John L. Ryan

Bacterial endotoxins and host immune responses.

Publisher Summary This chapter discusses the nature of endotoxins and their interactions with cells of the immune system. The direct interaction of endotoxin with B lymphocytes leading to the formation of antibodies to endotoxin as well as a spectrum of other immunoglobulin molecules is discussed. In addition, the interactions of endotoxins with T cells and macrophages and the associated immunoregulatory effects are discussed. The central role of the lipid A portion of the endotoxin molecule in cellular activation and the molecular events occurring at the cell surface in the course of stimulation are presented. The potential of endotoxins as therapeutic manipulators of the immune response in man is also evaluated. Major direct interactions of endotoxins with B lymphocytes have been documented, leading to synthesis and secretion of antibody directed not only against antigenic determinants on the endotoxin molecules themselves, but also with specificities characteristic of the complete repertoire of variable region gene products. Knowledge of the immunobiology of endotoxins has prompted investigations in their therapeutic uses in man as both antigenically distinct and cross-reactive immunogens in protection against gram-negative infections.

Gangliosides reduce leakage of aqueous-space markers from liposomes in the presence of human plasma

We have studied the role of glycolipids in reducing leakage of aqueous-space markers from liposomes, composed primarily of egg phosphatidylcholine, in the presence of human plasma. Liposomes were either small unilamellar (SUV) or large unilamellar (LUV). Leakage of liposome contents as affected by the incorporation into the liposomal bilayer of mono-, di-, or trisialogangliosides (GM, GD, GT) at different molar ratios in the presence or absence of cholesterol was examined. Leakage from liposomes decreased with increasing ganglioside sialic acid. Asialogangliosides had no effect on calcein leakage in the presence of plasma. The stabilizing effect of gangliosides and cholesterol was synergistic, and SUV containing 10 mol% GT and 33 mol% cholesterol had a half-life for leakage of calcein in plasma at 37 degrees C approaching 24 hours. LUV in the presence of plasma retained their contents longer than SUV, and gangliosides had an additional stabilizing effect. Phosphatidylserine and sulfatides were also capable of substituting for gangliosides in stabilizing liposomes to plasma-induced leakage. It appears that gangliosides stabilize liposomes in plasma at least in part through their ability to impart surface negative charge.

Cefoperazone versus combination antibiotic therapy of hospital-acquired pneumonia

Cefoperazone monotherapy was compared with combination antibiotic therapy in a randomized prospective evaluation of patients with hospital-acquired pneumonia. Cefoperazone was as effective as either clindamycin/gentamicin or cefazolin/gentamicin (cure rate: 45 of 52 cefoperazone-treated patients [87 percent], versus 44 of 61 combination-therapy patients [72 percent], p = 0.069). With the exception of hypoprothrombinemia in those patients who did not receive prophylactic vitamin K, there was no difference in the incidence of side effects. In addition, no difference was noted in the incidence of superinfections or secondary pneumonias. When antibiotic costs, administration costs, and laboratory costs were considered, cefoperazone monotherapy was the least expensive antibiotic regimen. Cefoperazone is a suitable alternative to combination antibiotic therapy for the treatment of hospital-acquired pneumonia.

Optimal therapy for enterococcal endocarditis

Enterococcal endocarditis accounts for an increasing proportion of cases of endocarditis in recent years. The combination of a penicillin and an aminoglycoside has become an accepted standard of treatment for this disease. However, the optimal choice of antibiotics, duration of therapy, and timing of surgical intervention remain controversial. This study reviews the presentation, clinical course, treatment, and outcome in 37 patients with 42 separate episodes of enterococcal endocarditis at four Yale University hospitals. Patients treated with aminoglycosides and penicillins or vancomycin had significantly better outcomes than those who did not receive aminoglycosides. However, the duration of aminoglycoside therapy (more than four versus less than four weeks) did not appear to affect outcome significantly. These results suggest that excellent cure rates may be achieved after treatment for less than four weeks with an aminoglycoside in combination with penicillin or vancomycin, thus potentially avoiding significant renal and vestibular toxicity.

Acylation of monocyte and glomerular mesangial cell proteins. Myristyl acylation of the interleukin 1 precursors.

Acylation of cellular proteins with the fatty acids myristate or palmitate represents an important mechanism for the co- or posttranslational modification of proteins. Lipid A, the biologically active component of bacterial endotoxin, exerts a number of biochemical effects on responsive cell types. Evidence is presented that lipid A stimulates the synthesis and subsequent myristyl acylation of intracellular monocyte and glomerular mesangial cell proteins. Two of the myristylated monocyte proteins were identified by specific immunoprecipitation as the 33-kD IL 1 alpha and beta precursors; a similar myristylated protein was found in mesangial cells. The 17-kD secretory form of monocyte IL 1 beta did not contain covalently linked myristate. Myristyl acylation of the IL 1 precursor proteins may facilitate the processing or membrane localization of these proteins, which lack characteristic hydrophobic signal sequences. The acylated 33-kD IL 1 alpha may remain preferentially associated with the membrane in an active form, whereas limited proteolysis may convert the biologically inactive IL 1 beta precursor into the extracellular, nonacylated, active 17-kD protein.

Cefoperazone versus ceftazidime monotherapy of nosocomial pneumonia

Cefoperazone and ceftazidime monotherapy were compared in a randomized, prospective evaluation of patients with nosocomial pneumonia. These antibiotics were equally effective, with an overall successful treatment rate of 45 of 62 (73 percent) for cefoperazone-treated patients and 50 of 63 (79 percent) for ceftazidime-treated patients (p = 0.41). There was no difference in the incidence of side effects (including hypoprothrombinemia), superinfections, or colonization of the oropharynx with yeast, enterococcus, Staphylococcus aureus, or resistant gram-negative bacilli. When antibiotic administration, and laboratory costs are considered, cefoperazone is less expensive than ceftazidime. Both cefoperazone and ceftazidime are effective therapy for nosocomial pneumonia.

Incorporation of LPS in liposomes diminishes its ability to induce tumoricidal activity and tumor necrosis factor secretion in murine macrophages.

We investigated the effect of lipopolysaccharide (LPS) incorporated into phospholipid vesicles (liposomes) on the induction of macrophage‐mediated tumor cytotoxicity and tumor necrosis factor (TNF) secretion. The incorporation of Salmonella minnesota rough (Re)‐LPS into multilamellar or small unilamellar vesicles (liposomes) resulted in an 100‐to 1,000‐fold reduction in its potency to activate both the macrophage cell line RAW 264.7 and murine thioglycolate elicited peritoneal macrophages to become cytotoxic for L929 and P815 tumor cells. Liposomal LPS was also a 100‐ to 1,000‐fold less potent inducer of TNF secretion from RAW 264.7 cells. Cytokines secreted by the activated macrophages contributed to the cytotoxic effect on the L929 cells but not the P815 cell line. Human recombinant TNF was not cytotoxic for either cell line but was cytostatic for the L929 cell line. Morphological examination of the cells after uptake of fluorescent, free, and liposomal LPS revealed that both forms were internalized by the endocytic pathway. This, together with the considerably reduced potency of liposomal LPS to induce tumor cytotoxicity and TNF secretion, suggests that the interaction of the hydrophobic part of the lipid A moiety of LPS with the macrophage plasma membrane is needed to optimally activate these cells. Incorporation of LPS into liposomes effectively abrogates this interaction.

Ganglioside alterations in stimulated murine macrophages

A two-dimensional thin-layer chromatographic technique has been used to separate and display gangliosides from murine peritoneal macrophages in different functional states. Resident macrophages have a relatively simple ganglioside pattern with about 15 resorcinol-positive spots. Gangliosides from resident cells contained mostly (90%) N-glycolylneuraminic acid. Thioglycolate-elicited and Corynebacterium parvum-activated macrophages have much more complex patterns with about 40 resorcinol-positive spots. Although ganglioside sialic acid content of stimulated macrophages was only slightly higher than that of resident cells, it consisted of nearly equal amounts of N-acetyl- and N-glycolylneuraminic acid. The shift in the ganglioside sialic acid type and the expression of different gangliosides in macrophages upon stimulation may help explain some of the differences in function and responsiveness noted in these macrophage populations.

Selective Anti-Inflammatory Action of Interleukin-11 in Murine Lyme Disease: Arthritis Decreases while Carditis Persists

The role of interleukin (IL)-11, a cytokine with potent anti-inflammatory properties, in murine Lyme disease was investigated. Borrelia burgdorferi-infected mice treated with IL-11 developed less arthritis than did control animals. In contrast, IL-11 blocking antibodies increased Lyme arthritis. Murine Lyme carditis was not affected by either IL-11 or IL-11 antibodies. Administration of IL-11 was associated with increased production of mRNA for IL-12 and inducible nitric oxide synthase but not interferon-gamma or IL-4 in B. burgdorferi-infected mice, suggesting a predominant effect of IL-11 on the innate immune response. These data show that IL-11 selectively reduced joint but not cardiac inflammation caused by B. burgdorferi in mice.

Enterococcal meningitis: Combined vancomycin and rifampin therapy

Intrathecal vancomycin and oral rifampin have been used together to successfully treat a patient with enterococcal meningitis who was allergic to penicillin and who had failed a course of treatment with chloramphenicol. This therapy was tolerated very well and represents an alternate mode of therapy which should be considered in penicillin allergic patients with enterococcal meningitis.