Identification of unique truncated KC/GRO beta chemokines with potent hematopoietic and anti-infective activities.

SK&F 107647, a previously described synthetic immunomodulatory peptide, indirectly stimulates bone marrow progenitor cells and phagocytic cells, and enhances host defense effector mechanisms in bacterial and fungal infection models in vivo. In vitro, SK&F 107647 induces the production of a soluble mediator that augments colony forming cell (CFU-GM) formation in the presence of CSFs. In this paper we purified and sequenced the stromal cell-derived hematopoietic synergistic factors (HSF) secreted from both murine and human cell lines stimulated with SK&F 107647. Murine HSF is an N-terminal 4-aa truncated form of the CXC chemokine, KC, while human HSF was identified as an N-terminal 4-aa truncated form of the CXC chemokine, GRO beta. In comparison to their full-length forms, truncated KC and truncated GRO beta were 10 million times more potent as synergistic growth stimulants for CFU-GM. Enhanced potency of these novel truncated chemokines relative to their full-length forms was also demonstrated in respiratory burst assays, CD11b Ag expression, and intracellular killing of the opportunistic pathogen, Candida albicans. Administration of truncated KC significantly enhanced survival of mice lethally infected with C. albicans. The results reported herein delineate the biological mechanism of action of SK&F 107647, which functions via the induction of unique specific truncated forms of the chemokines KC and GRO beta. To our knowledge, this represents the first example where any form of KC or GRO beta were purified from marrow stromal cells. Additionally, this is the first demonstration of in vivo efficacy of a CXC chemokine in an animal infectious fungal disease model.

Treatment of experimental gram-negative and gram-positive bacterial sepsis with the hematoregulatory peptide SK&F 107647.

SK&F 107647, a novel synthetic low-molecular-weight peptide, has demonstrated potent antiinfective activities in murine models of fungal and viral infection. To determine if the hematoregulatory activities of SK&F 107647 could offer protection over conventional antibiotic therapy or as a single agent in animal models of bacterial sepsis, rats were implanted intraperitoneally with a live bacteria-containing fibrin-thrombin clot. Rats pretreated subcutaneously or orally with SK&F 107647 and then infected with either a gram-negative (Escherichia coli) or a gram-positive (Staphylococcus aureus) bacteria-containing clot demonstrated significantly improved survival over control formulation-treated animals. Treated animals showed increased effector cell activation, measured by CD11b expression on neutrophils and monocytes, and up to 1000-fold reduction in the number of E. coli recovered from blood. Thus, the hematoregulatory activities of SK&F 107647 can increase natural host resistance to infections caused by both gram-negative and gram-positive bacteria.

Efficacy of the hematoregulatory peptide SK&F 107647 in experimental systemic Candida albicans infections in normal and immunosuppressed mice.

SK&F 107647, a novel synthetic dimeric pentapeptide, has been shown to be a potent hematoregulatory agent. The potential for the hematoregulatory factors elicited by SK&F 107647 to confer protection in experimental models of systemic Candida albicans infection was evaluated in immunosuppressed and immunocompetent mice. Prophylactic treatment with recombinant human interleukin-1 (rhIL-1), recombinant human granulocyte colony stimulating factor (rhG-CSF), or the hematoregulatory peptide SK&F 107647 significantly increased survival times in gamma irradiated immunosuppressed as well as non-irradiated immunocompetent mice challenged with a lethal dose of C. albicans. Protection was also observed in athymic nu/nu "nude" mice. Additionally, significant increases in survival in non-irradiated immunocompetent mice dosed by oral gavage were observed. These results indicate that SK&F 107647 can significantly enhance natural host resistance to experimental C. albicans infections both in immunosuppressed and immunocompetent mice.

Synthesis, specificity, and antifungal activity of inhibitors of the Candida albicans delta 24-sterol methyltransferase.

A series of side chain modified analogues of cholesterol and lanosterol (1-10) have been synthesized and evaluated as inhibitors of the Candida albicans delta 24-sterol methyltransferase. Two sterol substrate analogues 1 and 2 which contained a 24-thia substituent were relatively modest inhibitors of the enzyme (Ki = 1.5-72 microM). Compounds which mimic the carbocation intermediates proposed for the methyltransferase reaction, including sulfonium salts 4-6, amidines 7 and 8, and imidazoles 9 and 10 were substantially more potent inhibitors (Ki = 5-500 nM). All of the sterol analogues examined displayed less than 10-fold selectivity for inhibition of the methyltransferase versus the rat liver delta 24-sterol reductase. The sterol analogues were tested for in vitro antifungal activity against C. albicans, Candida tropicalis, and Torulopsis glabrata. The minimum inhibitory concentrations versus C. albicans correlated well with the Ki values for methyltransferase inhibition, and the potency of several compounds approached that of amphotericin B, although only modest fungicidal activity was observed.

Divergent patterns of pulmonary blastomycosis induced by conidia and yeasts in athymic and euthymic mice.

Athymic nude (nu/nu) mice are uniformly more susceptible than euthymic nu/+ mice to lethal infection with intranasally inoculated Blastomyces dermatitidis, whether infection is initiated by yeasts or conidia. Conidial infection requires a high inoculum size; the disease produced is prolonged and disseminated. Yeasts are infective at a low inoculum size and produce a rapidly fatal pneumonia. Thymus transplantation is more protective for conidia-infected than yeast-infected nude mice, presumably because the disease course is long enough for an effect to become demonstrable. Yeast inocula multiply more rapidly in the lungs than do conidial inocula. This may relate to the greater susceptibility of conidia to heterophils evoked in the airways, and the fact that yeasts derived from conidial inocula must survive in the face of an established inflammatory reaction. When yeasts and conidia are inoculated simultaneously, the disease produced is less severe than when yeasts are inoculated alone, presumably because of a more intense and diffuse inflammatory response engendered by the conidia. Suppression of conidia-derived yeast replication is demonstrable for at least 1 wk in nu/nu mice and for 2 to 3 wk in nu/+ mice. The latter delay appears attributable to the intact immune system in nu/+ mice, and the probability that cellular immunity limits the subsequent replication of yeasts. Eventually, the immune response fails to control yeast replication, and the mice succumb. These studies provide further insights into the role of the thymus in host defense against B. dermatitidis and the basis for the differential pace of infection when mice are infected with yeasts or conidia.

Influence of fungal surface components on the interaction of Coccidioides immitis with polymorphonuclear neutrophils.

Polymorphonuclear neutrophils (PMNs) possess phagocytic and fungicidal activity against Coccidioides immitis that declines during maturation from arthroconidia to round cells, is lost throughout spherule maturation, and returns when endospores are released from ruptured spherules. Studies of PMN chemiluminescence, iodination, and degranulation give similar results. Phagocytosis of forms other than spherules is strain dependent and enhanced by immune serum. The absence of adequate PMN-spherule interaction may be attributed to the production of an extracellular fibrillar matrix, glycoprotein in composition, that restricts intimate PMN-spherule contact. When the spherule ruptures, PMNs enter to phagocytose endospores that are themselves invested by a matrix derived from the inner spherule wall. The immunochemical relationship between the outer matrix and the inner matrix remains to be discovered. Nevertheless, presence of the outer matrix may help to explain the long-standing histopathologic observation that PMNs fail to attack spherules until they release their endospores.

Intracellular and extracellular defenses of human phagocytes against Blastomyces dermatitidis conidia and yeasts.

The lesions of blastomycosis are characterized by both suppuration and granuloma formation, but the relative roles of human neutrophils, monocytes, and macrophages against Blastomyces dermatitidis are poorly defined. Our studies reveal that B. dermatitidis yeasts are generally too large to be ingested by polymorphonuclear neutrophils (PMNs), and are killed predominantly by external PMN attachment and degranulation, whereas conidia are first ingested, then killed. PMN function is maximal in the presence of serum, divalent cations, and complement, and killing is more efficient for conidia (approximately 50%) than for yeasts (approximately 20%). PMNs that have degranulated, but remain attached to yeasts, block access by contiguous PMNs. When degranulated PMNs are removed, allowing access by fresh PMNs, there is a further increment in yeast killing. Both conidia and yeasts are killed by predominantly oxidative PMN mechanisms, with conidia being greater activators of the respiratory burst, and proportionately more influenced by oxidative inhibitors. Peripheral blood monocytes can kill conidia (approximately 35%), but are feebly active against yeasts (approximately 5%). Monocyte-derived macrophages kill approximately 90% of conidia and 40% of yeasts. The dramatic susceptibility of conidia, the infective particles of B. dermatitidis, to nonspecific phagocytic host defenses may help to explain the relative rarity of blastomycosis as a clinical problem. The presence of PMNs in lesions of blastomycosis may indicate an active, although limited, role of these cells in host defense against B. dermatitidis yeasts.

Patterns of death, complication, and error in the management of motor vehicle accident victims: implications for a regional system of trauma care.

A nonautopsy, retrospective analysis of severe motor-vehicle accident trauma can provide valuable information in regard to volume of trauma and quality of care. In a 6-county region surrounding a large metropolitan area trauma care, as reviewed by this method, had deficiencies at all levels of delivery. Patients were taken to the nearest hospital. Hospitals then had not been classified or designated according to capability. Twenty-five per cent of the fatalities and 16% of all outcomes were considered inappropriate for the severity of injury incurred. A regional trauma system with categorization and designation of hospitals providing trauma care would have eliminated or improved these deficiencies, resulting in improved outcomes for a significant percentage of these patients.