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.

Administration of group A streptococcal cell walls to rats induces an interleukin 2 deficiency.

Intraperitoneal administration of group A streptococcal cell walls to Lewis rats induces a chronic arthritis, whereas the Fischer strain is resistant to the development of the lesion. Spleen cells from cell wall-treated rats (Lewis and Fischer) are deficient in the synthesis of IL-2. Using an mAb directed against the rat IL-2-R, the present studies indicate that the expression of IL-2-R on spleens of cell wall-treated rats is normal. However, the addition of exogenous IL-2 to spleen cells cultured with Con A does not stimulate the mitogenic response.

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.

Antifolate drug interactions: enhancement of growth inhibition due to the antipurine 5,10-dideazatetrahydrofolic acid by the lipophilic dihydrofolate reductase inhibitors metoprine and trimetrexate.

The presence of low concentrations of the lipophilic dihydrofolate reductase inhibitors metoprine or trimetrexate, which cause little inhibition in the growth of cultured hepatoma cells in combination with weakly inhibiting concentrations of 5,10-dideazatetrahydrofolate, exhibit greater activity than would be predicted by the activity of the individual components. Growth inhibition by this inhibitor of glycineaminoribonucleotide transferase alone or in the presence of the reductase inhibitors is prevented by hypoxanthine indicating that the combination of drugs is enhancing the activity of 5,10-dideazatetrahydrofolate against purine biosynthesis. H35 hepatoma cells resistant to methotrexate (100-fold) as a result of a transport defect are 40-fold resistant to 5,10-dideazatetrahydrofolate suggesting that this analogue enters hepatoma cells at least in part by the reduced folate coenzyme-methotrexate transport system. The transport-resistant cells are also susceptible to enhanced inhibition of cell growth by low levels of reductase inhibitors in combination with 5,10-dideazatetrahydrofolate. These results have a corollary in an earlier study showing that the same concentrations of metoprine and trimetrexate could enhance the growth inhibition and cytotoxicity of the folate-based inhibitor of thymidylate synthase, 10-propargyl-5,8-dideazafolic acid (Galivan et al., Cancer Res., 47: 5256-5260, 1987). Combinations of 5,10-dideazatetrahydrofolic acid and 10-propargyl-5,8-dideazafolic acid are less growth inhibitory than that predicted by each of the folate analogues alone. It is possible that the effects of all these combinations are related to distortions in the folate pools caused by the folate analogues being used in combination. Two methods of analysis, one graphical and one mathematical, were used to analyze the drug interactions described in this presentation. The enhancement effect seen with the lipophilic dihydrofolate reductase inhibitors and 5,10-dideazatetrahydrofolate clearly represents a supraadditive or a synergistic drug interaction. In contrast the combination of the folate-based inhibitors of purine (5,10-dideazatetrahydrofolic acid) and thymidylate biosynthesis (N10-propargyl-5,8-dideazafolate) exhibit frank antagonism under certain conditions.

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.

Properties of a collagenase inhibitor partially purified from cultures of smooth muscle cells.

An inhibitor of mammalian collagenase has been partially purified from the spent medium of smooth muscle cells grown in culture. The inhibitor is a glycoprotein with an apparent molecular weight of 25,000. It is stable to heat, acid, and mercurials, but is destroyed by trypsin treatment and by reductive alkylation. The inhibitor interacts with active mammalian collagenase and this interaction results in the loss of enzymatic activity. This presumptive collagenase-inhibitor complex is stable to the treatment with mercurials and to trypsin. These latter observations suggest that this inhibitor is different from other collagenase inhibitors that are thought to be responsible for the latency of the enzyme.

In vivo chemotaxis of rat leukocytes in the presence of circulating chylomicrons.

Dietary-induced chylomicronemia was produced in rats to determine its effect on the chemotactic activity of 51Cr-labelled adoptively transferred isologous leukocytes. Rats fed a low protein high fat diet for 2 months had normal total plasma cholesterol and triglyceride levels, but increased amounts of chylomicrons. Circulating neutrophils and monocytes from animals on a normal diet were able to phagocytose chylomicrons from plasma of those animals on the special diet. Peritoneal exudate cells from the latter animals contained intracellular chylomicrons demonstratable both histologically and biochemically. Neutrophils and mononuclear cells from normal or special fed animals, when transferred to chylomicronemic recipients, had a reduced ability to accumulate at the site of a complement-dependent inflammatory reaction but circulated normally. The defective chemotactic activity observed could be duplicated when cells were allowed to ingest aggregated protein instead of chylomicrons. It was concluded that circulating leukocytes ingest chylomicrons from the plasma with a resultant reduction in their chemotactic activity. The results were discussed in relation to the increased incidence of infection in diabetes characterized by Type I and V hyperlipidemias. It was also suggested that phagocytosis of particulate substances by entrapped leukocytes in atherosclerotic lesions would induce phagocytic enzyme release which could contribute to the eventual destruction of the vascular wall.

Purification and properties of a collagenase inhibitor from cultures of bovine aorta.

Bovine medial explants in culture synthesize a potent inhibitor of mammalian collagenase but not of bacterial collagenase. This inhibitor has been partially purified and has an apparent molecular weight of 45,000. It is a glycoprotein and is stable to heat, trypsin, acid and mercurials. Inhibitory activity is destroyed on reductive alkylation. The inhibitor interacts with collagenase and this interaction leads to the loss of enzymatic activity. This inhibitor may play a physiological role in the control of collagen degradation in blood vessels.

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.

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.

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.

Connective tissue-degrading enzymes of human leukocytes.

Human leukocytes, when exposed to aggregated human gamma-globulin (AHGG) or immune complexes (isolated from RA synovial fluid) fixed to a cartilagenous surface, release neutral proteases that degrade the extracellular matrix of cartilage. The chondromucoprotein destruction by these proteases is suppressed by a variety of synovial fluids but is not susceptible to inhibition by trypsin, chymotrypsin, elastase inhibitors, or a combination of these agents. The inhibitory effect of synovial fluid can be reversed in the presence of increasing enzyme concentrations. Intact viable human polymorphonuclear leukocytes in the presence of AHGG also release a collagenase precursor that can be activated by limited proteolysis with trypsin or RA synovial fluids. Enzyme release (neutral proteases) by phagocytosing cells is inhibited by the antiinflammatory agents phenylbutazone and colchicine; these agents do not affect release of the collagenase precursor. However, the latent collagenase release is susceptible to inhibition when leukocytes are preincubated (prior to exposure to AHGG) with inhibitors of protein synthesis. Under these conditions, neutral protease release is unaffected.

Physiologic and pharmacologic alterations of rat leukocyte chemotaxis (Cx) in vivo.

The method of adoptively transferring 51Cr-labeled rat leukocytes iv to isologous recipients was used to quantitate the extravascular accumulation of specific cell types at the site of inflammation induced by local injection of various phlogistic agents. Experiments were designed to determine whether cellular accumulation could be modified at the level of the chemotactic factor, by serum components, or by alteration of the responding cell itself. The results indicated a selective attraction of mononuclear cells to the local injection site of BCG and of neutrophils to the injection site of aggregated but not monometric gamma-globulin. Thus, leukocytic accumulation was found to be dependent upon the local generation of specific reactants that were particular to the agent employed. Leukocytic accumulation could also be modified by serum factors. Cellular accumulation was inhibited when leukocytes were exposed to serum that contained phagocytosable particles or after phagocytosis in vitro prior to adoptive transfer. Chemotaxis of lymphocytes could be induced by their preincubation with sera from adjuvant arthritic animals. These observations were confirmed by in vitro studies and by the finding that 6 days after adjuvant injection , lymphocytes but not mononuclear cells accumulated at the noninjected extremity. In the final series of experiments, it was shown that BCG immunization was capable of inducing a unique population of peritoneal mononuclear cells that after adoptive transfer had an enhanced capacity to remain in the circulation, which, in turn, resulted in a functional increase in their accumulation at an inflammatory reaction site. In conclusion, these studies indicated that the chemotactic activity of adoptively transferred cells can be modified by changes in the chemotactic stimuli, can be enhanced or depressed by serum factors, and is a function of the physiologic capability of the cell population employed.

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.

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.

Type II collagen induced arthritis in rats: clinical and immunological studies.

Immunization of laboratory rats with native Type II collagen induces arthritis in approximately 40 percent of the individuals. Histological and radiological analysis have suggested that the lesion bears several similarities to human rheumatoid arthritis. Clinical studies conducted with this model indicate that it is responsive to the treatment with clinically-used NSAID'S, steroids and immunosuppressive agents. When rats with collagen arthritis were treated with DMARD's, only those treated with D-penicillamine showed clinical improvement (radiological evaluation). Type II collagen induced arthritis is complement-dependent and is an example of an immune complex mediated injury. Thus, passive arthritis can be induced in rats by the intravenous administration of affinity-purified anticollagen IgG. The passive lesion is transient and the administered IgG is detected on the articular cartilage of the hind paws. This articular cartilage also contains complement C3. Passive arthritis is also complement-dependent. Suppression of Type II collagen arthritis can be induced by the prior intravenous treatment of rats with either Type II collagen or its constituent peptide alpha 1, (II) CB10. Antigen-induced arthritis cannot be induced in those rats that have recovered from passive arthritis.

Studies on the effect of D-penicillamine on type II collagen-induced polyarthritis.

The effect of D-penicillamine on a preestablished lesion of type II collagen-induced polyarthritis is reviewed. D-penicillamine treatment (100 and 200 mg/kg/d)for 21 d had no significant effect on rat hind paw inflammation associated with this polyarthritis. At the lower dose, the drug caused an increase in circulating antibody titers to type II collagen. X-ray examination of the hind paws of D-penicillamine-treated animals showed a significant improvement in the joint lesions induced by type II collagen.