Elevated secretion of reactive nitrogen and oxygen intermediates by inflammatory leukocytes in hyperacute experimental autoimmune encephalomyelitis: enhancement by the soluble products of encephalitogenic T cells.

Perivascular lesions within the central nervous system (CNS) of rats with hyperacute experimental autoimmune encephalomyelitis (HEAE) contained large numbers of peripheral blood mononuclear cells (PBMC) and polymorphonuclear leukocytes (PMN), cells enzymatically capable of producing reactive nitrogen and oxygen intermediates (RNI and ROI), which, in excess, are mediators of tissue damage. PBMC and PMN isolated from the CNS and periphery of HEAE-affected rats secreted significantly (p less than 0.01-0.0001) elevated levels of ROI and RNI compared with that of similar cell populations from pertussis- and saline-treated control animals. Coincubation of systemically derived PBMC and PMN with antigen-stimulated myelin basic protein-specific T cell lines led to further increases in ROI and RNI output of between 15.3 and 83.1%, an effect that could be largely attributed to heat-labile, soluble products released by these T cell lines. Our studies suggest a putative neuropathological role for ROI and RNI in HEAE, which may be mediated via cytokines emanating from autoreactive T lymphocytes.

Identification of sulfated oligosaccharide-based inhibitors of tumor growth and metastasis using novel in vitro assays for angiogenesis and heparanase activity.

Inhibitors of tumor angiogenesis and metastasis are rapidly emerging as important new drug candidates for cancer therapy. To facilitate the identification of such drugs, we recently developed novel and rapid in vitro assays for human angiogenesis and for the extracellular matrix-degrading enzyme heparanase, which has been implicated in tumor metastasis. In this study, sulfated oligosaccharides, which are structural mimics of heparan sulfate, were investigated as drug candidates because these compounds may interfere with heparan sulfate recognition by many angiogenic growth factors and may inhibit cleavage of heparan sulfate by heparanase. In the preliminary screening studies, it was found that inhibitory activity in both assay systems was critically dependent on chain length and degree of sulfation, highly sulfated linear oligosaccharides of five or more monosaccharides in length being the most active. However, two sulfated oligosaccharides stood out as potential antitumor drugs, phosphomannopentaose sulfate (PI-88) and maltohexaose sulfate, both of these compounds having the important property of simultaneously being potent inhibitors of in vitro angiogenesis and heparanase activity. Due to the ease of manufacture of the starting material, phosphomannopentaose, PI-88 was studied in more detail. PI-88 was shown to inhibit the primary tumor growth of the highly invasive rat mammary adenocarcinoma 13762 MAT by approximately 50%, inhibit metastasis to the draining popliteal lymph node by approximately 40%, and reduce the vascularity of tumors by approximately 30%, all of these effects being highly significant. Acute hematogenous metastasis assays also demonstrated that PI-88 was a potent (>90%) inhibitor of blood-borne metastasis. Thus, by the use of novel in vitro screening procedures, we have identified a promising antitumor agent.

Differential effects of the anti-inflammatory compounds heparin, mannose-6-phosphate, and castanospermine on degradation of the vascular basement membrane by leukocytes, endothelial cells, and platelets.

Recent studies suggest that heparin, mannose-6-phosphate (M6P), and castanospermine (CS) may mediate their anti-inflammatory effects by inhibiting the passage of leukocytes through the subendothelial basement membrane (BM). In order to test this hypothesis, heparin, M6P, and CS were examined for their ability to prevent the in vitro degradation of a 35SO4-labeled extracellular matrix (ECM) by neutrophils, lymphocytes, endothelial cells (ECs), and platelets, the labeled ECM degradation products being analyzed by gel filtration chromatography. All three compounds inhibited 35SO4-labeled ECM degradation, but M6P and CS were cell-type specific in their effects. Heparin inhibited the heparanase activity of all cell types examined, confirming the results of previous studies using similar in vitro techniques. M6P selectively inhibited lymphocyte heparanase but not that of platelets, neutrophils, or ECs. CS selectively inhibited phorbol myristate acetate (PMA)-induced EC heparanase and sulfatase activity but did not affect the constitutive expression of degradative enzymes by non-stimulated ECs. These findings provide important clues to the mode of action of these compounds and the characteristic inflammatory pathology associated with the use of each anti-inflammatory agent. In particular, the data support the view that leukocytes markedly differ in the mechanisms they use to degrade BM/ECM to enable extravasation and that some degree of cooperation with EC is required in this process.

Antibody-mediated enhancement of hormone activity.

The enhancement of hormone activity by antibodies has been known for many years; however, investigation into the molecular basis of the phenomenon has only recently begun. A number of mechanisms for this enhancement, including "buffering" or slow release, bivalency and Fc region, and conformational and receptor "restriction" effects, have been documented or proposed. The availability of panels of monoclonal antibodies of distinct combining site specificity have aided in these studies and contributed substantially to our understanding of hormone-receptor interactions.

Antigenic structure of bovine growth hormone: location of a growth enhancing region.

Site-directed antisera generated by peptide immunization have been used to study the antigenicity of bovine growth hormone (bGH). Prediction of sequential antigenic sites has been performed using secondary structure information derived from the 'Protean' prediction routine. The structures predicted by this programme agree closely with the corresponding structure of GH recently derived from crystallographic studies. We have previously shown that the binding of monoclonal antibodies of particular epitope specificity to human or bovine GH results in significant enhancement of hormonal activity in vivo; however, the sites recognized by these antibodies were not known. Here we identify a sequence region, corresponding to a loop structure joining helices 3 and 4, which, is associated with the growth enhancement phenomenon. Antisera raised to either of two overlapping peptides (residues 120-140 and 134-154) significantly increase the biological activity of GH in vivo. Antisera directed to other regions on the GH molecule failed to demonstrate this property. Coincidentally, the sites recognized by the growth-enhancing anti-peptide antisera overlap with the site on GH which is highly susceptible to proteolytic cleavage; such cleavage has been shown in some cases to result in hormone enhancement.

Some roles of free radicals in malaria.

Malaria parasites are very vulnerable to oxidant stress during the part of their life cycle when they inhabit erythrocytes. As the infection progresses they also activate macrophages, one consequence of which is extracellular release of reactive oxygen species. For these reasons free radicals are frequently discussed in the literature on antimalarial drugs, malarial immunity, and disease pathogenesis. They are also central to arguments explaining how the genetic mutations that lead to sickle cell disease, thalassemia and glucose-6-phosphate dehydrogenase have become so common in tropical regions. This review summarizes how these links between free radicals and this disease came to be understood, and the present state of the field.

Bleomycin-detectable iron in plasma from Plasmodium vinckei vinckei-infected mice.

Plasma from mice heavily parasitized by Plasmodium vinckei vinckei was found to contain micromolar levels of iron as detected by the 'bleomycin assay' (slightly modified) of Gutteridge et al. [(1981) Biochem. J. 199, 263-265]. Uninfected mouse plasma contained little or no bleomycin-detectable iron. Plasma ultrafiltrate from infected mice contained no bleomycin-detectable iron, indicating that such iron was associated with the protein/macromolecule fraction. We speculate that this iron could catalyse reduction of peroxides in vivo and thus play a role in malaria pathology.

Flavins as potential antimalarials. 2. 3-Methyl-10-(substituted-phenyl) flavins.

A series of 3-methyl-10-(substituted-phenyl)flavins was prepared and tested for antimalarial activity against the lethal parasite Plasmodium vinckei in mice. Several of these analogues were found to be effective antimalarial agents. A quantitative structure-activity relationship study was undertaken with 44 analogues and no satisfactory relationship could be established.

Suppression of hyperacute and passively transferred experimental autoimmune encephalomyelitis by the anti-oxidant, butylated hydroxyanisole.

Butylated hydroxyanisole (BHA) was used to treat hyperacute, ordinary passive, and hyperacute passive experimental autoimmune encephalomyelitis (EAE) in the Lewis rat. The anti-oxidant, delivered via mini-osmotic pumps, reduced the incidence, severity and mortality in hyperacute EAE and also reduced the incidence, severity and duration of disease in passively induced EAE and hyperacute passive EAE. In all cases, cellular infiltration by both mononuclear and polymorphonuclear leukocytes were significantly reduced in treated rats. BHA appears therefore to act at the effector stage of EAE, reducing cellular infiltration in the brain and spinal cord and minimising clinical signs without blocking sensitisation or activation. This was supported by the finding that spleen cells from BHA-treated donors immunised for hyperacute EAE transferred disease at least as well as cells recovered from untreated donors.

Our shifting understanding of the role of nitric oxide in autoimmune encephalomyelitis: a review.

Nitric oxide was first described being produced in inflammatory cells involved in experimental autoimmune encephalomyelitis in 1992. Since then some 45 papers have appeared examining the role of NO in this central nervous system autoimmune inflammatory disease. Of the first 10 papers published all resulted in the interpretation that NO was a pathologic or "bad" molecule in the context of EAE. A few papers then began to appear suggesting that NO may not in fact always be a harmful molecule and by the end of 1997 early 1998, 22 papers suggested a destructive role for the molecule while three suggested it was protective. The past two years have seen a significant increase in reports supporting a protective mechanism for NO in EAE such that as of July 1999, 27 papers suggest a destructive and 15 a protective role for NO with a few uncommitted. This review sets out in a more or less chronological order the studies examining the role of NO in EAE and maps our changing understanding of the molecules role in this CNS inflammatory disease and by inference perhaps multiple sclerosis.

Nitric oxide is a potential down-regulating molecule in autoimmune disease: inhibition of nitric oxide production renders PVG rats highly susceptible to EAE.

Rat strains vary in their susceptibility to experimental autoimmune encephalomyelitis (EAE) and in many cases, factors other than MHC antigens are thought to play a role in this. We found that PVG rats, which have a very low susceptibility to EAE, were rendered highly susceptible to clinical disease when treated with N-methylarginine (NMA) an inhibitor of nitric oxide synthase (NOS). The clinical course of the ensuing disease in NMA-treated PVG rats was in most cases fulminating in nature and accompanied by some mortality. Following immunisation with myelin basic protein (MBP)-complete Freund's adjuvant (CFA), PVG rats developed higher serum levels of the surrogate markers of nitric oxide production, reactive nitrogen intermediates (RNI; nitrite and nitrate), than did their Lewis counterparts. This in vivo finding was reflected in vitro, where the levels of RNI produced in 24, 48 and 72 h IFN-gamma-stimulated spleen cell cultures for PVG rats were significantly higher than those for Lewis rats. A mechanism by which increased NO production might protect PVG rats against clinical EAE was suggested by the finding that lymph node cells, isolated from NMA-treated MBP-immunised PVG rats, proliferated in response to MBP at a rate approximately 3 x greater than those from MBP-immunised, saline treated rats. Thus, the greater number of MBP-specific T cells generated in the NOS inhibitor-treated vs. untreated rats could account for their increased susceptibility to developing clinical EAE. The findings in this study suggest that NO plays a role in protecting PVG rats against developing EAE.

TNF and inhibition of growth of Plasmodium falciparum.

The mechanism of intra-erythrocyte death of Plasmodium chabaudi in vivo has not yet been elucidated. Here we summarise recent experiments in which serum from mice undergoing a successful immune response to this parasite did not inhibit Plasmodium falciparum in vivo unless the P. chabaudi infection and TNF levels were high enough to cause illness in the host. This was true for the 556KA and DS strains of P. chabaudi in intact mice, but not for 556KA in nude mice, which did not generate inhibitory activity at any parasitaemia. Tumour necrosis factor (TNF) inhibits malaria parasites via some undefined secondary mediator. 10 mg of r hu TNF generated this inhibitory activity, as measured against P. falciparum in vitro, in the serum of mice only if they were pretreated with Corynebacterium parvum, which activates macrophages and sensitises the mice to the toxic effects of TNF. This implies a role for activated macrophages downstream from TNF in the process involved in intra-erythrocytic death of parasites.

TNF and Plasmodium berghei ANKA-induced cerebral malaria.

The cerebral pathology observed in Plasmodium berghei ANKA-infected CBA mice has been attributed to overproduction of TNF, the mice in which this syndrome is seen being those with the highest serum TNF levels. To investigate this further, we injected recombinant human TNF into malaria-primed mice to see if we could reproduce the cerebral changes observed in P. berghei ANKA infections. A range of doses, administered as a single or repeated injections, or via osmotic pumps, failed to reproduce these changes, but did induce hypoglycaemia, midzonal liver necrosis and neutrophil adhesion in pulmonary vessels. This pathology is seen in terminal Plasmodium vinckei infections, but absent in terminal P. berghei ANKA. In addition, the permeability of the blood-brain barrier to Evan's blue, which is present in P. berghei ANKA but not in normal or P. vinckei-infected mice, was not induced by exogenous TNF. Serum levels of TNF were measured in an ELISA assay, and found to be consistently higher in P. vinckei rather than P. berghei ANKA terminal infections. This is consistent with the pathological changes we could reproduce by injecting TNF. For these reasons we suggest that the cerebral pathology seen in mice infected with P. berghei ANKA may be governed by TNF produced locally by monocytes sequestered within the cerebral blood vessels, not simply by systemic levels of this cytokine.

Auto-oxidation of dialuric acid, divicine and isouramil. Superoxide dependent and independent mechanisms.

The toxicity of dialuric acid to pancreatic beta cells, and the haemolytic action of divicine and isouramil involve auto-oxidation and redox cycling reactions. Divicine and isouramil are produced on hydrolysis of the fava bean glycosides, vicine and convicine. The mechanism of auto-oxidation of the three compounds as well as the acid hydrolysis product of vicine (provisionally assigned the structure 2-amino-4,5,6-trihydroxypyrimidine) has been studied. All four pyrimidines auto-oxidized rapidly at neutral pH, generating H2O2 by an O2-dependent chain mechanism. Superoxide dismutase inhibited the initial oxidation, but inhibition was transitory, and after a lag period rapid oxidation occurred. The lag period varied with pH, temperature and pyrimidine concentration, and was much shorter for isouramil and divicine than for dialuric acid and acid-hydrolysed vicine. The initial rate of dialuric acid oxidation was greater and the acceleration less pronounced than with the other pyrimidines. A mechanism common to all four pyrimidines has been shown by kinetic analysis to account for nearly all the observations in the presence and absence of superoxide dismutase. Autocatalysis in the latter case is attributed mainly to the reactions reduced pyrimidine + oxidized pyrimidine in equilibrium 2 pyrimidine radical pyrimidine radical + O2----oxidized pyrimidine + O2- Rate constants for these and other reactions are reported. At pH 7.4 and 37 degrees the lag period before 100 microM acid-hydrolysed vicine underwent rapid oxidation was approx. 15 min. Isouramil and divicine at an equivalent concentration gave lags of less than 1 min, which became less at higher concentrations. Thus intracellular superoxide dismutase should provide only transitory protection against the oxidation products of dialuric acid, divicine or isouramil. Prolonged protection should only be achieved if accumulation of oxidized pyrimidine is also prevented.

Toxicity of certain products of lipid peroxidation to the human malaria parasite Plasmodium falciparum.

Aldehydes generated during radical-induced lipid peroxidation, in particular 4-hydroxynonenal, are known to inhibit growth of certain cells. To extend our arguments that free radicals might be involved in the host response against malaria parasites we tested 26 carbonyls (n-alkanals, C6-C11; 2-alkenals, C3-C9; 2,4-alkadienals, C7, C9, C10; 4-OH-2-alkenals, C6, C8, C9; 2-alkanones, C3-C9; and malonyldialdehyde) against Plasmodium falciparum in vitro. We had previously detected many of these substances in oxidant-stressed, malaria-infected erythrocytes. Three 2,4-alkadienals (C7, C9 and C10) and three 4-OH-2-alkenals (C6, C8 and C9), at 20-100 microM concentrations, markedly inhibited incorporation of [3H]-hypoxanthine by P. falciparum. Acrolein had low effect, and none of the other compounds (12 aldehydes and 7 ketones) were active at concentrations up to 100 microM. Malonyldialdehyde was without effect at concentrations up to 450 microM. The aldehydes found to be inhibitory against P. falciparum could contribute to both the non-antibody host responses against this parasite and the antimalarial effects of radical-generating compounds such as t-butyl hydroperoxide, hydrogen peroxide, alloxan, isouramil, divicine and primaquine.

Antimalarial action of flavin analogues seems not be due to inhibition of glutathione reductase of host erythrocytes.

A series of 10-(4'-chlorophenyl)-3-substituted flavins (1a-f) were examined with respect to their antimalarial properties. They were tested against Plasmodium falciparum in vitro and Plasmodium vinckei vinckei in vivo. The proposition that they might act through glutathione reductase (GR) (EC 1.6.4.2) inhibition has been studied. Inhibition of P. falciparum in vitro by these compounds shows only slight variation between analogues; in contrast, inhibition of human erythrocyte GR by members of the same series is highly variable, indicating that this is probably not their primary mode of antimalarial action. Results of the P. vinckei vinckei screen showed that 10-(4'-chlorophenyl)-3-methyl,3-ethyl and 3-propyl substituted flavins are active in vivo over the dose range screened (10-70 mg/kg).

Flavin analogs with antimalarial activity as glutathione reductase inhibitors.

10-(4'-Chlorophenyl)-3-methylflavin has antimalarial activity in vitro and in vivo (Cowden et al., J Med Chem 31: 799, 1988). This flavin analog and two of its derivatives were found to inhibit the antioxidant flavoenzyme glutathione reductase from human erythrocytes in its isolated form as well as in hemolysates. The mixed-type inhibition was completely reversible, the Ki-values being of the order of 1 microM. Surprisingly, the drugs were not competitive with FAD, but with GSSG, one of the enzyme's substrates. Malaria parasite glutathione reductase, extracted from Plasmodium falciparum, could also be inhibited by the compounds. Studies on the effects of the substances on P. falciparum in vitro, which were demonstrated morphologically and by growth inhibition, confirmed previous observations with 10-(4'-chlorophenyl)-3-methylflavin and showed similar parasiticidal characteristics for the two new derivatives. The activities of five other erythrocytic enzymes tested were not impaired by the drugs, nor was the nucleotide metabolism of erythrocytes and/or parasites significantly changed. Permeation into red blood cells was demonstrated for one compound by 19F-NMR-spectroscopy. Inhibition of glutathione reductase might contribute to, or account for, the antimalarial activity of this group of flavin analogs.

Castanospermine, an oligosaccharide processing inhibitor, reduces membrane expression of adhesion molecules and prolongs heart allograft survival in rats.

The inhibition of intracellular oligosaccharide processing is a new approach to immunosuppression in allotransplantation. The net effect of such inhibition is reduction in the membrane expression of certain glycoproteins. Hence cell-cell interaction in allorejection may be impaired in the presence of glycoprotein processing inhibitors because the expression of key ligand-receptor pairs of N-linked glycoproteins including adhesion molecules is inhibited. The aims of this study were to measure the immunosuppressive ability of castanospermine (CAST) in a rat heart allograft model, to measure its effect on membrane expression of adhesion molecules (LFA-1 alpha, LFA-1 beta, ICAM-1), class I and class II MHC antigens and on other T cell associated molecules (CD4, CD8, CD39, CD45, W3/13), to test its tolerogenic potential and its toxicity. Membrane expression of these molecules was measured by flow cytometry for single cells and by immunoperoxidase staining for the allograft. In grafted rats CAST significantly reduced the expression of LFA-1 alpha on lymphoid cells in the thymus, lymph node, spleen and heart allografts. ICAM-1 expression on endothelial cells of the allograft vasculature, class I and class II MHC expression on lymphoid cells in the thymus, class II MHC expression on lymphoid cells in the allograft; and CD4, CD8, CD45 and W3/13 expression on lymphoid cells in some organs. By contrast, in non-grafted rats CAST significantly upregulated expression of class I MHC and CD45 in the thymus, lymph node and spleen, ICAM-1 and CD4 on lymphoid cells in the spleen, but reduced expression of LFA-1 alpha on lymphoid cells in the thymus. It also prolonged rat heart allograft survival in a dose-dependent manner and with limited testing was relatively non-toxic. In conclusion, CAST is an immunosuppressive molecule which may work by downregulation of the ligand-receptor adhesion molecule pair, LFA-1 alpha-ICAM-1 although subtle downregulation of class I and II MHC, CD4 and CD8 molecules could also contribute to its immunosuppressive activity. Hence, both lymphocyte-endothelial cell binding and lymphocyte activation may be inhibited by CAST. This work suggests that CAST may hold significant potential as a transplant immunosuppressant probably as an adjuvant agent to inhibitors of interleukin 2 secretion.

Antimalarial activity of a riboflavin analog against Plasmodium vinckei in vivo and Plasmodium falciparum in vitro.

The riboflavin analog 10-(4'-chlorophenyl)-3-methylflavin was found to have significant activity against Plasmodium vinckei vinckei when administered orally and parenterally; it was active against P. falciparum in culture. It inhibited mouse erythrocyte glutathione reductase in a dose-dependent manner. When administered orally, 5-deazariboflavin was not active in vivo although it has been shown to have activity against P. falciparum in vitro.

Carbohydrates play a role in neurite outgrowth in vivo during development and regeneration.

We have used the alpha-glucosidase inhibitor, castanospermine, to investigate the possible involvement of N-linked glycosylation in the process of neurite outgrowth both during the development and regeneration of sympathetic nerve fibres into the iris of the rat. The effects on nerve growth were assessed qualitatively using catecholamine histochemistry and quantitatively by measuring the uptake of [3H]noradrenaline. Castanospermine was injected during the first, second or third postnatal week or during the second to fourth week after degeneration of adrenergic nerve terminals with the neurotoxin, 6-hydroxydopamine. Results showed that both the initial growth of sympathetic nerve fibres, as well as the regeneration of these fibres, was inhibited by castanospermine treatment. The inhibitory effects were restricted to injection of castanospermine at particular time periods during postnatal development and regeneration. These results suggest that N-linked carbohydrates may be important in particular stages of the processes of initial nerve growth and regeneration of sympathetic nerve fibres in the iris.