Structure and function of Parkin E3 ubiquitin ligase reveals aspects of RING and HECT ligases.

Parkin is a RING-between-RING E3 ligase that functions in the covalent attachment of ubiquitin to specific substrates, and mutations in Parkin are linked to Parkinson's disease, cancer and mycobacterial infection. The RING-between-RING family of E3 ligases are suggested to function with a canonical RING domain and a catalytic cysteine residue usually restricted to HECT E3 ligases, thus termed 'RING/HECT hybrid' enzymes. Here we present the 1.58 Å structure of Parkin-R0RBR, revealing the fold architecture for the four RING domains, and several unpredicted interfaces. Examination of the Parkin active site suggests a catalytic network consisting of C431 and H433. In cells, mutation of C431 eliminates Parkin-catalysed degradation of mitochondria, and capture of an ubiquitin oxyester confirms C431 as Parkin's cellular active site. Our data confirm that Parkin is a RING/HECT hybrid, and provide the first crystal structure of an RING-between-RING E3 ligase at atomic resolution, providing insight into this disease-related protein.

Suppression of acute experimental allergic encephalomyelitis with a small molecule inhibitor of alpha4 integrin.

PURPOSE: To determine the efficacy of a small molecule inhibitor of alpha4 integrin (CT301) at reversing the clinical, pathological and MR-detectable deficits associated with the acute phase of experimental allergic encephalomyelitis (EAE). MATERIALS AND METHODS: EAE was induced in 36 female Hartley guinea pigs, and the treatment period was from day 11 to day 17 post-immunization. Animals received either saline (n = 12), anti-alpha4 integrin antibody (AN100226m; n = 12) or CT301 (n = 12). T2-weighted fast spin echo and T1-weighted pre- and post-contrast scans were performed at the beginning (day 11) and end (day 18) of the treatment period, and scored for cerebral inflammation and gadolinium enhancement. T1-weighted images were further analyzed to quantify this enhancement as a measure of blood-brain barrier integrity. Dissected CNS was evaluated for inflammation and demyelination. RESULTS: CT301 successfully reversed two clinical indicators of disease over the course of the treatment period. These animals showed decreased T2-weighted abnormalities, as well as a reduction in gadolinium leakage on T1-weighted images. Meningeal and perivascular inflammation was decreased by anti-alpha4 integrin treatments. CONCLUSION: CT301 effectively reverses the clinical, pathological and MR-detectable deficits of acute EAE, and may therefore be a promising therapeutic agent in multiple sclerosis (MS).

Spontaneous remyelination following prolonged inhibition of alpha4 integrin in chronic EAE.

Inhibition of alpha(4)beta(1) integrin blocks immune cell influx into the CNS providing benefit to patients with multiple sclerosis and in animal model systems. We have used this mechanism to examine whether the presence of inflammatory cells suppresses spontaneous myelin repair in experimental autoimmune encephalomyelitis. We observed (1) 87% of plaques showed remyelination after 40 days of treatment; (2) myelin repair occurred in half of the total lesion area; (3) half of the animals regained motor function. There was no significant repair or gain of motor function in vehicle-treated animals. Therefore, prolonged inhibition of CNS inflammation, in the absence of targeted myelin repair, facilitates mechanisms of spontaneous remyelination.

Prolonged reversal of chronic experimental allergic encephalomyelitis using a small molecule inhibitor of alpha4 integrin.

CNS leukocytic invasion in experimental allergic encephalomyelitis (EAE) depends on alpha4beta1 integrin/vascular cell adhesion molecule-1 (VCAM-1) interactions. A small molecule inhibitor of alpha4beta1 integrin (CT301) was administered to guinea pigs in the chronic phase (>d40) of EAE for 10, 20, 30 or 40 days. CT301 elicited a rapid, significant improvement in the clinical and pathological scores that was maintained throughout the treatment period. A progressive loss of cells in the spinal cord of treated animals confirmed the resolution of inflammation associated with clinical recovery. Therefore, prolonged inhibition of alpha4beta1 integrin caused a sustained reversal of disease pathology in chronic EAE and may be similarly useful in MS.

The Src kinase p56(lck) up-regulates VLA-4 integrin affinity. Implications for rapid spontaneous and chemokine-triggered T cell adhesion to VCAM-1 and fibronectin.

In circulating lymphocytes, the VLA-4 integrin preexists in multiple affinity states that mediate spontaneous tethering, rolling, and arrest on its endothelial ligand, vascular cell adhesion molecule-1 (VCAM-1). The regulation and function of VLA-4 affinity in lymphocytes has never been elucidated. We show here that p56(lck), the major Src kinase in T cells, is a key regulator of high affinity VLA-4. This high affinity is essential for the rapid development of firm adhesion of resting T cells to VCAM-1 and to their extracellular matrix ligand, fibronectin. Lck-regulated VLA-4 function does not require intact TCR nor several key components of the TCR signaling pathway, including ZAP-70 and SLP-76. Furthermore, stimulation of p56(lck) by the phosphatase inhibitor, pervanadate, triggers firm VLA-4-dependent adhesion to VCAM-1. Although Lck is not required for chemokine receptor signaling to mitogen-activated protein kinase, the presence of Lck-regulated high affinity VLA-4 also facilitates firm adhesion triggered by the chemokine, SDF-1, at short-lived contacts. Surprisingly, bond formation rates, ability to tether cells to VLA-4 ligand, and VLA-4 tether bond stability under shear flow are not affected by VLA-4 affinity or Lck activity. Thus, the ability of high affinity VLA-4 to arrest cells on VCAM-1 under flow arises from instantaneous post-ligand strengthening rather than from increased kinetic stability of individual VLA-4 bonds. These results suggest that p56(lck) maintains high affinity VLA-4 on circulating lymphocytes, which determines their ability to strengthen VLA-4 adhesion and rapidly respond to proadhesive chemokine signals at endothelial sites.

Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease.

One hallmark of Alzheimer disease is the accumulation of amyloid beta-peptide in the brain and its deposition as plaques. Mice transgenic for an amyloid beta precursor protein (APP) mini-gene driven by a platelet-derived (PD) growth factor promoter (PDAPP mice), which overexpress one of the disease-linked mutant forms of the human amyloid precursor protein, show many of the pathological features of Alzheimer disease, including extensive deposition of extracellular amyloid plaques, astrocytosis and neuritic dystrophy. Active immunization of PDAPP mice with human amyloid beta-peptide reduces plaque burden and its associated pathologies. Several hypotheses have been proposed regarding the mechanism of this response. Here we report that peripheral administration of antibodies against amyloid beta-peptide, was sufficient to reduce amyloid burden. Despite their relatively modest serum levels, the passively administered antibodies were able to enter the central nervous system, decorate plaques and induce clearance of preexisting amyloid. When examined in an ex vivo assay with sections of PDAPP or Alzheimer disease brain tissue, antibodies against amyloid beta-peptide triggered microglial cells to clear plaques through Fc receptor-mediated phagocytosis and subsequent peptide degradation. These results indicate that antibodies can cross the blood-brain barrier to act directly in the central nervous system and should be considered as a therapeutic approach for the treatment of Alzheimer disease and other neurological disorders.

Alpha4beta1-integrin activation is necessary for high-efficiency T-cell subset interactions with VCAM-1 under flow.

OBJECTIVE: The purpose of this study was to examine the relationship between alpha4beta1-integrin state of activation on CD4+ T-cell subsets and their adhesive interaction to VCAM-1 under flow. METHODS: Human CD4+ memory and naive T-cells were freshly isolated and effector-helper T-cell subsets. Th1 and Th2 cells, were differentiated in vitro from CD4+ naive T-cells. The expression of activation/ligand induced epitopes on beta1-integrins of each T-cell subset was assessed using mAb HUTS21 and mAb 15/7. T-cell subsets attachment and rolling on VCAM-1 was determined under defined flow conditions and the rates of attachment (ka), accumulation, and instantaneous rolling velocities were correlated to their beta1-integrin activation epitope expression. RESULTS: A subset of memory T-cells constitutively express activation/ligand induced epitopes on beta1-integrins recognized by mAb HUTS21 and 15/7, whereas expression levels on naive T-cells is low or not detectable. Consistent with an activated phenotype, memory T-cells exhibit significantly higher rates of attachment and accumulation on VCAM-1 under flow as compared to naive T-cells. Interestingly, the expression of activation/ligand induced epitopes on beta1-integrins on Th2 cells and the ability of these cells to interact with VCAM-1 are comparable to memory T-cells. In contrast, Th1 cells did not interact as efficiently with VCAM-1, which correlated with lower expression of activation/ligand induced epitopes on these cells. VCAM-1 interactions are inhibited completely by pretreatment of the T-cells with blocking mAb to alpha4-integrins or beta1-integrins, indicating that alpha4beta1 is the predominant T-cell integrin involved. CONCLUSIONS: Memory T-cells express constitutively active alpha4beta1-integrins, as compared to naive T-cells, which mediate high rates of initial attachment and sustained high-affinity adhesive interactions with VCAM-1 under flow conditions in vitro. Similarly, in vitro differentiated Th2 cells but not Th1 cells, which also express elevated levels of activated alpha4beta1-integrins, are capable of sustaining high-affinity adhesive interactions with VCAM-1. The differences observed in beta1-integrin activation on T-cell subsets may underlie selective recruitment patterns of T-cell subsets in vivo.

Prevention and reduction of AD-type pathology in PDAPP mice immunized with A beta 1-42.

In AD certain brain structures contain a pathological density of A beta protein deposited into plaques. The effect of genetic mutations found in early onset AD patients was an overproduction of A beta 42, strongly suggesting that overproduction of A beta 42 is associated with AD. We hypothesized that an immunological response to A beta 42 might alter its turnover and metabolism. Young PDAPP transgenic mice were immunized with A beta 1-42, which essentially prevented amyloid deposition; astrocytosis was dramatically reduced and there was reduction in A beta-induced inflammatory response as well. A beta 1-42 immunization also appeared to arrest the progression of amyloidosis in older PDAPP mice. A beta immunization appears to increase clearance of amyloid plaques, and may therefore be a novel and effective approach for the treatment of AD.

alpha9beta1 integrin is expressed on human neutrophils and contributes to neutrophil migration through human lung and synovial fibroblast barriers.

Accumulation of leukocytes in inflamed tissue involves their migration through vascular endothelium and then in the connective tissue. Recently we utilized a barrier of human synovial, dermal, and lung fibroblasts (HSF, HDF, and HLF) grown on polycarbonate filters as a model of human polymorphonuclear leukocyte (PMN) migration through connective tissue. The beta2 integrins (CD 11/ CD18) and alpha4, alpha5, and alpha6beta1 (VLA-4, -5, and -6) integrins each contributed to this PMN migration. Here we report that on human blood leukocytes, alpha9beta1 (VLA-9) is expressed only on PMNs and that it is up-regulated after PMN activation. Based on monoclonal antibody (mAb) blocking studies, alpha9beta1 integrin contributed to C5a-induced PMN migration through fibroblast (HLF and HSF) barriers. This role was apparent only when alternate mechanisms such as CD18, alpha4, alpha5, and alpha6beta1 integrins were blocked and then mAb to alpha9beta1 integrin inhibited the residual PMN migration (by 40-50%) through the HLF or HSF barrier, resulting in > or = 75% inhibition overall. In contrast, PMN migration across interleukin-1-activated endothelium (HUVEC) in response to a C5a gradient, which is partly (30-40%) via CD11/CD18-independent mechanisms, was not inhibited by adhesion blocking by mAbs to alpha4, alpha5, alpha6, and alpha9beta1 even in combination. These results indicate that alpha9beta1 integrin on PMN may have a special role, in conjunction with other beta1 integrins, in mediating PMN migration in the extravascular space, and may contribute to differential neutrophil function within tissues.

Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse.

Amyloid-beta peptide (Abeta) seems to have a central role in the neuropathology of Alzheimer's disease (AD). Familial forms of the disease have been linked to mutations in the amyloid precursor protein (APP) and the presenilin genes. Disease-linked mutations in these genes result in increased production of the 42-amino-acid form of the peptide (Abeta42), which is the predominant form found in the amyloid plaques of Alzheimer's disease. The PDAPP transgenic mouse, which overexpresses mutant human APP (in which the amino acid at position 717 is phenylalanine instead of the normal valine), progressively develops many of the neuropathological hallmarks of Alzheimer's disease in an age- and brain-region-dependent manner. In the present study, transgenic animals were immunized with Abeta42, either before the onset of AD-type neuropathologies (at 6 weeks of age) or at an older age (11 months), when amyloid-beta deposition and several of the subsequent neuropathological changes were well established. We report that immunization of the young animals essentially prevented the development of beta-amyloid-plaque formation, neuritic dystrophy and astrogliosis. Treatment of the older animals also markedly reduced the extent and progression of these AD-like neuropathologies. Our results raise the possibility that immunization with amyloid-beta may be effective in preventing and treating Alzheimer's disease.

The integrin alpha9beta1 mediates adhesion to activated endothelial cells and transendothelial neutrophil migration through interaction with vascular cell adhesion molecule-1.

The integrin alpha9beta1 has been shown to be widely expressed on smooth muscle and epithelial cells, and to mediate adhesion to the extracellular matrix proteins osteopontin and tenascin-C. We have found that the peptide sequence this integrin recognizes in tenascin-C is highly homologous to the sequence recognized by the closely related integrin alpha4beta1, in the inducible endothelial ligand, vascular cell adhesion mole-cule-1 (VCAM-1). We therefore sought to determine whether alpha9beta1 also recognizes VCAM-1, and whether any such interaction would be biologically significant. In this report, we demonstrate that alpha9beta1 mediates stable cell adhesion to recombinant VCAM-1 and to VCAM-1 induced on human umbilical vein endothelial cells by tumor necrosis factor-alpha. Furthermore, we show that alpha9beta1 is highly and selectively expressed on neutrophils and is critical for neutrophil migration on VCAM-1 and tenascin-C. Finally, alpha9beta1 and alpha4 integrins contribute to neutrophil chemotaxis across activated endothelial monolayers. These observations suggest a possible role for alpha9beta1/VCAM-1 interactions in extravasation of neutrophils at sites of acute inflammation.

Differential regulation of beta1 integrins by chemoattractants regulates neutrophil migration through fibrin.

Chemoattractants differ in their capacity to stimulate neutrophils to adhere to and to migrate through matrices containing fibrin. Formyl methionyl leucyl phenylalanine (fMLP) stimulates neutrophils to adhere closely to, but not to migrate into, fibrin gels. Leukotriene B4 (LTB4) stimulates neutrophils to adhere loosely to and to migrate through fibrin gels. We report that alpha5beta1 integrins regulate the different migratory behaviors on fibrin gels of neutrophils in response to these chemoattractants. fMLP, but not LTB4, activated neutrophil beta1 integrins, as measured by binding of mAb 15/7 to an activation epitope on the beta1 integrins. Antibodies or peptides that block alpha5beta1 integrins prevented fMLP-stimulated neutrophils from forming zones of close apposition on fibrin and reversed fMLP's inhibitory effect on neutrophil chemotaxis through fibrin. In contrast, neither peptides nor antibodies that block beta1 integrins affected the capacity of LTB4-stimulated neutrophils to form zones of loose apposition or to migrate through fibrin gels. These results suggest that chemoattractants generate at least two different messages that direct neutrophils, and perhaps other leukocytes, to accumulate at specific anatomic sites: a general message that induces neutrophils to crawl and a specific message that prepares neutrophils to stop when they contact appropriate matrix proteins for activated beta1 integrins.

Endothelial selectins and alpha4 integrins regulate independent pathways of T lymphocyte recruitment in the pulmonary immune response.

The cell adhesion molecules (CAMs) required for T lymphocyte recruitment during pulmonary immune responses have not been defined. Our laboratories recently reported that intratracheal (IT) challenge of sensitized mice with SRBC induced prolonged expression of vascular P-selectin, E-selectin, and VCAM-1, particularly in areas of mononuclear leukocyte infiltration. A surge in the number of circulating T lymphocytes expressing selectin ligands preceded the peak accumulation of T cells in the lung. In addition, a significant percentage of the T cells recovered from the lung expressed selectin ligands as well. The current study demonstrates that cultured T lymphoblasts use both selectin ligands and alpha4 integrins to enter the airspace and interstitium during the response to SRBC. Fluorescently labeled T lymphoblasts, derived via activation on CD3 and growth in low dose IL-2, showed inflammation-specific recruitment into lungs harvested 24 h after cell infusion. Their flux paralleled the accumulation of host lymphocytes in the lung, with both peaking 2 to 4 days after SRBC challenge. Trafficking studies conducted over a 24-h period during peak lymphocyte accumulation in the lungs revealed preferential recruitment of labeled T lymphoblasts expressing P- and E-selectin ligands. In addition, mAb blockade of the alpha4 integrins and targeted deletion of an alpha(1,3)fucosyltransferase essential for selectin ligand synthesis each reduced labeled T lymphoblast trafficking to a significant degree. Furthermore, alpha4 integrin blockade reduced the trafficking of the selectin ligand-deficient cells into the airspace, confirming that its contribution is in part independent from the vascular selectins. These findings imply that both selectin ligands and alpha4 integrins participate in T lymphoblast recruitment during the pulmonary immune response to IT SRBC.

In vivo treatment with anti-alpha4 integrin suppresses clinical and pathological evidence of Borna disease virus infection.

Borna disease virus (BDV) infection of the rat brain induces a severe T-lymphocyte mediated inflammatory response that parallels the course of clinical Borna disease. In other models of CNS inflammation, the recruitment of T-lymphocytes from the circulation to sites of inflammation is believed to be directed, in part, by the cellular adhesion molecules alpha4 beta1 integrin (expressed on T-lymphocytes) and its ligand VCAM-1 (expressed on blood brain barrier endothelium). Since BDV-specific T-lymphocytes are known to express the alpha4 beta1 integrin, we examined the effect of in vivo treatment with an anti-alpha4 integrin monoclonal antibody (GG5/3) on the development of BDV-specific encephalitis and Borna disease. Here, we report that the inhibition of alpha4 integrin provided significant clinical benefit in slowing the progression of Borna disease. Antibody treatment greatly reduced the immune cell infiltrates in the CNS of BDV-infected animals, but we found that this inhibition of the immune response did not result in enhanced viral levels.

Intracellular calcium requirements for beta1 integrin activation.

Human polymorphonuclear leukocytes (PMNs) express beta1 integrins that mediate adhesion to extracellular matrix proteins following stimulation with agonists that induce an increase in intracellular calcium. The purpose of these studies was to determine the contribution made by alterations in intracellular calcium ([Ca++]i) to inside-out activation of beta1 integrins using dimethyl sulfoxide (DMSO)-differentiated granulocytic HL60 cells as a model of human PMNs. Activation of beta1 integrins was determined by measuring the expression of an activation-dependent epitope on the beta1 subunit that is recognized by monoclonal antibody (mAb) 15/7. Exposure of granulocytic HL60 cells to calcium ionophore ionomycin (800 nM) alone did not increase the binding of mAb 15/7 to the cell surface, nor did it increase beta1 integrin-mediated adhesion of the cells to fibronectin. Similarly, exposure of the cells to the direct protein kinase C (PKC) activator, dioctanoylglycerol (di-C8) at 100 microM, neither increased binding of mAb 15/7 to these cells nor adhesion to fibronectin. Simultaneous addition of di-C8 and ionomycin, however, caused a significant increase in the expression of the 15/7 epitope and cell adhesion, suggesting synergy between elevating [Ca++]i and stimulating PKC in beta1 integrin activation. Chelation of [Ca++]i with Quin-2 and EGTA reduced both basal (unstimulated) expression of the 15/7 epitope and basal adhesion of granulocytic HL60 cells to fibronectin. In addition, chelation of [Ca++]i caused a significant decrease in 15/7 binding and adhesion stimulated by low (1 ng/ml) concentrations of phorbol myristate acetate (PMA). The inhibitory effect of [Ca++]i chelation on beta1 integrin activation was reversed by repleting [Ca++]i with ionomycin in a Ca++-containing buffer, or by the addition of higher concentrations of PMA (10 ng/ml). These data suggest a role for [Ca++]i in inside-out activation of beta1 integrins, probably through a synergistic effect with PKC activation.

A role of chondroitin sulfate glycosaminoglycan binding site in alpha4beta1 integrin-mediated melanoma cell adhesion.

We have previously reported that alpha4beta1 (but not alpha5beta1) integrin-mediated melanoma cell adhesion is inhibited by removal of cell surface chondroitin sulfate glycosaminoglycan (CSGAG), suggesting that melanoma chondroitin sulfate proteoglycan plays a role in modulating the adhesive function of alpha4beta1 integrin. In the current study, we demonstrated that alpha4beta1 integrin binds to CSGAG. We have identified a peptide from within alpha4 integrin termed SG1 (KKEKDIMKKTI) that binds to cell surface melanoma chondroitin sulfate proteoglycan, indicating that SG1 represents a CSGAG binding site within the alpha4 integrin subunit. Soluble SG1 inhibits alpha4beta1 integrin-mediated human melanoma cell adhesion to CS1. Polyclonal antibody generated against the peptide inhibits melanoma cell adhesion to CS1, and the inhibition is reversed by Mn2+ and an activating monoclonal antibody anti-beta1 (8A2). Additionally, pretreatment of cells with anti-SG1 IgG inhibits the expression of the monoclonal antibody 15/7 epitope in the presence of soluble CS1 peptide, suggesting that anti-SG1 IgG prevents ligand binding by alpha4beta1 integrin. These results demonstrate that alpha4beta1 integrin interacts directly with CSGAG through SG1 site, and that this site can affect the ligand binding properties of the integrin.

Structure-function analysis of the integrin beta 7 subunit: identification of domains involved in adhesion to MAdCAM-1.

Beta 7 integrins serve special roles in mucosal immunity. Alpha 4 beta 7-mediated adhesion to mucosal addressin cell adhesion molecule-1 (MAdCAM-1) directs lymphocyte homing to the gut, and alpha E beta 7 mediates binding of lymphocytes to E-cadherin on epithelial cells. Since alpha 4 beta 7 mediates adhesion to MAdCAM-1 but alpha 4 beta 1 does not, we used beta 7/beta 1 chimeras to directly assess the importance of specific regions of beta 7 in MAdCAM-1 binding. We found a region of beta 7 (residues 46-386) that accounts for specificity of alpha 4 beta 7 binding to MAdCAM-1. We also used human/mouse and human/rat chimeric beta 7 subunits to map epitopes recognized by fifteen anti-beta 7 mAbs. Six of seven Abs that block adhesion to MAdCAM-1 and E-cadherin (Fib 21, 22, 27, 30, 504; Act-1) mapped to amino acid residues 176-250. Residues 176-250 lie within the region of beta 7 that specifies MAdCAM-1 binding and also within a region that has a predicted structure homologous to the metal ion-dependent adhesion site (MIDAS) domains of the integrin subunits alpha L and alpha M. Three new Abs that recognize beta 7 in the presence of Mn2+, but not Ca2+, and promote adhesion to MAdCAM-1, mapped to amino acids 46-149. One blocking and five other Abs mapped to other regions (amino acids 387-725). We conclude that a MIDAS-like domain serves a critical role in beta 7 integrin-mediated adhesion.

Humanization of a mouse antibody against human alpha-4 integrin: a potential therapeutic for the treatment of multiple sclerosis.

alpha 4 beta 1 integrin (VLA-4) is crucial for the adhesion of leukocytes to human vascular cell adhesion molecule-1 (VCAM-1) on inflamed endothelium. This cell adhesion event is the first step in leukocyte extravasation across the blood-brain barrier in inflammatory diseases of the central nervous system (CNS) such as experimental autoimmune encephalomyelitis (EAE). Prevention of leukocyte infiltration by antibodies against the alpha 4 integrin, which block the alpha 4 beta 1 integrin/VCAM-1 interaction, have been shown to suppress clinical and pathological features of EAE. In this study, two mouse monoclonal antibodies (MAb) directed against human alpha 4 integrin were analyzed in vitro for their ability to block the interaction of leukocytes with VCAM-1 under different assay conditions. The best blocking MAb, AN100226m, was humanized by complementarily-determining region grafting, associated with human C regions and expressed. We found that modification of two structural determinants (H27 and H29) for the heavy chain CDR1 loop in one hand, and modification of framework amino acid H38, H40 and H44 in the other hand, had no effect on antigen binding. In contrast, modification of a structural determinant (H71) for the heavy chain CDR2 loop resulted in loss of binding. The humanized antibody. AN100226, was equivalent to the murine antibody. AN100226m, in binding to alpha 4 beta 1 integrin and in blocking cell adhesion. More importantly, AN100226 was as effective as AN100226m in the reversal of active EAE in guinea pigs and thus may be useful in the treatment of autoimmune diseases such as multiple sclerosis. AN100226 is currently in phase II clinical trials in the UK for the treatment of multiple sclerosis exacerbations.

Evidence for a prolonged role of alpha 4 integrin throughout active experimental allergic encephalomyelitis.

The leukocyte integrin receptor, alpha 4 beta 1, and its endothelial cell ligand, vascular cell adhesion molecule 1, appear to be of critical importance in the leukocyte trafficking that accompanies CNS damage in experimental allergic encephalomyelitis (EAE). In this study, the persistence of the role for alpha 4 beta 1/VCAM-1 in EAE was established by observing antibody-mediated disease reversal up to 1 month following disease onset. Limited treatment with a monoclonal antibody against alpha 4 integrin, GG5/3, resulted in a significant decrease in both clinical and histopathologic signs. This was not observed in isotype control experiments. In the latter phase of progressive disease, widespread demyelination occurred in the animals that did not respond to 6 days of anti-alpha 4 treatment. These results demonstrate an essential role for alpha 4 beta 1 interactions throughout active EAE and illustrate the difference between reversible clinical deficits caused by edema and irreversible deficits associated with demyelination.