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.

A monoclonal antibody to alpha 4-integrin reverses the MR-detectable signs of experimental allergic encephalomyelitis in the guinea pig.

Experimental allergic encephalomyelitis (EAE) is a T cell-mediated autoimmune disease of the CNS characterized by blood-brain barrier breakdown, cerebral edema formation, lymphocyte infiltration, and demyelination, and is used as an animal model of multiple sclerosis (MS). MR imaging is important for the diagnosis of MS and for the evaluation of potential new therapies. In this study, T2-weighted and T1-weighted contrast-enhanced MR imaging was used to evaluate the effectiveness of an antiadhesion therapy in EAE. Leukocyte-endothelial adhesion at the blood-brain barrier is considered an essential step in the mediation of CNS leukocyte infiltration in EAE. AN100226m, a monoclonal antibody to alpha 4 integrin has been previously shown to reverse the clinical and histologic signs of EAE by blocking this interaction. In the present study, AN100226m treatment in acute EAE significantly decreased contrast enhancement of the CNS parenchyma indicating closure of the blood-brain barrier. The percentage of pixels due to leakage of contrast material in T1-weighted images decreased to < 4% in AN100226m-treated animals whereas it was increased to 15% in control animals (P < .05, Mann-Whitney rank sum test). A decrease in CNS abnormalities associated with cerebral edema and inflammation was also observed on T2-weighted images (P < .05, Mann-Whitney rank sum test). Thus, an antibody to alpha 4 integrin reversed the blood-brain barrier permeability changes characteristic of acute EAE. In addition, the further accumulation of inflammatory edema was prevented and preexisting edema was resolved.

A monoclonal antibody to alpha 4 integrin suppresses and reverses active experimental allergic encephalomyelitis.

In experimental allergic encephalomyelitis (EAE), circulating leukocytes enter the central nervous system (CNS) producing inflammation, myelin damage and paralysis. Prevention of leukocyte infiltration by an antibody against alpha 4 integrin suppressed clinical and pathological features of EAE in the guinea pig. Rapid clearance of leukocytes from the CNS and reversal of clinical findings were observed when anti-alpha 4 treatment was administered during active disease. Clinical improvement was accompanied by a marked decrease in abnormal pathological findings, including demyelination. Therefore anti-alpha 4 is an effective treatment of EAE and may be similarly useful in the treatment of autoimmune diseases such as multiple sclerosis.

Possible mechanism by which stress accelerates growth of virally derived tumors.

Stress accelerates the growth of certain types of tumors. Here we report a possible metabolic mechanism underlying this phenomenon. Some early features of transformation include increased number of glucose transporters and greatly enhanced rates of glucose uptake; this adaptation accommodates the vast energy demands needed for neoplastic growth. In contrast, glucocorticoids, a class of steroid hormones secreted during stress, inhibit glucose transport in various tissues; this is one route by which circulating glucose concentrations are raised during stress. We reasoned that should transformed cells become resistant to this inhibitory action of glucocorticoids, such cells would gain preferential access to these elevated concentrations of glucose. In agreement with this, we observed that Fujinami sarcoma virus-transformed fibroblasts became resistant to this glucocorticoid action both in vitro and in the rat. As a result, under conditions where glucocorticoids exerted catabolic effects upon nontransformed fibroblasts (inhibition of metabolism and ATP concentrations), the opposite occurred in the virally transformed cells. We observe that this glucocorticoid resistance upon transformation cannot be explained by depletion of glucocorticoid receptors; previous studies have suggested that transformation causes an alteration in trafficking of such receptors. Because of this resistance of transformed fibroblasts to the inhibitory effects of glucocorticoids upon glucose transport, glucose stores throughout the body are, in effect, preferentially shunted to such tumors during stress.

A cell culture model of the blood-brain barrier.

Endothelial cells that make up brain capillaries and constitute the blood-brain barrier become different from peripheral endothelial cells in response to inductive factors found in the nervous system. We have established a cell culture model of the blood-brain barrier by treating brain endothelial cells with a combination of astrocyte-conditioned medium and agents that elevate intracellular cAMP. These cells form high resistance tight junctions and exhibit low rates of paracellular leakage and fluid-phase endocytosis. They also undergo a dramatic structural reorganization as they form tight junctions. Results from these studies suggest modes of manipulating the permeability of the blood-brain barrier, potentially providing the basis for increasing the penetration of drugs into the central nervous system.

Glucocorticoids inhibit glucose transport and glutamate uptake in hippocampal astrocytes: implications for glucocorticoid neurotoxicity.

Glucocorticoids (GCs), the adrenal steroid hormones secreted during stress, can damage the hippocampus and impair its capacity to survive coincident neurological insults. This GC endangerment of the hippocampus is energetic in nature, as it can be prevented when neurons are supplemented with additional energy substrates. This energetic endangerment might arise from the ability of GCs to inhibit glucose transport into both hippocampal neurons and astrocytes. The present study explores the GC inhibition in astrocytes. (1) GCs inhibited glucose transport approximately 15-30% in both primary and secondary hippocampal astrocyte cultures. (2) The parameters of inhibition agreed with the mechanisms of GC inhibition of glucose transport in peripheral tissues: A minimum of 4 h of GC exposure were required, and the effect was steroid specific (i.e., it was not triggered by estrogen, progesterone, or testosterone) and tissue specific (i.e., it was not triggered by GCs in cerebellar or cortical cultures). (3) Similar GC treatment caused a decrease in astrocyte survival during hypoglycemia and a decrease in the affinity of glutamate uptake. This latter observation suggests that GCs might impair the ability of astrocytes to aid neurons during times of neurologic crisis (i.e., by impairing their ability to remove damaging glutamate from the synapse).

Glucocorticoids inhibit glucose transport in cultured hippocampal neurons and glia.

A classical action of glucocorticoids (GCs) is to inhibit glucose uptake into various peripheral tissues. Two recent reports suggest that GCs do the same in the brain. Because of the in vivo nature of those studies, it was impossible to determine whether this inhibition occurred at the blood-brain barrier, and/or within neurons and glia themselves. In order to answer this and other mechanistic questions, we examined the effects of GCs on glucose transport in primary brain cultures. We established that uptake of 14C-2-deoxyglucose into hippocampal cultures was linear over a 15-min period and was inhibited by D-glucose and the uptake inhibitor cytochalasin B. Using this system, we found the following. (1) Both corticosterone and dexamethasone inhibited uptake into cultures containing both neurons and glia. (2) The effect was dose-dependent; steroid concentrations in the nanomolar range inhibited uptake from 20 to 33%. The effect was time-dependent, with more than 4 h of steroid exposure needed for inhibition. (3) Non-GC steroids did not inhibit uptake. (4) The GC inhibition seemed to be mediated by the type II (glucocorticoid) corticosteroid receptor. The effect was blocked by a type II, but not a type I (mineralocorticoid) receptor antagonist. Moreover, corticosterone inhibited only at concentrations well above the Kd for the type I receptor. Finally, aldosterone inhibited transport when applied at concentrations that bound heavily to type II receptors. (5) Corticosterone did not inhibit uptake in hypothalamic, cerebellar or cortical cultures, despite the presence of corticosteroid receptors in these cultures. (6) GCs inhibited uptake in both neuron- and glia-enriched hippocampal cultures.

Dexamethasone causes translocation of glucose transporters from the plasma membrane to an intracellular site in human fibroblasts.

To investigate the mechanism by which glucocorticoids inhibit glucose transport in peripheral tissues, we have used a monoclonal antibody directed against the human glucose transporter to measure the relative amounts of glucose transporter polypeptide in various cell fractions of human foreskin fibroblasts after treatment with and without dexamethasone. In cells treated for 4 h with 100 nM dexamethasone, a decrease of 48% in glucose transport was accompanied by a decrease of 40% in the amount of glucose transporter polypeptide in a plasma membrane fraction enriched 10-fold in 5'-nucleotidase activity and a 78% increase in the amount of transporter polypeptide in a fraction of putative intracellular membranes, designated P2. There was no significant change in the amount of transporter polypeptide in whole cell lysates. Insulin (200 nM) stimulated glucose transport in basal fibroblasts by only 9%. However, addition of insulin for 30 min to cells that had been treated for 4 h with dexamethasone completely reversed the dexamethasone-induced decrease in glucose transport and also reversed the dexamethasone-induced changes in glucose transporter polypeptide content of the plasma membrane and P2 fractions. From these observations we conclude that dexamethasone decreases glucose transport by causing translocation of glucose transporters from the plasma membrane to an internal location and that insulin reverses the dexamethasone effect by reversing the translocation.

Biochemical changes in tissue catecholamines and serotonin in duodenal ulceration caused by cysteamine or propionitrile in the rat.

Previous structure-activity and pharmacologic studies with duodenal ulcerogens cysteamine and propionitrile implicating catecholamines in the pathogenesis of duodenal ulceration have now been followed up by dose- and time-response biochemical investigations to assess the importance of monoamines in the development of duodenal ulcers. The concentrations of norepinephrine (noradrenaline), dopamine, serotonin and their metabolites were measured in total brain, brain regions, stomach, duodenum, pancreas and adrenals in the rat. Turnover of catecholamines was determined in rats pretreated with the inhibitor of tyrosine hydroxylase alpha-methyl-p-tyrosine. The duodenal ulcerogens caused a dose- and time-dependent depletion of norepinephrine in virtually all the tissues examined. The effect was maximal 4 or 7 hr after cysteamine or propionitrile, and norepinephrine levels returned to normal in 24 hr. Dopamine changes were selective and often biphasic, e.g., elevation in adrenals, biphasic in brain cortex, hippocampus and midbrain, but uniformly decreasing in glandular stomach and duodenum. In the median eminence dopamine levels decreased by 181 and 324% at 15 and 30 min, respectively, after cysteamine, but neither dopamine nor 3,4-dihydroxyphenylacetic acid was modified in the periventricular nucleus. Serotonin levels were relatively stable, revealing slight elevations or no changes in most of the tissues. The turnover of norepinephrine was accelerated by both chemicals in virtually all brain regions, but dopamine turnover was affected only in a few areas, e.g., in the corpus striatum and medulla oblongata cysteamine decreased dopamine turnover, whereas propionitrile first (at 1 hr) accelerated then (at 8 hr) significantly suppressed it.(ABSTRACT TRUNCATED AT 250 WORDS)

Subacute and chronic action of acrylonitrile on adrenals and gastrointestinal tract: biochemical, functional and ultrastructural studies in the rat.

A single dose of acrylonitrile can produce fatal adrenal apoplexy within approximately 2 h. Our previous studies also indicate that multiple injections of the chemical cause acute hemorrhagic and occasional nonperforating duodenal ulcers. Other authors have reported increase in gut and lung neoplasia after chronic exposure. The present study was designed to elucidate the subacute and chronic actions of acrylonitrile on the adrenals, stomach and duodenum by correlating biochemical, functional and morphologic investigations, as well as to gain insight into the mechanisms of action of acrylonitrile. Rats were exposed to 0, 0.0001% (1 ppm), 0.002%, 0.01%, 0.05% or 0.2% acrylonitrile in drinking water, or to the same amount of the chemical given through daily gavage, for 7, 21 or 60 days. Acrylonitrile caused a time- and dose-dependent decrease in plasma corticosterone levels; aldosterone was affected only by the 'high' dose and prolonged time of exposure. Young rats were more susceptible than adults to this action of acrylonitrile. The adrenal cortex, especially the zona fasciculata, was atrophic in rats that had ingested the nitrile through drinking water. At 0.05% and 0.2%, it also caused decreased food intake and body weight gain. The adrenals were enlarged with a hyperplastic zona fasciculata after daily doses of a bolus of acrylonitrile. Ingestion of the chemical did not interfere with compensatory enlargement of the adrenal gland following unilateral adrenalectomy. On the other hand, the ACTH-induced elevation of corticosterone plasma concentration was significantly attenuated by acrylonitrile in drinking water. Electron microscopy of the adrenal glands revealed no consistent changes in the steroid-producing cells. We thus postulate that accelerated turnover of circulating corticoids and/or interference with the secretion or action of ACTH may primarily be responsible for the decreased plasma levels of corticosterone and aldosterone in rats that ingest acrylonitrile. The mucosa in the stomach at the junction of the forestomach and glandular region of animals that had ingested acrylonitrile was hyperplastic. The corpus also showed regional mucosal hyperplasia with the appearance of 'cobble-stoning'. These changes were preceded and associated with an elevated concentration of non-protein sulfhydryls mostly in the mucosa of the glandular stomach. A similar, less prominent elevation also occurred in the proximal duodenum. These alterations may resemble the preneoplastic combination of elevated glutathione and focal hyperplasia described in the liver with hepatocarcinogens.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of the adrenal cortex in gastric mucosal protection by prostaglandins, sulfhydryls, and cimetidine in the rat.

To investigate the possible role of hormones in gastric mucosal protection, the effect of prostaglandin F2 beta, dimercaprol, or cysteamine on ethanol-induced gastric erosions, and of cimetidine on gastric erosions caused by aspirin was studied in intact, adrenalectomized, medullectomized, ovariectomized, or thyroidectomized rats. Cimetidine was administered at a low dose that did not inhibit hydrogen ion secretion. Adrenalectomized animals failed to exhibit the usual mucosal protective response to prostaglandin F2 beta, sulfhydryls, or cimetidine. Ovariectomy or thyroidectomy did not influence mucosal protection with these agents. The inhibition by total adrenalectomy of mucosal protection was not reversed by large intragastric doses or by parenteral administration of prostaglandin F2 beta. Adrenal medullectomy alone significantly diminished (by approximately one-third) ethanol-induced gastric mucosal injury; prostaglandin F2 beta or sulfhydryl drugs produced significant additional protection. Replacement therapy with glucocorticoids (triamcinolone, corticosterone) but not with mineralocorticoids (deoxycorticosterone, 9 alpha-fluorocortisol) restored the cytoprotective effect of prostaglandin F2 beta and sulfhydryls in adrenalectomized rats. The generation of prostaglandin E2- and prostaglandin I2-like activity in the gastric mucosa was unaltered by adrenalectomy. These studies suggest a permissive role for glucocorticoids in gastric mucosal protection induced by prostaglandins, sulfhydryls, and cimetidine.

Direct suppression of natural killer activity in human peripheral blood leukocyte cultures by glucocorticoids and its modulation by interferon.

In vitro treatment of human peripheral blood leukocytes for 18 to 24 hr with physiological concentrations of glucocorticoids resulted in a marked decrease (up to 90%) in natural killer (NK) activity. The effect on NK activity was both dose and time dependent and was specific for glucocorticoids. Glucocorticoids had no effect when added directly to the 4-hr 51Cr release cytotoxicity assay, nor did they alter the susceptibility of K562 cells to NK-mediated cytolysis. Glucocorticoid-induced inhibition occurred in Percoll-fractionated peripheral blood leukocytes enriched for NK activity. Viabilities of steroid-treated and untreated cultures were similar. Mixing experiments failed to demonstrate the involvement of suppressor activity in the inhibition. Purified cloned human leukocyte interferon subtype A and inducers of interferon enhanced NK activity in the presence of glucocorticoid, although the levels of enhancement were lower than those produced by these agents in the absence of the steroid. Thus, glucocorticoids appear to suppress human NK activity by interacting directly with the NK effector cell, and our results obtained with physiological concentrations of these steroids suggest that they may play an important role in regulating NK activity in vivo. Additionally, these findings suggest a possible means for overriding this immunosuppressive side effect of glucocorticoid therapy by simultaneous treatment with interferon.

Acute toxicity of some silver salts of sulfonamides in mice and the efficacy of penicillamine in silver.

The silver salt of 2-metanilamido-5-chloropyrimidine (AgMCP) and the sodium, amminosilver and trimethylphosphite-silver salts of 3',5'-dichlorobenzenesulfonanilide (NaDBS, AgNH3DBS and AgP(OCH3)3DBS were synthesized as possible antibiotic of antiparasitic drugs. All the organosilver compounds were extremely water-insoluble. For animal studies these, and other reference compounds, were given as fine suspensions in an Emulphor-safflower oil mixture. The ip LD50's in mice in mmol/kg were: 1.67 for NaDBS, 0.22 for silver acetate (AgAc), 0.15 for AgP(OCH3)3DBS, 0.13 for AgMCP and 0.10 for AgNH3DBS. When given by mouth, 15 mmol AgAc/kg produced a high mortality, but none of the organosilver compounds caused death in maximal doses (1.9 to 2.6 mmol/kg) that could be given based on considerations of total volume and stability of the suspension. All the silver compounds, including AgAc, produced a similar toxic syndrome with initial hyperexcitability, ataxia, central nervous depression, labored breathing, loss of righting reflex and death. Most deaths occurred between 12 and 24 hours after dosing. In contrast, animals given NaDBS often died within 3 hours although the major signs were very similar to those produced by the silver compounds. When given ip as a single dose 30 minutes after AgAc, D-penicillamine was effective in reducing mortality, but it had no effect on the mortality of the organosilver compounds. Histological studies revealed similar patterns of silver deposition, especially in the liver and kidneys, at 6, 18 and 24 hours after the organosilver compounds and after AgAc. We conclude that the presence of silver contributes significantly to the acute toxicity of these sulfonamides although they may dissociate free silver less readily than does AgAc.

Pathogenesis of experimental adrenal hemorrhagic necrosis ("apoplexy"): ultrastructural, biochemical, neuropharmacologic, and blood coagulation studies with acrylonitrile in the rat.

The pathogenesis of experimental adrenal hemorrhagic necrosis produced by acrylonitrile in the rat was investigated by various morphologic, biochemical, and pharmacologic methods. One dose of this chemical injected intravenously caused 100 per cent incidence of adrenal hemorrhage and necrosis in 90 to 120 minutes. Electron microscopy, histochemistry, and light microscopy combined with colloidal carbon labeling suggested an early damage (30 minutes after administration of acrylonitrile) to the vascular endothelium in the adrenal cortex, prominent at 60 minutes, when lesion to the parenchymal cells was not visible. The use of extracellular diffusion tracer horseradish peroxidase further indicated that parenchymal cell injury was a late event. Damage to the vascular endothelium in the adrenal cortex was associated with retrograde embolization of medullary cells and cell fragments into the cortical capillaries. The ultrastructurally demonstrated platelet aggregation and fibrin precipitation at the sites of discontinuous vascular endothelium were accompanied by a decrease in circulating platelets and fibrinogen as well as prolongation of prothrombin, partial thromboplastin, and thrombin time. The concentration of dopamine, unlike that of noradrenaline, in the adrenals but not in the brain of rats injected with acrylonitrile showed a time-dependent elevation. Pretreatment with phenoxybenzamine (alpha-adrenergic antagonist) or labetalol (alpha- and beta-adrenergic blocker) or metyrapone (11-beta-hydroxylase inhibitor) and the depletion of catecholamines by reserpine or prior medullectomy prevented the chemically induced adrenal necrosis. These results indicate that the presence of a functional adrenal cortex is necessary for the development of cortical damage which is associated with early vascular lesion caused and/or modulated by vasoactive amines from the medulla and/or (metabolites of) acrylonitrile.