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1.
J Dent Res ; 97(1): 33-40, 2018 Jan.
Article in English | MEDLINE | ID: mdl-29053389

ABSTRACT

A valuable approach to understand how individual and population genetic differences can predispose to disease is to assess the impact of genetic variants on cellular functions (e.g., gene expression) of cell and tissue types related to pathological states. To understand the genetic basis of nonsyndromic cleft lip with or without cleft palate (NSCL/P) susceptibility, a complex and highly prevalent congenital malformation, we searched for genetic variants with a regulatory role in a disease-related tissue, the lip muscle (orbicularis oris muscle [OOM]), of affected individuals. From 46 OOM samples, which are frequently discarded during routine corrective surgeries on patients with orofacial clefts, we derived mesenchymal stem cells and correlated the individual genetic variants with gene expression from these cultured cells. Through this strategy, we detected significant cis-eQTLs (i.e., DNA variants affecting gene expression) and selected a few candidates to conduct an association study in a large Brazilian cohort (624 patients and 668 controls). This resulted in the discovery of a novel susceptibility locus for NSCL/P, rs1063588, the best eQTL for the MRPL53 gene, where evidence for association was mostly driven by the Native American ancestry component of our Brazilian sample. MRPL53 (2p13.1) encodes a 39S protein subunit of mitochondrial ribosomes and interacts with MYC, a transcription factor required for normal facial morphogenesis. Our study illustrates not only the importance of sampling admixed populations but also the relevance of measuring the functional effects of genetic variants over gene expression to dissect the complexity of disease phenotypes.


Subject(s)
Cleft Lip/genetics , Cleft Palate/genetics , Ribosomal Proteins/genetics , Adolescent , Child , Child, Preschool , Female , Genes/genetics , Genome-Wide Association Study , Humans , Infant , Infant, Newborn , Male , Mitochondrial Ribosomes/metabolism , Oligonucleotide Array Sequence Analysis , Polymorphism, Single Nucleotide/genetics , Quantitative Trait Loci/genetics , Young Adult
3.
Biochim Biophys Acta ; 1463(2): 323-32, 2000 Feb 15.
Article in English | MEDLINE | ID: mdl-10675510

ABSTRACT

The methyl-d(3) amide derivative of the polyene antibiotic amphotericin B was synthesized, assayed for biological activity, incorporated into mechanically aligned bilayers of dipalmitoylphosphatidylcholine (DPPC), and examined by deuterium and phosphorus NMR. The amide derivative has a lesser, but qualitatively similar, biological activity relative to amphotericin B. Incorporation of the amide derivative and ergosterol into aligned DPPC bilayers resulted in a single, stable bilayer phase, as shown by phosphorus NMR of the DPPC headgroups. Deuterium NMR spectra revealed one major (2)H quadrupolar splitting and one major (2)H-(1)H dipolar splitting in the liquid-crystalline phase, consistent with a high degree of alignment and a single, averaged physical state for amphotericin B methyl-d(3) amide in the bilayer. Variations of the quadrupolar and dipolar splittings as a function of macroscopic sample orientation and temperature indicated that the amide derivative undergoes fast rotation about a motional axis that is parallel to the bilayer normal.


Subject(s)
1,2-Dipalmitoylphosphatidylcholine/chemistry , Amphotericin B/analogs & derivatives , Amphotericin B/chemistry , Lipid Bilayers/chemistry , Deuterium , Ergosterol/chemistry , Models, Molecular , Molecular Conformation , Nuclear Magnetic Resonance, Biomolecular , Thermodynamics
4.
Biochem Biophys Res Commun ; 254(3): 605-13, 1999 Jan 27.
Article in English | MEDLINE | ID: mdl-9920786

ABSTRACT

Using differential display reverse transcription-PCR (DDRT-PCR) we have identified several sequences that are specifically expressed by Histoplasma capsulatum during infection of murine macrophages (MPhi). Here, we report the characterization of a clone, pHc12, identified as a differentially expressed gene 1 hour after infection of MPhi. Screening of a cDNA library of H. capsulatum allowed us to isolate a clone, pHc12-E, that contains the complete coding sequence. We show that after infection the level of transcription of this gene increases about 5 fold. Analysis of its sequence revealed the presence of an open reading frame of 890 aa (ORF890) that shares respectively 30 and 33% identity with human and Caenorhabditis elegans p100 kD and rat p105 kD co-activator proteins. Using the two-dimensional Hydrophobic Cluster Analysis (HCA) method, we showed that H. capsulatum ORF890 and p100 kD co-activator proteins are clearly related. The H. capsulatum protein consists of a four-fold repeated module (domains I to IV) like the p100 kD co-activator proteins, whose three-dimensional (3D) structure is related to staphylococcal thermonuclease, followed by a modified fifth "hybrid" domain which partially resembles the structure of the tudor domain found in multiple copies in the Drosophila melanogaster tudor protein. These data strongly suggest that ORF890 is homologous to human p100 kD and that this protein, named Hcp100, may play an essential role during infection by co-activating the expression of specific genes.


Subject(s)
Fungal Proteins/genetics , Histoplasma/metabolism , Macrophages/microbiology , Amino Acid Sequence , Animals , Blotting, Northern , Cloning, Molecular , DNA, Complementary , Fungal Proteins/chemistry , Humans , Mice , Molecular Sequence Data , Open Reading Frames , Sequence Homology, Amino Acid
5.
Med Mycol ; 36(4): 205-12, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9776836

ABSTRACT

A 425-bp insertion in Histoplasma capsulatum strain G186B, denoted as Hc.SSU.1, was identified as a group I intron, based on the presence of the conserved sequence elements P, Q, R and S and a predicted secondary structure consistent for group I introns. The Hc. SSU.1 sequence from strain G186B was identical to strain G184B but differed from strain FLs1 by five nucleotides. Hc.SSU.1 was most similar to the group I intron from the black mould Exophiala castellanii. Southern blot analysis suggests that the intron is not dispersed in the genome and that most, if not all 18S rRNA genes harbour the intron. Northern blots demonstrated absence of the intron from mature 18S rRNA. A Hc.SSU.1-specific PCR assay detected the intron in six of 37 isolates of Histoplasma. Hc.SSU.1-containing strains exhibited no significant differences in antimicrobial susceptibilities when compared to isolates not containing Hc.SSU.1. This investigation demonstrates the existence of group I intron sequences in the H. capsulatum genome and its evolutionary relationship among other group I intron sequences.


Subject(s)
DNA, Ribosomal/genetics , Histoplasma/genetics , Nucleic Acid Conformation , RNA, Ribosomal, 18S/chemistry , RNA, Ribosomal, 18S/genetics , Base Sequence , Conserved Sequence , DNA Transposable Elements , DNA, Fungal/genetics , Evolution, Molecular , Genes, Fungal , Histoplasma/isolation & purification , Histoplasma/pathogenicity , Humans , Introns , Models, Structural , Molecular Sequence Data , Phylogeny , Polymerase Chain Reaction , RNA, Fungal/chemistry , RNA, Fungal/genetics , Sequence Alignment , Sequence Homology, Nucleic Acid , Soil Microbiology
6.
Microb Pathog ; 25(2): 55-66, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9712685

ABSTRACT

Establishment of infection and disease implies modifications in the genetic programmes of the cell systems that are involved and the differential expression of genes in both parasite and host. In order to identify and isolate relevant genes of the fungus, Histoplasma capsulatum, in which expression is specifically induced during its interaction with murine macrophages (Mphi), we performed a comparative analysis of the pattern of gene expression of the fungus before and after exposure to, and internalization into Mphi by using differential display reverse transcriptase-PCR (DDRT-PCR). Using a limited set of primer combinations, six cDNA fragments of H. capsulatum were identified and isolated; five representing fungal genes in which expressions were enhanced during Mphi infection, whereas one mRNA fragment was down-regulated. Slot blots followed by Northern blot analyses confirmed that the transcripts detected with cDNA clones were over expressed after 1 h of Mphi infection, whereas no transcripts were detected with mRNA purified from H. capsulatum before infection. Sequence analyses and database searches revealed no significant homology to any known sequence for five of these clones. One of the clones showed homology to the rat p105 kD protein, and to the p100 kD co-activator proteins of human and Caenorhabditis elegans. To our knowledge, this is the first experimental evidence that specific genes are differentially expressed by a fungal pathogen when it is exposed to, and phagocytosed by Mphi. Furthermore, these results show that the DDRT-PCR procedure has adequate sensitivity to detect fungal genes induced during parasite-host interaction to identify potential new targets that can be used to develop new antifungal drugs.


Subject(s)
Gene Expression Regulation, Fungal , Genes, Fungal , Histoplasma/genetics , Macrophages/microbiology , Polymerase Chain Reaction/methods , Amino Acid Sequence , Animals , Base Sequence , Caenorhabditis elegans/genetics , Cells, Cultured , Conserved Sequence , DNA Primers , Hematopoietic Stem Cells/cytology , Histoplasma/physiology , Humans , Male , Mice , Molecular Sequence Data , Open Reading Frames , RNA-Directed DNA Polymerase , Rats , Sequence Alignment , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid
7.
Med Mycol ; 36 Suppl 1: 45-51, 1998.
Article in English | MEDLINE | ID: mdl-9988491

ABSTRACT

Heat shock proteins (hsps) are ubiquitous families of proteins, found in all organisms studied so far. They are highly conserved across the species barrier and serve fundamental functions in cell physiology. The term 'heat shock' was adopted because of the early observation of the heat-inducible nature of these proteins, although, as it is now realized that they can be induced by a variety of stressful stimuli, it is probably more appropriate to call them 'stress proteins'. The nomenclature of many hsps, for example hsp90, hsp70 and hsp60, reflects the approximate molecular mass of hsps within each of these families. For many bacterial and parasitic infections, hsps were first recognized as immunodominant antigens on immunoblots of extracts from the organism probed with immune sera, or in T-cell proliferation assays. They have now been identified in a range of fungal pathogens, again often linked to an immune response. In this symposium, we review the association of hsps with humoral immunity to candidosis and aspergillosis, cellular immunity to histoplasmosis, and the identification of hsp70 in another dimorphic fungus, Paracoccidioides brasiliensis. Finally, the crucial role of the membrane in setting the temperature of the heat shock response in yeasts is discussed.


Subject(s)
Fungal Proteins/immunology , Fungi/immunology , Heat-Shock Proteins/immunology , Mycoses/immunology , Animals , Antibodies, Fungal/biosynthesis , Antibodies, Fungal/immunology , Cloning, Molecular , Fungal Proteins/genetics , Fungi/physiology , Genes, Fungal , Heat-Shock Proteins/genetics , Hot Temperature , Humans , Immunity, Cellular , Membrane Lipids/physiology , Mycoses/microbiology
8.
J Clin Microbiol ; 35(5): 1071-6, 1997 May.
Article in English | MEDLINE | ID: mdl-9114383

ABSTRACT

Immunological cross-reactivity among fungi has hampered the development of specific serodiagnostic assays for histoplasmosis. We report the molecular cloning and characterization of a Histoplasma capsulatum cDNA (GH17) that encodes an antigen with immunodiagnostic potential. GH17 is an 810-bp cDNA which encodes a protein of 211 amino acid residues. The GH17 sequence has almost no significant homology with other sequences in GenBank. Southern blot analysis suggests that GH17 is confined to a single location in the genomic DNA of H. capsulatum. Immunoblots indicated that the protein product of GH17 (expressed as a 140-kDa beta-galactosidase fusion protein) was recognized by antibodies in 18 of 18 sera from histoplasmosis patients, but not by antibodies in sera from patients or animals infected with other fungi. GH17 was expressed in a prokaryotic expression vector, pPROEX-1, and recombinant protein was purified by preparative electrophoresis. Antibodies raised to this protein bound to a 60-kDa native antigen in immunoblots of H. capsulatum yeast antigen extract. These results suggest that GH17 encodes an H. capsulatum antigen that may be useful for the diagnosis of histoplasmosis in humans.


Subject(s)
Antigens, Fungal/genetics , Histoplasma/immunology , Histoplasmosis/diagnosis , Immunoassay/methods , Amino Acid Sequence , Antigens, Fungal/immunology , Base Sequence , Cloning, Molecular , DNA, Complementary/analysis , DNA, Recombinant , Histoplasmosis/immunology , Humans , Molecular Sequence Data
9.
Biochem Biophys Res Commun ; 230(2): 457-61, 1997 Jan 13.
Article in English | MEDLINE | ID: mdl-9016802

ABSTRACT

We have characterized a region of the promoter of a cloned delta9-desaturase gene (Ole1) of Histoplasma capsulatum, a dimorphic pathogenic fungus of humans. The product of the delta9-desaturase gene is involved in regulating membrane fluid state in animal cells and microorganisms. To identify sequences critical for Ole1 expression in both the saprobic mycelial and parasitic yeast phases of this organism, we performed a deletion analysis. Evidence is presented that a 240 nt region of the proximal promoter is involved in a phase-specific binding in vitro. By sequence analysis we have identified one likely regulatory element that coincides with an AP1 binding site (TGACTAA) that is located at -740 nt of 5'-upstream from the ATG. Using gel mobility shift assays, we show that this cis-acting element binds nuclear proteins extracted from the yeast and mycelial phases of H. capsulatum that may participate in control of expression of the delta9-desaturase gene.


Subject(s)
Gene Expression Regulation, Enzymologic , Genes, Fungal , Histoplasma/enzymology , Promoter Regions, Genetic , Stearoyl-CoA Desaturase/biosynthesis , Transcription Factor AP-1/metabolism , Transcription, Genetic , Base Sequence , DNA Footprinting , DNA, Fungal/chemistry , DNA, Fungal/isolation & purification , Gene Expression Regulation, Fungal , Histoplasma/genetics , Molecular Sequence Data , Sequence Homology, Nucleic Acid , Stearoyl-CoA Desaturase/genetics
10.
Proc Natl Acad Sci U S A ; 93(9): 3870-5, 1996 Apr 30.
Article in English | MEDLINE | ID: mdl-8632982

ABSTRACT

Addition of a saturated fatty acid (SFA) induced a strong increase in heat shock (HS) mRNA transcription when cells were heat-shocked at 37 degrees C, whereas treatment with an unsaturated fatty acid (UFA) reduced or eliminated the level of HS gene transcription at 37 degrees C. Transcription of the delta 9-desaturase gene (Ole1) of Histoplasma capsulatum, whose gene product is responsible for the synthesis of UFA, is up-regulated in a temperature-sensitive strain. We show that when the L8-14C mutant of Saccharomyces cerevisiae, which has a disrupted Ole1 gene, is complemented with its own Ole1 coding region under control of its own promoter or Ole1 promoters of H. capsulatum, the level of HS gene transcription depends on the activity of the promoters. Fluorescence anisotropy of mitochondrial membranes of completed strains corresponded to the different activity of the Ole1 promoter used. We propose that the SFA/UFA ratio and perturbation of membrane lipoprotein complexes are involved in the perception of rapid temperature changes and under HS conditions disturbance of the preexisting membrane physical state causes transduction of a signal that induces transcription of HS genes.


Subject(s)
Fatty Acids, Nonesterified/pharmacology , Heat-Shock Proteins/biosynthesis , Histoplasma/metabolism , Mitochondria/metabolism , Saccharomyces cerevisiae/metabolism , Stearoyl-CoA Desaturase/biosynthesis , Transcription, Genetic , Blotting, Northern , Genes, Fungal , Genetic Complementation Test , Histoplasma/drug effects , Hot Temperature , Intracellular Membranes/metabolism , Mitochondria/drug effects , Molecular Sequence Data , Oleic Acid , Oleic Acids/pharmacology , Oxidative Phosphorylation , Oxygen Consumption , Palmitic Acid , Palmitic Acids/pharmacology , Promoter Regions, Genetic , RNA, Messenger/biosynthesis , Saccharomyces cerevisiae/genetics , Stearic Acids/pharmacology , Transcription, Genetic/drug effects
11.
Lipids ; 30(10): 899-906, 1995 Oct.
Article in English | MEDLINE | ID: mdl-8538376

ABSTRACT

We have isolated and characterized the delta 9-desaturase gene (Ole1), which codes for a key enzyme involved in regulating membrane fluidity in animal cells and microorganisms, from two strains of Histoplasma capsulatum, one that is temperature-tolerant (G217B) and the other temperature-susceptible (Downs). These pathogenic fungi are dimorphic in that they undergo a morphologic transition from the mycelial to yeast-like form when the temperature of incubation is switched from 25 to 37 degrees C or when they infect a susceptible host. The coding sequences of the two genes, both containing an intron of 93 nucleotides, are virtually identical and analogous to the delta 9-desaturase gene of Saccharomyces cerevisiae and those of the rat, mouse and human. Ole1 transcription of the thermotolerant G217B and thermosensitive Downs strains is similar in yeast phase cells and during the temperature shift down from 34, 37, or 40 to 25 degrees C (yeast-to-mycelia transition). Nevertheless, the delta 9-desaturase gene is transcriptionally inactive in mycelia of G217B at 25 degrees C while it is actively transcribed in the Downs strain at the same temperature. These results are in agreement with the finding that membranes of the Downs strain have a higher level of oleic acid. The differential expression of delta 9-desaturase genes is discussed in relationship to differences in thermosensitivity in the fungal isolates and in regulating the level of expression of heat shock genes.


Subject(s)
Fatty Acid Desaturases/genetics , Genes, Fungal , Histoplasma/enzymology , Histoplasma/genetics , Hot Temperature , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Cloning, Molecular , Fatty Acid Desaturases/chemistry , Gene Expression , Heat-Shock Proteins/genetics , Humans , Mice , Molecular Sequence Data , Oleic Acid , Oleic Acids/metabolism , Rats , Saccharomyces cerevisiae/genetics , Stearoyl-CoA Desaturase , Transcription, Genetic
12.
J Med Vet Mycol ; 33(2): 123-5, 1995.
Article in English | MEDLINE | ID: mdl-7658303

ABSTRACT

One hundred and three sera drawn from 20 proven and 65 suspected cases of blastomycosis were examined concurrently with the enzymes immunoassay and microimmunodiffusion tests for the 'A' antibody specific for Blastomyces dermatitidis. Results indicated that all 20 proven sera were positive by both these tests. Thirteen of the 65 sera from suspected blastomycosis cases were positive by the enzyme immunoassay only, whereas none reacted positively in the micro-immunodiffusion test. Eighteen sera from apparently normal subjects, and patients with heterologous fungal and HIV infections were also tested by both tests. The sensitivity and specificity of the enzyme immunoassay test was 100% and 85.6%, respectively. The micro- immunodiffusion test was 100% sensitive and specific. In light of the fact that the enzyme immunoassay test is not entirely specific, a positive result should be confirmed by either a positive culture, histopathology or micro-immunodiffusion test.


Subject(s)
Antibodies, Fungal/blood , Blastomyces/immunology , Blastomycosis/diagnosis , Immunoenzyme Techniques , Blastomycosis/blood , Blastomycosis/immunology , Blastomycosis/microbiology , Humans , Immunodiffusion , Sensitivity and Specificity
13.
Arch Med Res ; 26(3): 293-6, 1995.
Article in English | MEDLINE | ID: mdl-8580683

ABSTRACT

The laboratory diagnoses or confirmation of fungal diseases are extremely important considerations in the proper and efficient management of patients experiencing these infections. When an infectious process is suspected, suitable material must be collected and submitted to the pathologist for microscopic examination and to the diagnostic laboratory for culture. The practice of modern medicine dictates that the turn-around time be reduced to a minimum so that specific therapy can be rapidly instituted. Unfortunately, the classical procedures used in processing specimens for isolation and identification of fungi implicated in these diseases are technically time consuming and on occasion labor-intensive. More importantly, fungi are slow growing and cultures are frequently held for 3 to 4 weeks before they are discarded as negative. With the exception of coccidioidomycosis, most attempts to develop serological tests for rapid diagnosis of fungal infections have been hampered by poor specificity caused by immunologic cross reactivity. To some extent, such problems can be circumvented by application of molecular biological techniques to these problems. For example, procedures have been developed that allow efficient screening of DNA expression libraries to identify useful recombinant antigens and production of selected antigens in quantity. This is potentially a practical approach using molecular biological techniques for development of sensitive, specific and practical reagents useful in the serological diagnosis of fungal infections. In addition, nucleic acid hybridization techniques based on the ability of complementary nucleic acid strands to specifically align and associate to form stable double-stranded complexes have been put to use to develop confirmation tests for a variety of important fungal isolates.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Mycoses/diagnosis , Antigens, Fungal , DNA, Fungal , Fungi/genetics , Fungi/isolation & purification , Gene Amplification , Humans , Nucleic Acid Hybridization
14.
Experientia ; 50(11-12): 1067-74, 1994 Nov 30.
Article in English | MEDLINE | ID: mdl-7988666

ABSTRACT

The heat shock (HS) response is a general homeostatic mechanism that protects cells and the entire organism from the deleterious effects of environmental stresses. It has been demonstrated that heat shock proteins (HSP) play major roles in many cellular processes, and have a unique role in several areas of cell biology, from chronic degenerative diseases to immunology, from cancer research to interaction between host and parasites. This review deals with the hsp70 gene family and with its protein product, hsp70, as an antigen when pathogens infect humans. Members of HSP have been shown to be major antigens of many pathogenic organisms when they experience a major temperature shift upwards at the onset of infection and become targets for host B and T cells.


Subject(s)
Antigens/immunology , HSP70 Heat-Shock Proteins/physiology , Parasitic Diseases/immunology , Animals , HSP70 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/immunology , Humans , RNA, Messenger/metabolism
15.
Mycoses ; 37(9-10): 313-6, 1994.
Article in English | MEDLINE | ID: mdl-7746288

ABSTRACT

A total of 178 sera, including 68 from proven cases of histoplasmosis (65 positive for the presence of Histoplasma capsulatum var. capsulatum antibodies and three positive for antigen), 93 from patients with suspected histoplasmosis but with no laboratory evidence of H. capsulatum var. capsulatum infection, 14 from humans with heterologous fungal and non-fungal infections and three from normal individuals, were tested for IgG H. capsulatum antibodies and M or M and H precipitins by enzyme immunoassay (EIA) (Meridian Diagnostics, Cincinnati, OH, USA) and microimmunodiffusion (MID) respectively. Sixty-three of the 68 histoplasmosis case sera demonstrated IgG antibody, and 65 of 68 demonstrated the presence of specific precipitins in the MID test. Nine positive case sera, when tested with the Laboratory Branch complement fixation (LBCF) test, reacted positively to whole yeast and histoplasmin antigens (titres 1:8 to 1:512). Three histoplasmosis case sera repeatedly tested negative for IgG, specific precipitins and complement-fixing antibodies, whereas they were positive for Histoplasma antigen. Eighteen of 95 sera from patients without evidence of histoplasmosis demonstrated IgG antibody in the EIA only. Among these positive sera, three out of three cases of aspergillosis and three out of five cases of blastomycosis were confirmed. Sera from HIV-infected and healthy individuals did not show IgG or M and/or H antibodies to H. capsulatum. Ninety-three sera were negative by both EIA and MID. The EIA for IgG was less sensitive (97%) than MID (100%). The specificity of EIA and MID was 84% and 100% respectively.


Subject(s)
Antibodies, Fungal/blood , Histoplasma/immunology , Histoplasmosis/immunology , Immunoenzyme Techniques , Complement Fixation Tests , Histoplasmosis/microbiology , Humans , Immunodiffusion , Sensitivity and Specificity
16.
Trends Microbiol ; 2(4): 110-4, 1994 Apr.
Article in English | MEDLINE | ID: mdl-8012752

ABSTRACT

Considerable information has accumulated recently about specific genes of Histoplasma capsulatum that are expressed during the process of adaptation when the organism undergoes morphological transition at the onset of infection. The study of these genes is crucial to identify targets for the development of novel antifungal agents.


Subject(s)
Histoplasma/physiology , Histoplasmosis/microbiology , Histoplasma/cytology , Histoplasma/genetics , Humans
17.
Antimicrob Agents Chemother ; 38(2): 294-9, 1994 Feb.
Article in English | MEDLINE | ID: mdl-8192455

ABSTRACT

Amphotericin B (AmB) with deoxycholate (Fungizone) and AmB incorporated into mixed micelles (AmB-mixMs) composed of egg lecithin with glycocholate, deoxycholate, or taurocholate were compared as treatments for murine infections. For mice infected with Candida albicans, treatment consisted of a single intravenous injection; for mice infected with Cryptococcus neoformans, treatment consisted of two intravenous injections. The maximal tolerated doses of AmB as Fungizone were 1.25 mg/kg of body weight in mice with candidiasis and 2.5 mg/kg of body weight in mice with cryptococcosis. The AmB-mixMs were nontoxic to mice at doses of 80 and 100 mg/kg of body weight and were therapeutically more active than the maximal tolerated dose of Fungizone in both models of infection. However, when Fungizone or AmB-mixMs were administered at equivalent doses of AmB, AmB-mixMs were more active in treating murine candidiasis, whereas Fungizone was more active in treating murine cryptococcosis.


Subject(s)
Amphotericin B/administration & dosage , Bile Acids and Salts , Candidiasis/drug therapy , Cryptococcosis/drug therapy , Micelles , Phosphatidylcholines , Amphotericin B/pharmacology , Amphotericin B/toxicity , Animals , Dose-Response Relationship, Drug , Drug Carriers , Drug Evaluation, Preclinical , Female , Mice , Mice, Inbred Strains
18.
Antimicrob Agents Chemother ; 38(2): 300-6, 1994 Feb.
Article in English | MEDLINE | ID: mdl-8192456

ABSTRACT

The cellular activities of amphotericin B (AmB) used as Fungizone were compared with those of AmB complexed to either egg lecithin and glycocholate (Egam) or egg lecithin and deoxycholate (Edam). Under conditions in which leakage of K+ from erythrocytes and cultured L cells treated with Fungizone was almost complete, Egam and Edam containing concentrations of AmB severalfold greater than the concentration of AmB in Fungizone had no effect but retained the ability to decrease the level of retention of K+ in fungal cells. Analysis by absorption and circular dichroism spectroscopy demonstrated that when these formulations containing AmB at concentrations of less than 10(-5) M were added to buffer, the AmB dissociated slowly as monomers from Egam or Edam and dissociated rapidly as a mixture of monomers and self-associated species from Fungizone. We propose that in Egam and Edam, the absence of free AmB in the self-associated form reduces the toxicity of AmB to mammalian cells, whereas the presence of monomeric AmB results in the retention of the antifungal activities of these complexes.


Subject(s)
Amphotericin B/administration & dosage , Amphotericin B/pharmacokinetics , Bile Acids and Salts , Micelles , Mycoses/drug therapy , Phosphatidylcholines , Amphotericin B/toxicity , Animals , Cells, Cultured , Circular Dichroism , Drug Carriers , Drug Stability , Erythrocytes/drug effects , Erythrocytes/metabolism , Fibroblasts/drug effects , Fibroblasts/metabolism , Humans , Lipoproteins/pharmacology , Mice , Potassium/metabolism , Potassium/pharmacokinetics , Spectrophotometry , Therapeutic Equivalency
19.
Mycoses ; 36(1-2): 31-4, 1993.
Article in English | MEDLINE | ID: mdl-8316259

ABSTRACT

A total of 143 cerebrospinal and serum samples, from proven and suspected cases of cryptococcosis, were concurrently examined using a recently introduced enzyme immunoassay (EIA Premier, Meridian Diagnostics, Inc., Cincinnati, OH, USA) and three latex agglutination (LA) procedures (Immunomycologics, Inc., Norman, OK, USA; IBL, Inc., Cranbury, NJ, USA and a non-commercial LA test). Of these 143 specimens, 115 were negative for cryptococcal antigen (CrAg) with the EIA and LA tests. The remaining 28 specimens were evaluated by the LA tests, and all were positive for CrAg (with titres ranging from 1:2 to 1:8192). Of these 28 LA-positive specimens, 26 were also tested by the EIA. This procedure detected CrAg in 23 specimens (88.5%), with antigen levels ranging from 1:4 to 1:266,857. There were 3 LA-positive specimens (tires 1:4 to 1:32) which were negative by the EIA procedure (10.7%). One LA-negative specimen demonstrated CrAg (titre 1:30) by the EIA procedure. The sensitivity of the EIA and LA tests was 85.2 and 100%, respectively. The specificity of the LA test was 100%, whereas that of the EIA was 97%. The agreement among laboratories for testing the specimens with the three LA tests was 100%.


Subject(s)
Antigens, Fungal/analysis , Cryptococcus neoformans/immunology , Immunoenzyme Techniques , Cryptococcosis/diagnosis , Evaluation Studies as Topic , Humans , Latex Fixation Tests , Sensitivity and Specificity
20.
Antimicrob Agents Chemother ; 36(9): 1805-9, 1992 Sep.
Article in English | MEDLINE | ID: mdl-1416871

ABSTRACT

An international collaborative study of broth dilution (MIC) and disk diffusion susceptibility testing of fluconazole was conducted by using a chemically defined medium (High-Resolution Antifungal Assay Medium; Oxoid Ltd., Basingstoke, United Kingdom) and standard test methods performed in eight reference laboratories. Ten yeast isolates were tested by each test method in duplicate on each of 3 separate days. The intralaboratory reproducibility of the MIC test was excellent; 95.7% of the replicate tests (n = 220) were within 2 doubling dilutions of the other values in the set for the eight laboratories. The intralaboratory reproducibility of the disk test was also good, with 91% of the replicate tests (n = 234) agreeing with each other within an arbitrarily chosen value of 4 mm. Interlaboratory agreement of MIC test results was acceptable, with 84% of the MICs agreeing within 2 doubling dilutions. In contrast, the interlaboratory agreement of the disk test was not good, with only 59% of test results agreeing within 4 mm. Comparison of the rank order of MICs obtained in each laboratory with the reference rank order gave an agreement of 70 to 80% (median, 80%) with the MIC test and 70 to 90% (median, 80%) with the disk test. These preliminary results are encouraging for the development of standardized testing methods for testing fluconazole.


Subject(s)
Fluconazole/pharmacology , Fungi/drug effects , Culture Media , International Cooperation , Microbial Sensitivity Tests
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