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.

Isolation and characterization of a sigB deletion mutant of Staphylococcus aureus.

The sigB gene of Staphylococcus aureus, coding for the alternate sigma factor B, has been deleted by allelic replacement mutagenesis. The mutant grew as well as the parent in vitro, although it was deficient in clumping factor, coagulase, and pigment. In two murine and one rat infection model the mutant showed no reduction in virulence.

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.

In vivo modulation of hematopoiesis by a novel hematoregulatory peptide.

The hematoregulatory peptide dimer, HP5B, enhances myelopoiesis by stimulating stromal cell cytokine production. However, the disulfide bridge of this peptide is susceptible to reduction, leading to the formation of monomeric pentapeptide, HP5, a direct-acting inhibitor of myelopoiesis. We have replaced the disulfide (S-S) bond of HP5B dimer with an isosteric ethylene (CH2-CH2) group, creating a new, nonreducible, metabolically more stable peptide (SK&F 107647). This novel peptide was tested in vitro and in vivo for hematopoietic effects. In vitro, SK&F 107647 has no direct colony-stimulating activity (CSA). Stimulation of murine stromal cells with SK&F 107647 results in production and release of CSA at concentrations as low as 0.01 ng/mL, at least 10-fold lower than observed with HP5B dimer. Injection of SK&F 107647 in normal mice results in a two- to six-fold increase in serum CSA, which becomes maximal at 6 hours postinjection. Administration of peptide daily over 4 days (q.d. x 4) by both parenteral and oral routes results in significant increases in absolute numbers of granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells, as well as stimulating their cell cycle rates. A doubling in day 8 CFU-S was also observed in SK&F 107647-treated mice. Continuous subcutaneous (s.c.) infusion of SK&F 107647 in femorally cannulated rats demonstrated modest but significant elevation of peripheral blood neutrophil and monocyte counts within 7 days. SK&F 107647 represents a novel synthetic hematoregulatory peptide that shares biological and/or modulatory activities with natural hematopoietic cytokines.

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.

The critical role of CO2 in the morphogenesis of Coccidioides immitis in cell-free subcutaneous chambers.

In order to ascertain the factors controlling conversion of Coccidioides immitis arthroconidia to endosporulating spherules, studies were conducted with the use of subcutaneously implanted dialysis chambers in mice. The dialysate had the following characteristics: pH approximately 7.36; CO2 partial pressure, approximately 50 mm Hg; O2 partial pressure, approximately 140 mm Hg; protein content, approximately 20 mg/ml; and no cells. When chambers were inoculated with arthroconidia, endosporulating spherules developed. Introduction of syngeneic phagocytes had no effect. When dialysate or autologous serum was studied in vitro in room air, arthroconidia converted to mycelia unless CO2 supplementation (CO2 partial pressure, 20-80 mm Hg) was provided, in which case endosporulating spherules developed. The effect of CO2 could not be reproduced with either NaHCO3 or other buffers. These studies indicate that CO2 at a partial pressure found in normal host tissues is essential for formation of endosporulating spherules and that host phagocytes are not needed for such conversion in tissue fluids, either in vivo or in vitro.