Daniel S. Fletcher

Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase

Mice deficient in inducible nitric oxide synthase (iNOS) were generated to test the idea that iNOS defends the host against infectious agents and tumor cells at the risk of contributing to tissue damage and shock. iNOS-/-mice failed to restrain the replication of Listeria monocytogenes in vivo or lymphoma cells in vitro. Bacterial endotoxic lipopolysaccharide (LPS) caused shock and death in anesthetized wild-type mice, but in iNOS-/-mice, the fall in central arterial blood pressure was markedly attenuated and early death averted. However, unanesthetized iNOS-/-mice suffered as much LPS-induced liver damage as wild type, and when primed with Propionobacterium acnes and challenged with LPS, they succumbed at the same rate as wild type. Thus, there exist both iNOS-dependent and iNOS-independent routes to LPS-induced hypotension and death.

Resistance to fever induction and impaired acute-phase response in interleukin-1β-deficient mice

We used gene targeting in embryonic stem cells to introduce an IL-1 beta null allele in mice. The IL-1 beta-deficient mice develop normally and are apparently healthy and fertile. The IL-1 beta null mice responded normally in models of contact and delayed-type hypersensitivity or following bacterial endotoxin LPS-induced inflammation. The IL-1 beta-deficient mice showed equivalent resistance to Listeria monocytogenes compared with wild-type controls. In contrast, when challenged with turpentine, which causes localized inflammation and tissue injury, the IL-1 beta mutant mice exhibited an impaired acute-phase inflammatory response and were completely resistant to fever development and anorexia. These results highlight a central role for IL-1 beta as a pyrogen and a mediator of the acute-phase response in a subset of inflammatory disease models, and support the notion that blocking the action of a single key cytokine can alter the course of specific immune and inflammatory responses.

Pyrroles and other heterocycles as inhibitors of P38 kinase

Investigation of furans, pyrroles and pyrazolones identified 3-pyridyl-2,5-diaryl-pyrroles as potent, orally bioavailable inhibitors of p38 kinase. 3-(4-pyridyl-2-(4-fluoro-phenyl)-5-(4-methylsulfinylphenyl)-pyrrol e (L-167307) reduces secondary paw swelling in the rat adjuvant arthritis model: ID50 = 7.4 mg/kg/b.i.d.

Interaction of human Clq with insoluble immunoglobulin aggregates.

A system of interaction between125I-Clq and IgG aggregates insolubilized by glutaraldehyde, which was designed as a model for studying the initial event in activation of the complement sequence, is described. Binding of Clq was shown to be rapid, specific, reversible and saturable, with an average dissociation constant in the order of 1 × 10−8M. The binding data produced non-linear Scatchard plots. The binding was favored at an ionic strength below μ = 0.15. a pH range of 5.5–8.5. and was sensitive to low concentrations of organic solvents. Among many compounds which interfered with Clq-IgG aggregate interaction, an anionic compound, suramin, was shown to be a good inhibitor of Clq binding with a sigmoidal inhibition curve and with a KD of Clq-suramin complex in the order of 5 × 10−5M. Dissociation of radiolabeled Clq by succinylation caused a loss of binding activity but the succinylated Clq retained the capacity to inhibit125I-Clq binding to IgG aggregates. The characteristic radioiodination pattern of Clq subunits was shown to be significantly changed when iodination was carried out on the IgG aggregates-bound Clq in such a fashion as to imply an IgG aggregates-induced conformational change in Clq, suggesting the usefulness of this system in exploring the possible mechanism underlying immune-complex mediated Cl activation.

IL-1β mediates induction of hepatic type 1 plasminogen activator inhibitor in response to local tissue injury

Type 1 plasminogen activator inhibitor (PAI-1), a major physiological inhibitor of plasminogen activation, is an important component of the hepatic acute phase response. We studied the acute phase regulation of murine hepatic PAI-1 in response to systemic toxicity and local tissue injury in both wild-type mice and in mice in which the interleukin (IL)-1β gene had been inactivated by gene targeting. Endotoxin induced plasma PAI-1 antigen levels and PAI-1 mRNA accumulation in liver to the same extent in both wild-type and IL-1β-deficient mice. In contrast, turpentine increased plasma PAI-1 and hepatic PAI-1 mRNA accumulation in wild-type mice but not in IL-1β-deficient mice. Intraperitoneal injection of murine IL-1β rapidly increased plasma PAI-1 and hepatic PAI-1 mRNA in both wild-type and IL-1β-deficient mice. These results suggest that IL-1β is a critical inducer of hepatic PAI-1 gene expression during the acute phase response to local tissue injury. In situ hybridization studies revealed that hepatocytes are the cells primarily responsible for the hepatic expression of the PAI-1 gene induced by lipopolysaccharide and turpentine.

Prevention of human leukocyte elastase-mediated lung damage by 3-alkyl-4-azetidinones

Abstract Simple 3-alkyl-4-azetidinones have been previously reported as potent inhibitors of human leukocyte elastase (HLE). Further modification of these simple monocyclic β-lactams has led to development of substituted 4-azetidinones that both inhibit HLE in a time dependent manner and, like previously reported modified cephalosporin sulfones, prevent HLE-induced lung damage in hamsters.

Quantitation of immune complexes by competitive inhibition of binding of Clq to insoluble IgG aggregates

An assay system for the binding of Clq to insoluble IgG aggregates was found useful for the quantitation of immune complexes in biological fluids. The assay, both easily and rapidly performed, is based on the competition of Clq binding substances with IgG aggregates. Serum from rheumatoid arthritis patients showed an increase in Clq binding substances over normal serum and this increase could be abolished by pretreatment of the serum with D-penicillamine.

Incorporation of l-leucine and d-glucosamine into skin fibroblasts derived from cystic fibrosis and normal individuals

Abstract Cultured skin fibroblasts from patients with diagnosed cystic fibrosis were compared with those from normal individuals in respect to incorporation of l -leucine or d -glucosamine into the subcellular components, to uptake of α-isobutyric acid and calcium, and to (Na+-K+)ATPase activity, etc. No significant difference which could be attributed to the disease-specific basic metabolic abnormality was demonstrated. Cystic fibrosis skin fibroblasts may not express the primary genetic defect at the level of generalized transport mechanism and overall protein metabolism.

Role of polymorphonuclear leukocytes in connective tissue breakdown during the reverse passive Arthus reaction.

The reverse passive Arthus (RPA) reaction performed in the skin of rats was modified to allow for the determination of polymorphonuclear leukocyte (PMN) infiltration and hemorrhage, as well as changes in vascular permeability. After initiation of the RPA reaction, PMN infiltration, monitored by measurement of tissue myeloperoxidase (MPO, EC 1.11.1.7) content, increased dramatically with time. Depending on the experimental conditions used, PMN accumulation reached a maximum 2-10 hr after increased vascular permeability (125I-labeled albumin content) had peaked. Hemorrhage (59Fe-labeled erythrocyte accumulation) began to occur only after significant levels of PMN were reached and continued to increase proportionately to the level of PMN infiltration attained. Indomethacin administered 30 min prior to initiating the RPA reaction had no effect on vascular permeability increase but suppressed both PMN accumulation and hemorrhage development about 50%. When indomethacin was given 2 hr after the RPA reaction was begun, no effect on any of the RPA variables was noted. Dexamethasone suppressed the increase in vascular permeability (53%), PMN accumulation (78%), and hemorrhage (90%) when given 30 min prior to initiation of the reaction. Dexamethasone given 2 hr after initiating the RPA suppressed the entire reaction, but to a lesser extent. Catalase, as well as trasylol, alpha-1-antiproteinase and soybean trypsin inhibitor, inhibited PMN accumulation as well as hemorrhage when given intravenously at plus 2 hr. These results indicate that the damage to blood vessels during a severe RPA reaction is a direct consequence of PMN activity.

Inhibition of immune complex-mediated activation of complement. Effects of agents modulating activation of, and the activated C1 complex.

AbstractSeveral known chemical compounds were shown to selectively inhibit the interaction between immune aggregates and Clq, the activation of Clr-Cls complex by immune aggregate-bound C1q, and the esterolytic activity of the activated Cls,