Effect of CL 184,005, a platelet-activating factor antagonist in a murine model of Staphylococcus aureus-induced gram-positive sepsis.

Experiments using a murine model of heat-killed Staphylococcus aureus-induced gram-positive bacterial sepsis indicate that the lethal bacterial effects can be prevented if mice are pretreated with CL 184,005, a platelet-activating factor (PAF) antagonist. CL 184,005 was ineffective when administered after bacterial challenge. Plasma of mice pretreated with CL 184,005 contained significantly less tumor necrosis factor (TNF), suggesting that CL 184,005 interferes with TNF synthesis induced by S. aureus. Spleen-associated TNF protein was also decreased by pretreatment with CL 184,005. Although TNF levels were significantly decreased in mice treated with CL 184,005, interleukin-6 levels in serum were significantly increased. Athymic mice were also susceptible to the lethal effects of S. aureus, suggesting that T cells were not involved. When rats rendered hypotensive with S. aureus were treated with CL 184,005, their blood pressure was normalized. Mice treated with enterotoxin B were not protected if they were pretreated with CL 184,005; however, TNF levels in these mice were significantly lower, suggesting that mediators other than PAF and TNF may contribute to the lethal effects of enterotoxin.

Age-dependent structural changes in intact human lenses detected by synchrotron radiation X-ray scattering. Correlation with Maillard reaction protein fluorescence.

To understand alterations in the spatial organization of the crystallins, the major determinant of lens transparency, the x-ray scattering by intact normal human lenses from individuals 6-82 years of age was measured using synchrotron radiation. The angular dependence of the integrated scattering intensity is consistent with short-range order within the crystallin assemblies. A significant change in the scattering patterns of the lenses occurs after 55 years of age, in parallel with an increase of the fluorescence of the urea-insoluble crystallin fraction. This correlation suggests a gradual derangement of the short-range order as a result of cross-linking of the crystallin subunits by advanced Maillard reaction products that are generated by the continuous reaction of sugars, such as glucose or fructose, with proteins.

Studies on the effect of CL 306,293, a substituted quinoline carboxylic acid, on the clinical disease induced in mice with LP-BM5 virus.

CL 306,293, a substituted quinoline carboxylic acid, is a potent inhibitor of dihydroorotic acid dehydrogenase, an enzyme essential for the biosynthesis of pyrimidines. In mammalian cell culture, the agent exhibits antiproliferative properties that can be reversed by the addition of uridine. CL 306,293 inhibits the development of the clinical disease in a murine model of immunodeficiency induced by a mixture of LP-BM5 retroviruses. In infected mice, the agent prevents the development of hypergammaglobulinemia, lymphadenopathy, splenomegaly and induction of an IL-2 deficiency. The CD4/CD8 ratio and the number of B cells in the lymph nodes are decreased if the infected animals are treated with CL 306,293. CL 306,293 was more efficacious and potent than 3'-azido-3'-deoxythymidine. The beneficial effects of CL 306,293 observed in this model are most probably related to its antiproliferative properties.

Preclinical studies with platelet-activating factor antagonists in models of septic shock.

Since the isolation and elucidation of the structure of platelet-activating factor (PAF) in the late 1970's, several preclinical studies have suggested that PAF is a key mediator of septic shock induced in animals by either endotoxin or by Gram-negative bacteria. A number of PAF antagonists have been sythesized that protect animals from the lethal effects of endotoxin. Some of these antagonists are in early stages of clinical development. The most advanced cadidate is BN 52021, a ginkgolide, that is in Phase II/III clinical trials in patients with septic shock. Preliminary results with BN 52021 indicate that it is efficacious and significantly reduces mortality associated with Gram-negative sepsis. Pivotal trials with BN 52021 aer ongoing. The present review summarizes the biological effects of PAF and the effect of PAF antagonists in animal models of septic shock. The interrelationship of PAF and tumor necrosis factor (another key mediator of septic shock) is also discussed.

Pluronic F 127 liquid sensitizes mice to low doses of Escherichia coli lipopolysaccharide.

BACKGROUND AND METHODS: In murine models of endotoxemia, large amounts of lipopolysaccharide have to be administered to induce mortality. If mice are pretreated with D-galactosamine, the amount of lipopolysaccharide required to induce mortality is significantly lowered. Pluronic F 127 liquid is a relatively non-toxic copolymer that exhibits reverse gelation properties. Thus, it is a liquid at cold temperature and a gel at body temperature. The present studies were performed to ascertain whether the reverse gelation properties of Pluronic F 127 liquid could be used in devising a model of septic shock where a sustained delivery of lipopolysaccharide occurred. In evaluating this model, dose-response studies were conducted with lipopolysaccharide when a) it was administered intraperitoneally in saline or in Pluronic F 127 liquid, and b) it was administered intravenously to mice that had been pretreated with saline or Pluronic F 127 liquid. Mortality was followed for up to 72 hrs. RESULTS: Various doses of Escherichia coli lipopolysaccharide dissolved in saline or in Pluronic F 127 liquid were administered intraperitoneally to mice. The lethal dose of lipopolysaccharide required to kill 50% of the mice (LD50) administered in Pluronic F 127 liquid was approximately ten- to 15-fold less than the values obtained for lipopolysaccharide administered in saline. This decrease in the LD50 of lipopolysaccharide was also observed if the mice were treated intraperitoneally with Pluronic F 127 liquid and challenged 6 hrs later with iv lipopolysaccharide. The concentrations of tumor necrosis factor and interleukin-6 in the plasma were significantly higher when a low dose of lipopolysaccharide was administered to mice that had been pretreated with Pluronic F 127 liquid. While there was no effect on the liver enzymes, Pluronic F 127 liquid caused an increase in the plasma triglycerides. CONCLUSIONS: The data reported in this paper indicate that the LD50 of lipopolysaccharide is significantly decreased if it is administered in Pluronic F 127 liquid or administered to mice that have been pretreated with the Pluronic F 127 liquid. Thus, Pluronic F 127 liquid appears to sensitize mice to low levels of lipopolysaccharide. Unlike the D-galactosamine model, lipopolysaccharide can be administered as late as 6 hrs after treatment with Pluronic F 127 liquid. While the mechanisms by which Pluronic F 127 liquid sensitizes mice is not known, plasma triglycerides were increased in mice treated with this agent, suggesting that tissues responsible for the synthesis and/or degradation of triglycerides play a role in this sensitization process.

Studies of the effect of a platelet-activating factor antagonist, CL 184,005, in animal models of gram-negative bacterial sepsis.

The effect of CL 184,005, a potent and specific platelet-activating factor antagonist, has been examined in a variety of animal models relevant to gram-negative bacterial sepsis. Pretreatment of mice with CL 184,005 protected them from the lethal effects of platelet-activating factor. When rats or primates rendered hypotensive with endotoxin were treated with CL 184,005, blood pressure was normalized. Pretreatment of rats with CL 184,005 protected them from the gastrointestinal lesions induced by endotoxin. Pretreatment of rats and mice with CL 184,005 protected them from the lethal effects of endotoxin. Plasma tumor necrosis factor levels in endotoxin-treated mice were lower when the mice were pretreated with CL 184,005. These observations suggest that CL 184,005 may be potentially useful in the treatment of gram-negative bacterial sepsis, and the agent is undergoing clinical evaluation.

Fructose-induced fluorescence generation of reductively methylated glycated bovine serum albumin: evidence for nonenzymatic glycation of Amadori adducts.

In vitro glycation of bovine serum albumin by fructose (fructation) induces fluorescence generation about 10-times faster than glucose (G. Suárez et al. (1989) J. Biol. Chem. 264, 3674-3679). In order to gain further insight into possible mechanisms that would explain this difference, the protein was glycated with either glucose or fructose and then reincubated in the absence of sugars. In contrast to the previous findings, albumin that had been glycated with glucose generated fluorescence at a higher rate during the sugar-free incubation. However, when partially glycated BSA was reincubated with sugars under conditions where de novo glycation was prevented by reductive methylation of amino groups fructose induced fluorescence to a much larger extent than glucose. These results are consistent with the notion of covalent addition of sugars to Amadori groups, the earliest stable products of the Maillard reaction. A chemical pathway is proposed where pyrrolic structures result from the double sugar adducts by aldol condensation and dehydration. These structures might be precursors of fluorophores.

Antiinflammatory and antiarthritic properties of a substituted quinoline carboxylic acid: CL 306,293.

CL 306,293, a substituted quinoline carboxylic acid at a daily oral dose between 1.5 and 3.0 mg/kg suppressed the inflammation and joint destruction (radiological criteria) associated with both developing and established adjuvant arthritis. When a weekly oral dosing regimen was used, joint destruction was attenuated when this agent was administered at a dose of 50 to 200 mg/kg. Inflammation associated with a delayed type hypersensitivity reaction in dogs was suppressed at a daily dose of 0.25 mg/kg or a weekly dose of 1 mg/kg. At efficacious doses, CL 306,293 had no effects on cyclooxygenase or lipoxygenase activities nor did it have an effect on carrageenin induced paw edema. In acute tests, the compound was not ulcerogenic. The above observations indicate that the antiinflammatory effects of CL 306,293 are distinct from those observed with nonsteroidal antiinflammatory agents. Mechanistic studies conducted and to be published indicate that CL 306,293 down regulates T cell function and this mechanism may account, at least in part, for the antiinflammatory and antiarthritic properties observed in animal models of inflammation and joint destruction.

Platelet-activating factor or a platelet-activating factor antagonist decreases tumor necrosis factor-alpha in the plasma of mice treated with endotoxin.

When L-platelet-activating factor (PAF) or alprazolam (a PAF antagonist) was administered to lipopolysaccharide (LPS)-treated mice, the level of plasma tumor necrosis factor (TNF alpha) determined by either ELISA or a cytotoxic assay using WEHI cells was significantly lowered. The inactive stereoisomer, D-PAF, was not effective in lowering plasma TNF alpha levels in LPS-treated mice. The decrease in plasma TNF alpha induced by L-PAF or alprazolam was partly reversed by indomethacin. Despite a decrease in plasma TNF alpha, L-PAF or alprazolam caused an increase in the amount of TNF alpha mRNA present in the kidneys and the livers of LPS-treated mice, suggesting that a posttranscriptional event leading to the synthesis or release of TNF alpha was inhibited by these agents.

Studies on the homing of Mycobacterium-sensitized T lymphocytes to the synovium during passive adjuvant arthritis.

The migration of intravenously administered adjuvant sensitized T lymphocytes to the knee synovium of recipient rats undergoing passive adjuvant arthritis has been followed. Using fluorescein isothiocyanate (FITC)-labeled adjuvant-sensitized T cells and anticollagen IgG, the present studies demonstrate the presence of fluorescent cells in the inflamed knee synovium of recipient rats undergoing passive arthritis. Proliferation studies indicate that synovial cells from these rats respond to Mycobacterium tuberculosis (MT). Since cross-reactivity between Mycobacterial antigens and cartilage proteoglycans has been previously demonstrated, it is suggested that adjuvant-sensitized T cells that are injected into naive rats migrate to the synovium, proliferate in response to cartilage proteoglycan, and initiate passive arthritis.

Modulation of nicotinamide adenine dinucleotide and poly(adenosine diphosphoribose) metabolism by calicheamicin gamma 1 in human HL-60 cells.

The mechanism of calicheamicin gamma 1-mediated cytotoxicity was studied in human promyelocytic HL-60 leukemic cells. Calicheamicin gamma 1 caused an increase in poly(ADP-ribose) polymerase activity in HL-60 cells parallel to cell death. This effect of the drug correlated with a decrease in intracellular NAD+ level. 3-Aminobenzamide, an inhibitor of poly(ADP-ribosylation), prevented the calicheamicin gamma 1-triggered cytotoxicity in a dose-dependent manner. Simultaneous with the reversal of cytotoxicity, the addition of 3-aminobenzamide to drug-treated cells also inhibited the increase in poly(ADP-ribosylation) and the reduction in cellular NAD+ content. These results indicate that poly(ADP-ribosylation) activation and the subsequent perturbations in NAD(+)-dependent metabolic reactions are associated with the cytotoxic properties of the antitumor antibiotic calicheamicin gamma 1.

M protein deficient streptococcal cell walls can induce acute and chronic arthritis rats.

Cell walls from M+ and M- protein variants of group A streptococci were examined for their arthritogenicity in female Lewis rats. Intraperitoneal administration of both of these sonicated cell wall preparations caused a severe acute and chronic arthritis in recipient rats. Histological evaluation of the hind paw of these rats indicated synovial lining hyperplasia, cell infiltration in the subsynovial space, pannus formation, and erosions of bone and cartilage. Joint pathology was similar in the hind paws of rats immunized with cell walls prepared from either the M+ or the M- protein variants. Cell-mediated immunity was also similar when lymph nodes were exposed to cell walls derived from these two preparations. A recombinant M6 protein from streptococci did not elicit a proliferative response from lymph nodes prepared from arthritic rats. These observations indicate that the M protein that has previously been implicated in auto-immunity does not have a critical role in the pathogenesis of streptococcal cell wall arthritis in rats.

Streptococcal cell wall arthritis. Passive transfer of disease with a T cell line and crossreactivity of streptococcal cell wall antigens with Mycobacterium tuberculosis.

Primary lymph node cells derived from streptococcal cell wall arthritic rats or those derived from adjuvant arthritic rats proliferated in response to cell wall antigens derived from either streptococcal cell walls or those from M. tuberculosis. In addition, two T cell lines have been isolated from lymph nodes of rats during the chronic phase of streptococcal cell wall arthritis. These T cell lines transfered clinical disease to naive syngeneic irradiated recipients, and they proliferated in the presence of cell wall antigens derived from streptococci or antigens derived from Mycobacterium but failed to proliferate in the presence of the 65-kD antigen (containing the sequence TFGLQLELT) derived from Mycobacterium. These observations indicate that T cells play a crucial role in the pathogenesis of streptococcal cell wall arthritis and suggest that antigenic crossreactivity exists between cell walls of group A streptococci and antigens derived from Mycobacterium. The 65-kD Mycobacterium protein is not involved in the observed antigenic crossreactivity.

Nonenzymatic glycation of bovine serum albumin by fructose (fructation). Comparison with the Maillard reaction initiated by glucose.

Nonenzymatic glycation by glucose (glucation) was compared with glycation by fructose (fructation). The rate and extent of protein-bound fluorescence generation upon fructation was about 10 times that upon glucation. In contrast, nonenzymatically glucated bovine serum albumin (BSA) released about twice as much formaldehyde upon periodate oxidation as did nonenzymatically fructated BSA. However, the rate of blocking of amino groups was similar in both proteins. Periodate oxidation of borohydride-reduced glycated BSA led to regeneration of amino groups with preservation of fluorescence. From the ratio between the decrease in formaldehyde-releasing ability and the regenerated amino groups, formaldehyde molar yields of 0.47 and 0.8 were computed for fructose- and glucose-derived Amadori groups, respectively. This is consistent with participation of both carbon 1 and carbon 3 in the Amadori rearrangement from fructose. The formaldehyde releasing ability of nonenzymatically fructated BSA attains asymptotic maximum values earlier than that of nonenzymatically glucated BSA. Thus, the higher rate of fluorescence generation in nonenzymatically fructated BSA could be explained by a faster conversion of its Amadori groups. Since fluorescence generation through the Maillard reaction has been correlated with long term complications of diabetes mellitus, the participation of nonenzymatic fructation in this pathological state deserves further exploration. This is especially relevant in tissues where fructose levels increase in diabetes as a result of the operation of the sorbitol pathway.

Methotrexate in rheumatoid arthritis: studies with animal models.

The present studies have shown that low doses of methotrexate can suppress the inflammation and joint destruction associated with animal models of arthritis. The antiinflammatory effects of methotrexate are probably related to its inhibitory effect on chemotaxis. At the low doses used, methotrexate does not induce systemic immunosuppression. In methotrexate-treated rats, an improvement in IL-2 synthesis is observed and increases in IL-2 levels are expected to improve cell mediated immunity. Suppressor cells appear to be very sensitive to methotrexate. Macrophage function is modulated by methotrexate. All of these effects including the effects on joint destruction are probably due to inhibition of DHFR activity of critical cells that are involved in the pathogenesis of rat arthritis induced either by adjuvant or by streptococcal cell walls. Some of these effects have been extended to human arthritis but additional studies are required to understand how low dose methotrexate exerts its beneficial effects in humans.

Passive collagen arthritis induced by anticollagen IgG.

Studies conducted in rats and mice indicate that passive arthritis can be transferred to naive recipients with anticollagen IgG. The passively transferred disease is less severe and is transient. Rats that recover from passive arthritis are resistant to a second phase of clinical disease when administered either anticollagen IgG or type II collagen. Suppressor T cells appear to be responsible for this resistance. Passive arthritis induced by anticollagen IgG is a complement dependent lesion. Deposition of IgG on the cartilage and host complement C3 and C5 activation are essential for the induction of passive disease. Inflammatory cells are necessary for the demonstration of passive arthritis; mice deficient in inflammatory cells or defective in this cell population are resistant to passive arthritis. Monoclonal antibodies reactive to type II collagen or to a renatured TCA fragment can also induce passive arthritis. The disease is subclinical and can be detected only after histological analysis of the joints.

Synthesis and biological properties of 5,10-dideaza-5,6,7,8-tetrahydrofolic acid.

The synthesis of the antifolate 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF) has been modified. It is prepared from 2-acetamido-6-formyl-4(3H)-pyrido[2,3-b]pyrimidone and [P-(N-[1,3-bis(ethoxycarbonyl)propan-1-yl]aminocarbonyl)] phenylmethyl]-triphenylphosphonium bromide. The synthesis proceeds via a sodium hydride promoted Wittig condensation in 1-methyl-2-pyrrolidone followed by catalytic reduction, mild base hydrolysis, and acid precipitation of the product. Synthesis of DDATHF is achieved in a total of seven steps from commercially available reagents. DDATHF is transported effectively into CCRF-CEM cells and inhibits growth of both human (CEM) and murine (L1210) cells in culture. Studies reported here support the view that methotrexate and DDATHF are transported via a shared transport mechanism.

Methotrexate suppresses passive adjuvant arthritis: studies on the metabolism of methotrexate in mononuclear cells derived from normal and adjuvant arthritic rats.

Passive transfer of adjuvant arthritis by spleen cells is suppressed by methotrexate. Mononuclear cells derived from spleens of normal and adjuvant arthritic Lewis rats were incubated with [3H]-methotrexate and harvested at various periods of time. The amount of methotrexate and its various polyglutamates were quantitated. The results of these studies indicate that the kinetics of uptake of methotrexate by mononuclear cells from normal and adjuvant arthritic rats are similar. However, the amount of methotrexate polyglutamates accumulating in the mononuclear cells of adjuvant arthritic rats was significantly lower than that observed in mononuclear cells derived from normal rats.

Administration of an aldose reductase inhibitor induces a decrease of collagen fluorescence in diabetic rats.

As a consequence of an increased flux through the sorbitol pathway fructose levels rise in various tissues in diabetes. Also, in vitro nonenzymatic fructosylation of protein induces the generation of fluorescence at a rate 10 times greater than glucosylation. The administration of sorbinil, an aldose reductase inhibitor known to lower tissue fructose concentration, to experimental diabetic rats led to a decrease in the fluorescence related to advanced Maillard products in their skin collagen. This effect is consistent with the in vivo occurrence of nonenzymatic fructosylation of collagen. A potential pathogenetic role for this posttranslational modification in diabetic complications should be considered.