Helicobacter pylori growth and urease detection in the chemically defined medium Ham's F-12 nutrient mixture.

Obstacles continue to hinder in vitro studies of the gastric human pathogen Helicobacter pylori, including difficulty culturing the organism in the absence of serum or blood, rapid loss of viability following exponential growth due to autolysis, and the necessity for using high starting inocula. We demonstrate that H. pylori grows in the chemically defined broth medium Ham's F-12 nutrient mixture (F-12) in the absence of fetal bovine serum (FBS); this represents a breakthrough for studies in which serum components or proteins interfere with interpretation of results. Cultures can be continually passaged in fresh, FBS-free F-12 medium at an initial inoculum of only approximately 10(3) CFU/ml. All H. pylori strains (n = 21), including fresh clinical isolates, grew in serum-free F-12. H. pylori grew poorly in the related medium, F-10, unless additional zinc was supplied. Enhanced growth of H. pylori in F-12 broth was obtained by addition of bovine serum albumin (BSA) (1 mg/ml), beta-cyclodextrin (200 microg/ml), or cholesterol (50 microg/ml). H. pylori also grew in several simplified versions of F-12 broth lacking glucose and most vitamins but containing hypoxanthine, pyruvate, and all 20 amino acids. On F-12 medium solidified with agar, H. pylori only grew when BSA (98% pure; 1 mg/ml), cholesterol (50 microg/ml), beta-cyclodextrin (200 microg/ml), or FBS (2 to 4%) was added; addition of urea and phenol allowed colorimetric detection of urease activity. Thus, F-12 agar plus cholesterol or beta-cyclodextrin represents the first transparent chemically defined agar and the first urease indicator agar for H. pylori. Several lines of evidence suggested that BSA itself is not responsible for H. pylori growth enhancement in F-12 containing BSA or FBS. Taken together, these innovations represent significant advances in the cultivation and recovery of H. pylori using chemically defined media. Use of F-12 or its derivatives may lead to improved understanding of H. pylori metabolism, virulence factors, and transmission, and result in improved recovery and identification of H. pylori from clinical specimens.

Cytokine induction by the immunomodulators imiquimod and S-27609.

Imiquimod (R-837, S-26308) and the analogue S-27609 were evaluated for cytokine induction in human blood cells. Both compounds induced interferon-alpha (IFN), tumor necrosis factor-alpha (TNF), interleukin (IL)-1 beta, and IL-6 with S-27609 being 5 to 10 times more potent. Imiquimod and S-27609 also induced IL-1 alpha, IL-1 receptor antagonist, IL-10, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte CSF (G-CSF), and macrophage inflammatory protein-1 alpha. The profile of cytokines induced by imiquimod and S-27609 was different from those seen with lipopolysaccharide and polyinosinic-polycytidylic acid. Kinetic studies with both imiquimod and S-27609 revealed induction of cytokines as early as 1-4 h after stimulation. Although most of the cytokines produced by S-27609 were secreted, significant concentrations of IL-1 alpha and IL-1 beta remained intracellular. Monocytes were largely responsible for the cytokines produced. Finally, S-27609-induced mRNA expression for TNF, IFN, and IL-8, and this induction did not require protein synthesis. Taken together, these studies extend previous findings by showing induction of additional cytokines and providing insight into the mechanism of cytokine induction by these molecules.

Cellular requirements for cytokine production in response to the immunomodulators imiquimod and S-27609.

Imiquimod (R-837) and its analog, S-27609, belong to a class of imidazoquinolinamines that have potent antitumor and antiviral effects in animals. Much of their biologic activity is a result of the induction of cytokines, including interferon-alpha (IFN-alpha), tumor necrosis factor alpha (TNF), and others. In this study, the cells responsible for S-27609- and imiquimod-induced cytokine production were characterized. E rosette+ T cells were not the major cell population responsible for IFN-alpha and TNF in response to S-27609 or imiquimod. In contrast, E rosette- cells and unseparated PBMC produced similar concentrations of IFN-alpha and TNF in response to S-27609 and imiquimod. Elimination of monocytes by treatment with the lysosomotropic agent L-leucine methyl ester (LME) or depletion using antibody to CD14 and immunomagnetic beads abrogated IFN-alpha and TNF production induced by S-27609, imiquimod, or LPS but not poly(I)/(C). LME treatment also abolished interleukin (IL)-1 alpha, IL-beta, IL-6, and IL-8 production stimulated by S-27609 and imiquimod. Removal of HLA-DR+ or CD36+ monocytes also caused a significant reduction in S-27609- and imiquimod-induced IFN-alpha and TNF. Elimination of B cells, NK cells, and dendritic cells did not significantly reduce cytokine induction in response to S-27609. Thus, the cell population responsible for the majority of cytokine release in human PBMC in response to S-27609 and imiquimod is a E rosette-, CD14+, CD36+, HLA-DR+ monocyte.

Cytokine induction in mice by the immunomodulator imiquimod.

Imiquimod has been identified as a potent antiviral and antitumor agent in animal models. The biological activity associated with imiquimod has been attributed to its induction of interferon (IFN)-alpha. The present studies evaluated imiquimod administered orally for its ability to stimulate production of IFN and other cytokines in mice. The cytokine profile induced by imiquimod was compared with other known immunomodulators. Imiquimod was found to stimulate increased serum IFN in mice. Daily dosing of imiquimod for five consecutive days led to diminished production of IFN in mice as measured after the final dose. Elevated levels of serum tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 but not IL-1 alpha were found in serum from mice treated with imiquimod. Imiquimod produced significantly higher levels of IFN but lower levels of TNF and IL-6 and IL-1 alpha than lipopolysaccharide. Polyinosinic acid:polycytidylic acid induced significantly higher amounts of IFN but lower levels of TNF and IL-6 than imiquimod. Imiquimod stimulated significantly higher levels of IFN when compared with 2-amino-5-bromo-6-phenyl-4(3H)-pyrimidinone (ABPP) and similar levels of IFN when compared with tilorone. Neither ABPP nor tilorone induced TNF or IL-6. Finally, imiquimod stimulated TNF, IFN, and IL-6 production in cultures of mouse spleen and bone marrow cells. These studies demonstrate that imiquimod induces not only IFN but other cytokines as well, all of which may contribute to its biological activity.