Monoclonal antibodies to human laminin alpha 4 chain globular domain inhibit tumor cell adhesion and migration on laminins 411 and 421, and binding of alpha 6 beta 1 integrin and MCAM to alpha 4-laminins

Peer reviewe

Investigations of a Possible Chemical Effect of Salvadora persica

Salvadora persica is commonly used chewing sticks in many parts of the world as an oral hygiene tool. This study measured the amount of benzyl isothiocyanate (BITC) released into the mouth and assessed its retention time in saliva. The study also tested if the released amount of BITC could potentially be antibacterial or cytotoxic. Twelve subjects brushed their teeth with fresh Miswak once, twice, and four times. The amount of BITC in the saliva and in the used brushes was quantified using gas chromatography-mass spectrometry. The antibacterial effect of BITC and Miswak essential oil (MEO) was tested against Haemophilus influenzae, Aggregatibacter actinomycetemcomitans, and Porphyromonas gingivalis. The cytotoxic effect on gingival fibroblasts and keratinocytes was tested using MTT. The highest amount of the active compounds was detected in saliva after using the Miswak tip for once and immediately. It significantly decreased when the Miswak tip was used more than once and thus after 10 min. The growth of the tested bacteria was inhibited by MEO and BITC in a dose dependent manner, P. gingivalis being the most sensitive. MTT assay showed that BITC and MEO were cytotoxic towards gingival fibroblasts while oral keratinocytes showed resistance. This study suggests that the Miswak tip should be cut before each use to ensure the maximum effect

A Novel Monoclonal Antibody to Human Laminin alpha 5 Chain Strongly Inhibits Integrin-Mediated Cell Adhesion and Migration on Laminins 511 and 521

Peer reviewe

Expression, recognition and usage of laminin-8 (a4b1g1, Lm-411) by monocytes and neutrophils

Laminins (LNs) are a family of large alphabetagamma heterotrimeric glycoproteins found in all basement membranes (BMs). They are expressed in a tissue- and developmental stagespecific manner and implicated in vital cellular functions, including cell adhesion, migration and signaling. So far, eleven laminin chains (5alpha, 3beta and 3gamma) that assemble into 15 isoforms (LN-1 to 15, Lm-111 to Lm-523) have been identified. LN-8 (alpha4beta1gamma1, Lm-411) is a major LN isoform of vascular endothelial BM. Its expression and functional importance in blood platelets and lymphocytes have been documented. However, expression, recognition and utilization of LNs by blood monocytes and neutrophils are poorly understood. Monocytes and neutrophils originate from a common myeloid progenitor cell and are crucial cellular elements in both innate and adaptive immunity. In response to inflammatory signals, these leukocytes extravasate and migrate to the affected tissue. Leukocyte extravasation is a multi-step process involving sequential participation of various adhesion molecules. While the initial steps, such as leukocyte interaction with endothelium, are well characterized, the subsequent steps of leukocyte extravasation, such as migration through the vascular BM, are not well defined. In this thesis, efforts have been made to detect, isolate and functionally characterize LN-8 in monocytes and neutrophils, and to define the role of alpha4 LNs in leukocyte migration and extravasation. First, we determined the chain specificity of 16 commonly used monoclonal antibodies (mAbs) to human LN by ELISA, Western blotting, and immunoprecipitation using recombinant (r) LNbeta1 and LNgamma1 chains. In addition, eight novel mAbs to LNalpha4 chain were generated and characterized. By immunohistochemistry, differential distribution of LNalpha4 chain in developing and adult human tissues was found. LNalpha4 was mainly localized in tissues of mesodermal origin, such as endothelial BM. By indirect immunofluorescence, LN-8 chains were detected in permeabilized monocytes and neutrophils, and intact LN-8 was isolated from these cells. This LN isoform was synthesized by monoblastoid cells and secreted by stimulated neutrophils. mAbs to LNalpha4 chain inhibited neutrophil migration through human serum albumin coated inserts, suggesting participation of the endogenous LN-8 in the cell migration. Monoblastoid JOSK-I cells adhered constitutively to rhLN-8 via alpha6beta1 and, to a lower extent, beta2 integrins, whereas stimulated neutrophils adhered to rhLN-8, rhLN-10 (alpha5beta1gamma1, Lm-511), and mouse LN-1 (alpha1beta1gamma1, Lm-111) via alphaMbeta2 integrin. rhLN-8 strongly promoted monocyte and neutrophil migration both in the absence and presence of chemoattractants, and the migration-promoting activity on neutrophils was mediated via alphaMbeta2 and, to a lower extent, 01 integrins. Compared to rhLN-8, several commercial LN preparations isolated from human placenta displayed in general lower migration-promoting activity on neutrophils. These preparations contained fragmented LN chains, a mixture of LN isoforms, and/or containing fibronectin. In LNalpha4 deficient mice, neutrophil recruitment to inflamed tissue was significantly impaired. rhLN-8 also protected neutrophils against spontaneous apoptosis. Altogether, the results from these studies indicate that both monocytes and neutrophils express LN-8, and that this laminin isoform can be secreted by the cells. In addition, LN-8 plays a major role in the physiology of myeloid cells, including their adhesion, migration, extravasation and survival. The results also indicate that alphaMbeta2 integrin may constitute a novel receptor for LN-8 and other LN isoforms

mAb 8G9 to LMα5 chain strongly inhibits binding of isolated α3β1 integrin to immobilized laminins 511 and 521.

<p>A) α3β1 integrin binds rhLMs 511 and 521, but not rhLM411. B) Effect of mAbs to LMα5 chain on the binding of α3β1 integrin to rhLM511. C) Effect of mAbs to LMα5 chain on the binding of α3β1 integrin to rhLM521. Statistical analyses (Student's <i>t</i>-test) including mean and SD were calculated, as well as level of significance of integrin binding to rhLMs 411, 511 and 521 when compared to HSA (A), and effect of antibodies when compared to mouse IgG on integrin binding to rhLMs 511 (B) and 521 (C) (*, p<0.05; **, p<0.01; ***, p<0.001).</p

mAb 8G9 to LMα5 chain strongly inhibits α3β1/α6β1 integrin-mediated cell adhesion to laminin-511 and, together with LMα5 mAb 4C7, to laminin-521.

<p>A) KG1C glioma, B) BE melanoma and C) MDA-MB-231 breast carcinoma cells were used. Cells were allowed to adhere to surfaces coated with rhLMs 511 or 521 (20 μg/ml) for 1 hour at 37°C. Statistical analyses (Student's <i>t</i>-test) including mean and SD were calculated, as well as level of significance comparing antibodies to mouse IgG (*, p<0.05; **, p<0.01; ***, p<0.001).</p

mAb 8G9 to LMα5 chain strongly inhibits α3β1/α6β1 integrin-mediated cell migration on laminin-511 and, together with LMα5 mAb 4C7, on laminin-521.

<p>A) KG1C glioma, B) BE melanoma and C) MDA-MB-231 breast carcinoma cells were used. Cells were allowed to migrate for 18 hours at 37°C through 8-μm pore-membranes coated with rhLMs 511 or 521 (20 μg/ml) on the lower surface. Statistical analyses (Student's <i>t</i>-test) including mean and SD were calculated, as well as level of significance comparing antibodies to mouse IgG (*, p<0.05; **, p<0.01; ***, p<0.001).</p

mAb 4E10 to a LMβ1 chain epitope near the globular domain of laminin-511 hinders the binding of mAb 8G9.

<p>A) mAb 4C7 and other LMα5 antibodies minimally affect the binding of mAb 8G9 to rhLM511. B) mAb 4E10 largely competes with the binding of mAb 8G9 to rhLM511. Statistical analyses (Student's <i>t</i>-test) including mean and SD were calculated, as well as level of significance of the effect of the mAbs on the binding of biotinylated-8G9 to rhLM511 when compared to mouse IgG. Competing antibodies were used at 50x higher concentration than biotinylated-8G9 (*, p<0.05; **, p<0.01; ***, p<0.001).</p

Reactivity of novel mAbs to LMα5 chain as measured by ELISA, Western blotting and immunoprecipitation.

<p>A) Reactivity of the antibodies with rhLMs 411, 511 and 521 by ELISA. B) Reactivity of the antibodies with rhLM511 by Western blotting under reducing conditions. C) Ability of the antibodies to immunoprecipitate laminin-511 from A549 cells' conditioned medium and detection of the LMα5 chain by Western blotting with mAb 4B5. Bands of 300/350 kDa corresponding to LMα5 chain were detected with some of the antibodies.</p