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Cytokine antibody arrays were used to establish the profiles of cytokine release from THP-1 monocytes exposed to different amphotericin B (AMB) drug delivery systems. Fungizone (FZ) and Amphotec (ABCD) caused the release of significantly more inflammatory molecules and the release of inflammatory molecules at higher levels than either AmBisome (L-AMB) or Abelcet (ABLC) after 6 h of treatment. Specifically, tumor necrosis factor alpha (TNF-␣), interleukin-8 (IL-8), GRO-(␣␤␥), monocyte chemoattractant protein-1 (MCP-1), RANTES, IL-10, and IL-6 were detected and semiquantified with a chemiluminscence imaging system. TNF-␣, IL-8, and MCP-1 were the most predominant; however, little if any TNF-␣ was present in ABLC-or L-AMB-treated cultures. The TNF-␣ and IL-8 levels determined by quantitative enzyme-linked immunosorbent assay correlated with the relative cytokine levels measured by using the antibody arrays. Although the viabilities of THP-l monocytes in all AMB-treated cultures were similar by trypan blue exclusion, the amount of lactic dehydrogenase released was significantly larger in FZ-and ABCD-treated cultures than in L-AMBand ABLC-treated cultures, indicating more membrane perturbations with those formulations. Membrane cation channel formation was also measured in model cholesterol-containing large unilamellar vesicles to directly assess the ion channel formation ability of the system. Only FZ and ABCD induced significant ion currents at concentrations less than 1.5 ؋ 10 ؊5 M. These results may help provide rationales for the immediate cytokine-mediated side effects observed with FZ and ABCD and the reduced side effects observed with L-AMB and ABLC.Amphotericin B (AMB) is a polyene antifungal antibiotic and for a number of years has been the "gold standard" for the treatment of systemic fungal infections. Three new lipid-based formulations, Abelcet (ABLC), Ambisome (L-AMB), and Amphotec (ABCD), have been developed in an attempt to obtain improved efficacies, therapeutic indices, and tolerabilities compared to those of the traditional AMB formulation, Fungizone (FZ). In clinical trials, improvements in renal tolerability were shown for all three lipid formulations; however, the levels of acute toxicity, including fever, chills, hypotension, and nausea, vary with each formulation (8, 23). L-AMB has been shown to promote the least adverse effects, while immediate reactions to ABCD are often as frequent and severe as those to FZ (23). Clinical toxicity has been associated with increases in plasma tumor necrosis factor alpha (TNF-␣) and interleukin-6 (IL-6) levels during infusion of the drug (1), while in vitro studies with the human THP-1 monocytic cell line have further identified and characterized a number of proinflammatory cytokines that are induced and released following exposure to FZ (5,19,20). Because the clinical effects of the four AMB formulations are different, we hypothesized that each AMB formulation would produce a different cytokine profile. Using recently developed antibody arrays (13, 27) and t...

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The magnitude of the genetic relatedness of the two antigenic subtypes of equine herpesvirus 1 (EHV-1) was determined by DNA-DNA reassociation kinetics. Denatured, labeled viral DNA from one EHV-1 subtype was allowed to reassociate in the presence or absence of the unlabeled heterologous viral DNA. The initial rate of reassociation of either labeled viral DNA was increased by the presence of the heterologous viral DNA to an extent indicating 10 to 20% homology between the two EHV-1 genomes. Similar estimates of the amount of homology between the genomes of the two EHV-1 subtypes were obtained by determining the maximum fraction of labeled viral DNA that could be made resistant to S1 nuclease by hybridization with a large molar excess of the unlabeled, heterologous viral DNA. Analysis of the thermal stability of the subtype 1-subtype 2 heteroduplex DNA indicated approximately 30%o base pair mismatching within the hybrid DNA molecules. Cross-hybridization of 2p_

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The effects of deoxycholate amphotericin B (DAmB), amphotericin B lipid complex (ABLC), and amphotericin B colloidal dispersion (ABCD) on mRNA profiles from 218 genes in treated THP-1 monocytes were compared. Sixty-one genes were up-regulated and 8 were down-regulated by one or more of the AmB formulations. Fifty-three genes were up-regulated by DAmB while 24 and 18 genes were up-regulated by ABCD and ABLC, respectively. DAmB and ABCD up-regulated many pro-inflammatory genes, whereas ABLC did not. All three formulations up-regulated multiple categories of genes including the anti-apoptosis gene BIRC3 and the chemokine CXCL9 (MIG). Based on representative genes, DAmB activated more signaling pathways than ABCD or ABLC. Quantitative real time PCR confirmed array up-regulation of representative pro-inflammatory genes IL-8, CCL20 (MIP-3alpha), and CXCL2 (MIP-2alpha). Our study represents the first larger scale comparative gene expression profiling which may provide additional rationales for clinical side effects of each amphotericin B formulation.