 54% at 3, 6 and 12 months. MRI imaging studies in 11 patients one year after they began treatment showed no new lesions. Two patients suffered severe anaphylactic shock and another one had an outbreak of urticaria. The presence of neutralising antibodies was the reason for suspending treatment in 6.6% of the patients. CONCLUSIONS: The treatment's effectiveness and safety in our patient group suggest that natalizumab is a treatment for refractory patients or those with aggressive types of multiple sclerosis, although we do not yet know about its long-term effects and the evolution of the appearance of neutralising antibodies.

Vasculitis in systemic lupus erythematosus (SLE)--assessment of peripheral blood mononuclear cell activation and the degree of endothelial dysfunction: initial report.

BACKGROUND: Inflammatory-immune changes in the vascular endothelium are one of the main factors initiating vessel wall damage. Enhanced expression of endothelial adhesion molecules and their receptors on the surface of circulating leukocytes seems to play an important role in the pathogenesis of vasculitis. Increasing evidence indicates endothelial cell activation/damage in SLE. In patients with SLE complicated by vasculitis, enhanced expression of integrin activation markers on the surface of peripheral blood mononuclear cells (PBMCs) has been reported. It seems relevant to assess the mechanisms of inflammatory response involving PBMCs and endothelial cells at particular stages of SLE microangiopathy. AIM: The main aim was to assess the surface expressions of the integrin adhesion molecules VLA-4 (CD49d) and LFA-1 (CD11a) on PBMCs as well as the number of circulating endothelial cells (CECs) in patients with SLE and complications related to inflammatory microangiopathy and to determine whether these parameters vary depending on disease activity. PATIENTS: Twenty-nine women with SLE (mean age: 38.72+/-10.23 years) were divided into subgroup I: those with severe disease activity according to the modified disease activity index SLEDAI, characterized by the presence of inflammatory microangiopathy-related complications such as systemic central nervous system affection and/or vasculitis and/or nephritis (15 women, mean age: 38.33+/-11.02 years), and subgroup II: patients with mild or moderate disease activity according to SLEDAI and without vascular complications (14 women, mean age: 39.14+/-9.72 years). METHODS: Expressions of VLA-4 and LFA-1 on the surface of peripheral blood lymphocytes and monocytes were assessed by flow cytometry using monoclonal antibodies. CECs (a marker of endothelial damage) were isolated from peripheral blood with anti-CD146(S-Endo 1)-coated immunomagnetic Dynabeads. Tests for the lupus anticoagulant, antinuclear antibody, anti-dsDNA, and anticardiolipin antibody were performed in every study subject by ELISA. Erythrocyte sedimentation rate and serum levels of fibrinogen, C-reactive protein, the complement components C3 and C4, urea, creatinine, and uric acid were determined by standard methods. Peripheral blood counts and a general urinalysis were also performed. RESULTS: The mean CEC count was significantly higher in SLE patients than in the control group (15.29+/-12.10 vs. 3.08+/-1.46 cells/ml, p<0.001). CEC counts was notably elevated in patient subgroup II compared with the control group (9.14+/-5.16 vs. 3.08+/-1.46 cells/ml, p<0.05) and in subgroup I compared with subgroup II (21.03+/-13.96 vs. 9.14+/-5.19 cell/ml, p<0.05). In patients with severe SLE flares, CEC count visibly correlated with disease activity assessed by SLEDAI score (R=0.92, p<0.001). The expressions of VLA-4 and LFA-1 on peripheral blood lymphocytes in both patient subgroups were significantly higher than in the control group (subgroup I vs. controls: 1.70+/-1.56 vs. 0.39+/-0.26%, p<0.05, and 1.97+/-2.60 vs. 0.67+/-0.83%, p<0.05; subgroup II vs. controls: 1.71+/-1.04 vs. 0.39+/-0.26%, p<0.001, and 3.32+/-2.48 vs. 0.67+/-0.83%, p<0.05, for VLA-4 and LFA-1, respectively). There was no significant difference between the two subgroups of patients (1.70+/-1.56 vs. 1.71+/-1.04%, p>0.05, and 1.97+/-2.60 vs. 3.32+/-2.48%, p>0.05, respectively). Similarly, the surface expression of LFA-1 on circulating monocytes in patients in both subgroups was notably enhanced over that of the control group (91.44+/-16.00 vs. 84.95+/-19.86%, p<0.05, and 90.11+/-10.34 vs. 84.95+/-19.86%, p<0.05, in subgroups I and II respectively) and was comparable in both subgroups of patients (91.44+/-16.00 vs. 90.11+/-10.33%, p>0.05). The surface expression of VLA-4 on peripheral blood monocytes was considerably higher in patients with severe disease activity than in the control group and in patients with less active disease (77.10+/-13.56 vs. 64.90+/-19.13%, p<0.05, and 77.10+/-13.56 vs. 63.40+/-20.95%, p<0.05, respectively). However, there was no significant difference between patients with mild or moderate disease activity and the control group (63.40+/-20.95 vs. 64.90+/-19.13%, p>0.05). CONCLUSIONS: 1) The number of CECs increases in the course of SLE and correlates with disease activity, indicating progressive endothelial damage.2) The expressions of VLA-4 and LFA-1 on the surface of peripheral blood lymphocytes as well as that of LFA-1 on circulating monocytes are enhanced in SLE patients regardless of disease activity. 3) The expression of VLA-4 on the surface of circulating monocytes is enhanced only in patients with severe disease activity, characterized by the presence of complications connected with inflammatory microangiopathy, which may indicate that the upregulation of VLA-4 expression in monocytes plays a leading role in the pathogenesis of vasculitis in SLE.

Assessment of selected adhesion molecules and lymphocyte subpopulations in children with IgA nephropathy.

PURPOSE: The aim of the study was to assess the expression of selected adhesion molecules on mononuclear cells of peripheral blood and lymphocyte subpopulations in children with IgA nephropathy (IgAN). MATERIAL AND METHODS: 14 children with IgAN and 20 healthy controls were included in the study. Flow cytometry was used to determine the expression of such adhesion molecules as L selectin (CD62L), VLA-4 integrin (CD49d), intracellular molecule ICAM-1 (CD54) and cytotoxic lymphocyte molecule CTLA-4 (CD152), as well as the lymphocyte antigens: CD3, CD4, CD8, CD19, CD1656 (NK), CD4 and CD8 RO+ and RA+. RESULTS: The findings revealed that the expression of the adhesion molecules VLA-4 and CTLA-4 did not differ from that of the healthy controls (p > 0.05). However, the expression of CD62L (L-selectin) was increased (p < 0.05). The expression of ICAM-1 was reduced, but not significantly, compared to the control group (p > 0.05). We found a decrease in the expression of NK cells (CD1656) and CD4/CD8 ratio, and an increase in CD8 cells (p < 0.05). In the group of 9/14 children, with proteinuria over 1.0 g/24 hours, a decreased expression of CD4 was additionally found (p < 0.05). CONCLUSIONS: The children with IgAN show: 1. Changes in peripheral lymphocyte subpopulations involving an increase in CD8 cells and a decrease in CD1656(NK) cells, a reduction in the CD4/CD8 ratio, and additionally in cases with proteinuria a reduction in CD4 cell count, 2. Increased expression of L-selectin (CD62L) on peripheral blood mononuclear cells.

Decreased expression of membrane alpha4beta1, alpha5beta1 integrins and transferrin receptor on erythroblasts in splenectomized patients with beta-thalassemia intermedia. Parallel assessment of serum soluble transferrin receptors levels.

Dysfunction of cell membrane is a recognized consequence of the pathogenetic process underlying the beta-thalassemia syndromes and it is reasonable to hypothesize that surface structures crucial for the development of erythroid lineage may also be affected. The study included six adult splenectomized patients with beta-thalassemia intermedia. Expression of alpha4beta1 integrin (CD49d/CD29), alpha5beta1 integrin (CD49e/CD29) and transferrin receptor (CD71) on peripheral blood and bone marrow erythroblasts and on erythroid precursors grown in vitro was studied by flow cytometry and immunocytochemistry. Serum soluble transferrin receptor levels (sCD71) were also measured with enzyme-linked immunosorbent assay. In beta-thalassemic patients, significant reduction of CD49d, CD29 and CD71 expression was found in peripheral blood nucleated red cells, compared to patients presenting with erythroblasts in the circulation because of other diseases. Marrow erythroblasts were also deficient for the same molecules against the erythroblasts in iron deficiency anemia. All molecules tested were greatly diminished on erythroid precursors developed in vitro from the patients' cells. Serum sCD71 levels were much higher in thalassemic patients in comparison to both patients with iron deficiency anemia and healthy individuals. The loss of certain integrins and CD71 from erythroid precursors in beta-thalassemia intermedia could be attributed to a generalized membrane dysfunction, perhaps affecting the integrity of their transmembrane domains. The elevation of serum sCD71 levels may be the result of the increased red cell lineage turnover or, alternatively, may indicate increased shedding from the cells to prevent iron overload. In any case, further molecular study of the membrane components is warranted to provide a better understanding of the pathogenetic process in beta-thalassemia syndromes.

An assessment of the mechanistic differences between two integrin alpha 4 beta 1 inhibitors, the monoclonal antibody TA-2 and the small molecule BIO5192, in rat experimental autoimmune encephalomyelitis.

Integrin alpha 4 beta 1 plays an important role in inflammatory processes by regulating the migration of lymphocytes into inflamed tissues. Here we evaluated the biochemical, pharmacological, and pharmacodynamic properties and efficacy in experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, of two types of alpha 4 beta 1 inhibitors, the anti-rat alpha 4 monoclonal antibody TA-2 and the small molecule inhibitor BIO5192 [2(S)-[[1-(3,5-dichloro-benzenesulfonyl)-pyrrolidine-2(S)-carbonyl]-amino]-4-[4-methyl-2(S)-(methyl-[2-[4-(3-o-tolyl-ureido)-phenyl]-acetyl]-amino)-pentanoylamino]-butyric acid]. TA-2 has been extensively studied in rats and provides a benchmark for assessing function. BIO5192 is a highly selective and potent (KD of <10 pM) inhibitor of alpha 4 beta 1. Dosing regimens were identified for both inhibitors, which provided full receptor occupancy during the duration of the study. Both inhibitors induced leukocytosis, an effect that was used as a pharmacodynamic marker of activity, and both were efficacious in the EAE model. Treatment with TA-2 caused a decrease in alpha 4 integrin expression on the cell surface, which resulted from internalization of alpha 4 integrin/TA-2 complexes. In contrast, BIO5192 did not modulate cell surface alpha 4 beta 1. Our results with BIO5192 indicate that alpha 4 beta 7 does not play a role in this model and that blockade of alpha 4 beta 1/ligand interactions without down-modulation is sufficient for efficacy in rat EAE. BIO5192 is highly selective and binds with high affinity to alpha 4 beta 1 from four of four species tested. These studies demonstrate that BIO5192, a novel, potent, and selective inhibitor of alpha 4 beta 1 integrin, will be a valuable reagent for assessing alpha 4 beta 1 biology and may provide a new therapeutic for treatment of human inflammatory diseases.

Comparative assessment of the ligand and metal ion binding properties of integrins alpha9beta1 and alpha4beta1.

Integrins alpha9beta1 and alpha4beta1 form a distinct structural class, but while alpha4beta1 has been subjected to extensive study, alpha9beta1 remains poorly characterized. We have used the small molecule N-(benzenesulfonyl)-(L)-prolyl-(L)-O-(1-pyrrolidinylcarbonyl)tyrosine (3) to investigate the biochemical properties of alpha9beta1 and directly compare these properties with those of alpha4beta1. Compound 3 has a high affinity for both integrins with K(D) values of < or =3 and 180 pM for alpha9beta1 in 1 mM Mn2+ (activating) and 1 mM Ca2+ and 1 mM Mg2+ (nonactivating) conditions and < or =5 and 730 pM for alpha4beta1 under the corresponding conditions. Ca2+ treatment promoted the binding of 3 to both integrins (EC50 = 30 microM Ca2+ in both cases). Compound 3 binding to both integrins was also stimulated by the addition of the activating monoclonal antibody TS2/16. These findings indicate that the mechanisms by which metal ions and TS2/16 regulate ligand binding to alpha9beta1 and alpha4beta1 are similar. The binding of 3 to both integrins induced the mAb 9EG7 LIBS epitope, a property consistent with occupancy of the receptor's ligand binding site by 3. But whereas EGTA treatment inhibited the binding of 9EG7 to alpha4beta1, it stimulated the binding of 9EG7 to alpha9beta1. The 9EG7 and TS2/16 effects point to contributions of the beta1-chains on binding. Cross-linking data revealed that the integrin alpha-chains are also involved in binding the small molecule, as stable linkages were observed on both the alpha9 chain of alpha9beta1 and the alpha4 chain of alpha4beta1. Extensive structure-activity analyses with natural and synthetic ligands indicate distinct features of the ligand binding pockets. Most notable was the estimated >1000-fold difference in the affinity of the integrins for VCAM-1, which binds alpha4beta1with an apparent K(D) of 10 nM and alpha9beta1 with an apparent K(D) of >10 microM. Differences were also seen in the binding of alpha9beta1 and alpha4beta1 to osteopontin. Compound 3 competed effectively for the binding of VCAM-1 and osteopontin to both integrins. While these studies show many similarities in the biochemical properties of alpha9beta1 and alpha4beta1, they identify important differences in their structure and function that can be exploited in the design of selective alpha9beta1 and alpha4beta1 inhibitors.

A homogeneous fluorometric assay for measuring cell adhesion to immobilized ligand using V-well microtiter plates.

We have developed a homogeneous high-capacity assay format for measuring integrin- and selectin-dependent cell binding to immobilized ligand using V-well microtiter plates. 2',7'-Bis(2-carboxyethyl)-5-(and-6)-carboxylfluorescence, acetoxymethylester-labeled cells are added to ligand-coated V-shaped microtiter wells. Bound cells are separated from free cells using centrifugal force to produce shear stress. Nonadherent cells accumulate in the nadir of the well and are measured using a fluorescence plate reader. Antibody or low-molecular-weight inhibitors of either the ligand or the cell surface receptor result in less cell binding, more cells in the pellet, and increased signal. The optimization and validation of the very late antigen-4/vascular cell adhesion molecule-1 assay is described in detail. We demonstrate that this assay can be rapidly adapted to measure other integrin- and selectin-mediated interactions. This assay format has several advantages over conventional assays. The centrifugal process is biologically relevant and eliminates the washing steps to remove nonadherent cells that can cause well-to-well and plate-to-plate variation. Because the assay is robust with a high signal-to-noise ratio and low variability, it is ideally suited for studying multiple parameters of cell adhesion and for high capacity screening.

Very late antigen (VLA) expression by normal and neoplastic human plasma cells; including an assessment of antibodies submitted to the Vth International Workshop on Leucocyte Differentiation Antigens using human myeloma cell lines.

The biology of normal plasma cells and the pathophysiology of human multiple myeloma remain poorly understood. Functional assays are scarce and at present cell phenotyping is providing the most information about how human plasma cells may behave. Three different types of human plasma cells: normal, fresh neoplastic myeloma cells and plasma cell lines, have been studied for their reactivity with antibodies to the beta-1 integrins (Very Late Antigens; VLAs), including a panel obtained from the Vth International Workshop on Leucocyte Differentiation Antigens. Most plasma cell targets express VLA-4 (CD49d positive) and the common beta chain recognized by CD29. CD49e (VLA-5) was occasionally positive. Other VLAs were not usually expressed. These data suggest the wide use by plasma cells of VLA-4, possibly as a ligand with fibronectin and high endothelial venules (HEV). Of other adhesion structures expressed by plasma cells, only CD44 is seen as frequently, and this is also a HEV ligand.