Comparative action of 8 azole derivatives against Candida albicans: fungistatic action and cytologic study by scanning electron microscopy

The authors compared the in vitro antifungal activity of eight imidazole derivatives (clotrimazole, econazole, isoconazole, ketoconazole, miconazole, oxiconazole, terconazole, tioconazole) against 42 strains of Candida albicans by the agar dilution method using casitone medium. The geometric (G) mean MIC values, the MIC 90 and the MIC 50 values and the corresponding standard deviations of each antifungal agent were determined. The G-MIC values were found to be in the range of 0.008-0.390 micrograms ml-1. The effects of these eight antifungal agents on the ultrastructure of C. albicans yeast cells and spheroplasts were studied by scanning electron microscopy (SEM). The results showed a good correlation between the lesions observed and the structure of the imidazole derivatives tested. On the basis of the SEM results, the compounds could be divided into three groups: (1) ketoconazole and terconazole; (2) econazole, isoconazole, miconazole, oxiconazole and tioconazole; (3) clotrimazole.     

Torulopsis glabrata. Morphologic characteristics, biotyping and sensitivity to isolates of antifungal agents in vitro]

The authors described morphological and biochemical properties of twenty strains of Torulopsis glabrata and two strains of T. candida and T. sphaerica, mostly of human origin. By means of eight biotyping tests based on the evaluation of so-called resistograms the isolates of T. glabrata were divided into eight biotypes. The authors discussed the problem of possible use of biotyping of T. glabrata in investigations of the epidemiology and pathogenesis of mycoses caused by this microorganism. In investigations of the sensitivity of strains of Torulopsis spp. to nine antimycotic agents in vitro it was revealed that the isolates were sensitive to all polyene antibiotics (amphotericin B, nystatin and pimaricin) and to 5-fluorocytosine. The sensitivity to azole chemotherapeutic agents (clotrimazole, econazole, miconazole, ketoconazole and itraconazole) was more varied: some strains were resistant to clotrimazole and econazole.     

Modification of polymorphonuclear leucocyte function by imidazoles

The effect of five imidazole derivatives (metronidazole, tinidazole, clotrimazole, miconazole and ketoconazole) on human polymorphonuclear leucocytes (PMNL) was examined in vitro. Metronidazole and tinidazole had no apparent effect on either PMNL chemotactic response or PMNL fungal/bacterial killing. In contrast, clotrimazole, miconazole and ketoconazole inhibited PMNL chemotaxis. In addition, miconazole and ketoconazole were shown to depress the ability of PMNL to kill bacteria and fungi.     

Synergic effect of 5-fluorocytosine and imidazole derivatives against yeast strains resistant to 5-fluorocytosine

Antifungal agents associations are widely used in therapy of deep mycotic diseases, particularly amphotericin B-5-fluorocytosine association. Synergistic effect has also been described between 5-fluorocytosine and imidazole derivates. The authors have tested here eventual synergy between 5-fluorocytosine and imidazole derivatives (miconazole, ketoconazole, fluconazole, itraconazole) against 57 years isolates resistant to 5-fluorocytosine by a semi-automated methods in liquid medium (Yeast Nitrogen Base and Brain Heart Infusion). The synergistic effect between 5-fluorocytosine and antifungal imidazoles varies widely with the drug tested. It's more frequent with ketoconazole. Itraconazole and fluconazole present very little synergistic effects in vitro.     

Overview of medically important antifungal azole derivatives.

Fungal infections are a major burden to the health and welfare of modern humans. They range from simply cosmetic, non-life-threatening skin infections to severe, systemic infections that may lead to significant debilitation or death. The selection of chemotherapeutic agents useful for the treatment of fungal infections is small. In this overview, a major chemical group with antifungal activity, the azole derivatives, is examined. Included are historical and state of the art information on the in vitro activity, experimental in vivo activity, mode of action, pharmacokinetics, clinical studies, and uses and adverse reactions of imidazoles currently marketed (clotrimazole, miconazole, econazole, ketoconazole, bifonazole, butoconazole, croconazole, fenticonazole, isoconazole, oxiconazole, sulconazole, and tioconazole) and under development (aliconazole and omoconazole), as well as triazoles currently marketed (terconazole) and under development (fluconazole, itraconazole, vibunazole, alteconazole, and ICI 195,739).     

Tablet sensitivity testing on pathogenic fungi.

A diffusion method for determining the sensitivity of pathogenic fungi to therapeutic agents is described using tablets containing the following antibiotics: amphotericin B, clotrimazole, econazole, fluorocytosine, and miconazole. The composition of the media used, standardisation of inocula, incubation time, and temperature are detailed.     

Action of isoconazole on Candida albicans. Study by scanning and transmission electron microscopy

Candida albicans yeast cells were treated prior to their growth phase with isoconazole at the following concentrations: 0.1 microgram/ml, 1 microgram/ml, 10 micrograms/ml), isoconazole induces a blockade of cell diprecipitates bearing witness to the presence of polysaccharides were noted in the cytoplasma and against the membrane wall, but cell division was apparently unaffected. At low concentrations (1 microgram/ml, 10 microgram/ml), isoconazole induced a blockade of cell division by its fungicidal action on synthesis and organisation of the membrane wall. At higher concentrations (50 microgram/ml; 100 micrograms/ml), isoconazole induces total necrosis and death. These findings confirm the analogy between the fungicidal actions of isoconazole and miconazole; however, the site of action of imidazole derivatives (wall or plasma membrane) is discussed.     

Human galectin-3 promotes Trypanosoma cruzi adhesion to human coronary artery smooth muscle cells

Human galectin-3 binds to the surface of Trypanosoma cruzi trypomastigotes and human coronary artery smooth muscle (CASM) cells. CASM cells express galectin-3 on their surface and secrete it. Exogenous galectin-3 increased the binding of T. cruzi to CASM cells. Trypanosome binding to CASM cells was enhanced when either T. cruzi or CASM cells were preincubated with galectin-3. Cells stably transfected with galectin-3 antisense show a dramatic decrease in galectin-3 expression and very little T. cruzi adhesion to cells. The addition of galectin-3 to these cells restores their initial capacity to bind to trypanosomes. Thus, host galectin-3 expression is required for T. cruzi adhesion to human cells and exogenous galectin-3 enhances this process, leading to parasite entry.     

beta-Chemokines enhance parasite uptake and promote nitric oxide-dependent microbiostatic activity in murine inflammatory macrophages infected with Trypanosoma cruzi.

In the present study, we describe the ability of Trypanosoma cruzi trypomastigotes to stimulate the synthesis of beta-chemokines by macrophages. In vivo infection with T. cruzi led to MIP-1alpha, RANTES, and JE/MCP1 mRNA expression by cells from peritoneal inflammatory exudate. In addition, in vitro infection with T. cruzi resulted in expression of beta-chemokine MIP-1alpha, MIP-1beta, RANTES, and JE mRNA by macrophages. The expression of the beta-chemokine MIP-1alpha, MIP-1beta, RANTES, and JE proteins by murine macrophages cultured with trypomastigote forms of T. cruzi was confirmed by immunocytochemistry. Interestingly, macrophage infection with T. cruzi also resulted in NO production, which we found to be mediated mainly by beta-chemokines. Hence, treatment with anti-beta-chemokine-specific neutralizing antibodies partially inhibited NO release by macrophages incubated with T. cruzi parasites. Further, the addition of the exogenous beta-chemokines MIP-1alpha, MIP-1beta, RANTES, and JE/MCP-1 induced an increased T. cruzi uptake, leading to enhanced NO production and control of parasite replication in a dose-dependent manner. L-NMMA, a specific inhibitor of the L-arginine-NO pathway, caused a decrease in NO production and parasite killing when added to cultures of macrophages stimulated with beta-chemokines. Among the beta-chemokines tested, JE was more potent in inhibiting parasite growth, although it was much less efficient than gamma interferon (IFN-gamma). Nevertheless, JE potentiates parasite killing by macrophages incubated with low doses of IFN-gamma. Together, these results suggest that in addition to their chemotactic activity, murine beta-chemokines may also contribute to enhancing parasite uptake and promoting control of parasite replication in macrophages and may play a role in resistance to T. cruzi infection.     

Glycosylphosphatidylinositols are required for the development of Trypanosoma cruzi amastigotes.

Induction of a glycosylphosphatidylinositol (GPI) deficiency in Trypanosoma cruzi by the heterologous expression of Trypanosoma brucei GPI-phospholipase C (GPI-PLC) results in decreased expression of major surface proteins (N. Garg, R. L. Tarleton, and K. Mensa-Wilmot, J. Biol. Chem. 272:12482-12491, 1997). To further explore the consequences of a GPI deficiency on replication and differentiation of T. cruzi, the in vitro and in vivo behaviors of GPI-PLC-expressing T. cruzi were studied. In comparison to wild-type controls, GPI-deficient T. cruzi epimastigotes exhibited a slight decrease in overall growth potential in culture. In the stationary phase of in vitro growth, GPI-deficient epimastigotes readily converted to metacyclic trypomastigotes and efficiently infected mammalian cells. However, upon conversion to amastigote forms within these host cells, the GPI-deficient parasites exhibited a limited capacity to replicate and subsequently failed to differentiate into trypomastigotes. Mice infected with GPI-deficient parasites showed a substantially lower rate of mortality, decreased tissue parasite burden, and a moderate tissue inflammatory response in comparison to those of mice infected with wild-type parasites. The decreased virulence exhibited by GPI-deficient parasites suggests that inhibition of GPI biosynthesis is a feasible strategy for chemotherapy of infections by T. cruzi and possibly other intracellular protozoan parasites.     

Trypanosoma cruzi attachment to lymphocyte muscarinic cholinergic and beta adrenergic receptors modulates intracellular signal transduction.

Plasma membrane vesicles of Trypanosoma cruzi (PMVs) formed saturation binding isotherms with naive murine T lymphocytes. Parasite membrane attachment to the muscarinic cholinergic receptors of Lyt 2.2+T cells (suppressor cells) resulted in the synthesis of cGMP, attenuation of cAMP levels and in the secretion of prostaglandin E2, an immunoregulator effector substance. These T suppressor cell signals were blunted by atropine and by monospecific antibody against T. cruzi surface epitopes. The interaction of T. cruzi PMVs with the beta adrenergic receptors of Lyt L3T4+T cells (helper cells) resulted in the synthesis of cAMP and in the attenuation of cGMP levels. T helper cells did not secrete prostaglandin E2 when T. cruzi PMVs were added to this system. These T helper cell signals were blunted by propranolol and by monospecific antibody against T. cruzi surface epitopes. The interaction of T. cruzi with T lymphocytes may result, therefore, in the down-regulation of the immune response induced by prostaglandin E2 T suppressor cell secretion and by cAMP inhibition of proliferation of T helper cells.     

Cloning and characterization of a gene encoding phosphatidyl inositol-specific phospholipase C from Trypanosoma cruzi.

A gene encoding phosphatidyl inositol-4,5-bisphosphate phospholipase C (PLC) was cloned from the protozoan parasite Trypanosoma cruzi. A partial cDNA encoding putative PLC was obtained by a polymerase chain reaction (PCR) using degenerate oligonucleotide primers corresponding to conserved regions of PLCs. A 2178-bp protein coding region of the T. cruzi PLC gene, composed from cDNA and genomic clones, encodes a putative PLC with a calculated molecular mass of 82,032 Da and an isoelectric point of 5.93. The deduced amino acid sequence of T. cruzi PLC exhibited 23-42% overall identities with the PLCs from other organisms. Among them, PLC from Ictalurus punctatus revealed the highest identity to T. cruzi PLC. The percentage identities of the entire proteins and the catalytic X/Y domains suggested that T. cruzi PLC is more evolutionarily related to the PLCs of higher eukaryotes than to those of lower unicellular eukaryotes. The tetrad analysis of the segregants of the Saccharomyces cerevisiae PLC1/plc1::HIS3 diploid strain transformed with the T. cruzi PLC-expressing plasmid showed that expression of T. cruzi PLC suppressed the growth defect caused by the plc1 disruption in yeasts. Temperature-sensitive phenotype of the S. cerevisiae plc1-mutant haploid strain was also suppressed by the expression of T. cruzi PLC. The phosphatidyl inositol-4,5-biphosphate (PtdIns(4,5)P2) hydrolyzing activity of T. cruzi PLC was demonstrated in the lysate from the plc1-temperature sensitive yeast mutant strain transformed with the T. cruzi PLC-expressing plasmid. The yeast-expressed T. cruzi PLC showed an absolute Ca2+ dependence which was similar to mammalian PLC isoforms: the half-maximal activity at 0.5-1 x 10(-5) M Ca2+ and the maximal activity at 1-2 x 10(-4) M Ca2+.     

Characterization of bifunctional sphingolipid Δ4-desaturases/C4-hydroxylases of trypanosomatids by liquid chromatography-electrospray tandem mass spectrometry

Six genes encoding putative sphingolipid desaturases have been identified in trypanosomatid genomes: one in Trypanosoma brucei (TbSLdes protein), one in Trypanosoma cruzi (TcSLdes) and four in Leishmania major (LmSLdes1-4), tandemly arrayed on chromosome 26. The six amino acid sequences showed the three characteristic histidine boxes, with a long spacer between the first and second box, as in fungal desaturases and bifunctional desaturases/hydroxylases, to which they are phylogenetically related. We functionally characterized the trypanosomatid enzymes by their expression in Saccharomyces cerevisiae sur2Δ mutant, which lacks C4-hydroxylase activity. The sphingoid base profile (dinitrophenyl derivatives) of each yeast mutant transformed with each one of the different parasite genes was analyzed by HPLC, using a sur2Δ mutant expressing the Schyzosaccharomyces pombe sphingolipid desaturase (SpSLdes) as positive control. TbSLdes was capable of desaturating endogenous sphingolipids at levels comparable to those found in SpSLdes. By contrast, L. major and T. cruzi enzymes showed either no or negligible activities. Using the HPLC system coupled to electrospray tandem quadrupole/time of flight mass spectrometry we were able to detect significant levels of desaturated and hydroxylated sphingoid bases in extracts of all transformed yeast mutants, except for those transformed with the empty vector. These results indicate that S. pombe, T. brucei, T. cruzi and L. major enzymes are all bifunctional. Using the same methodology, desaturated and hydroxylated sphingoid bases were detected in T. cruzi epimastigotes and L. major promastigote cells, as described previously, and in T. brucei procyclic and bloodstream forms for the first time.     

A monoclonal antibody with specificity for Trypanosoma cruzi, central and peripheral neurones and glia.

Of 26 hybridomas secreting anti-Trypanosoma cruzi antibodies, two reacted with murine brain and spinal cord extracts but not other tissues. Both antibodies (5H7 and 3H3) had identical isotypes (IgM) and specificities as judged by western blotting. Antigens of molecular weight 58,000 and 37,000 in mouse brain and spinal cord, and 58,000 and 35,000 in T. cruzi were detected. Different tissue antigens were recognized by a previously reported T. cruzi mammalian neural tissue cross-reacting monoclonal antibody CE5; on immunofluorescence 5H7 stained some glia, and some central and peripheral neurones in rat tissue sections. Pooled sera from mice chronically infected with T. cruzi competed with binding of 5H7 to T. cruzi antigens.     

Captopril increases the intensity of monocyte infection by Trypanosoma cruzi and induces human T helper type 17 cells

The anti-hypertensive drug captopril is used commonly to reduce blood pressure of patients with severe forms of Chagas disease, a cardiomyopathy caused by chronic infection with the intracellular protozoan Trypanosoma cruzi. Captopril acts by inhibiting angiotensin-converting enzyme (ACE), the vasopressor metallopeptidase that generates angiotensin II and promotes the degradation of bradykinin (BK). Recent studies in mice models of Chagas disease indicated that captopril can potentiate the T helper type 1 (Th1)-directing natural adjuvant property of BK. Equipped with kinin-releasing cysteine proteases, T. cruzi trypomastigotes were shown previously to invade non-professional phagocytic cells, such as human endothelial cells and murine cardiomyocytes, through the signalling of G protein-coupled bradykinin receptors (B(2) KR). Monocytes are also parasitized by T. cruzi and these cells are known to be important for the host immune response during infection. Here we showed that captopril increases the intensity of T. cruzi infection of human monocytes in vitro. The increased parasitism was accompanied by up-regulated expression of ACE in human monocytes. While T. cruzi infection increased the expression of interleukin (IL)-10 by monocytes significantly, compared to uninfected cells, T. cruzi infection in association with captopril down-modulated IL-10 expression by the monocytes. Surprisingly, studies with peripheral blood mononuclear cells revealed that addition of the ACE inhibitor in association with T. cruzi increased expression of IL-17 by CD4(+) T cells in a B(2) KR-dependent manner. Collectively, our results suggest that captopril might interfere with host-parasite equilibrium by enhancing infection of monocytes, decreasing the expression of the modulatory cytokine IL-10, while guiding development of the proinflammatory Th17 subset.     

Immunization against Trypanosoma cruzi: adjuvant effect of glucan

Trypanosoma cruzi, the causative agent of Chagas' disease, infects humans and animals in tropical, subtropical and some temperature regions of the western hemisphere. At present, there is no effective vaccine for T. cruzi infection. Glucan, a beta-1,3 polyglucose biological response modifier, possesses significant adjuvant activity. The present study investigated the adjuvant activity of particulate glucan when combined with a vaccine of glutaraldehyde-killed T. cruzi culture forms. ICR/HSD mice (20 g) were injected s.c. with glutaraldehyde-killed T. cruzi on days 21, 14 and 7 prior to challenge with 50 T. cruzi blood forms. Particulate glucan (1 mg/mouse) was administered s.c. either alone or in conjunction with T. cruzi vaccine. Isovolumetric dextrose served as control. Dextrose, glucan or T. cruzi vaccine as single treatment regimens showed 100% mortality with 20.5, 21.4 and 21.6 day median survival times, respectively. In contrast, glucan administered with T. cruzi vaccine showed an 85% (P less than 0.01) survival at 275 days post-challenge. In addition, the number of T. cruzi observed in the blood of glucan--T. cruzi immunized mice was lower than the appropriate controls. However, immunized mice which survived at 275 days were positive for the presence of T. cruzi by xenodiagnosis. Histopathologic evaluation of glucan--T. cruzi mice revealed no parasites or cardiac pathology, but a mild splenic hyperplasia and inflammation of skeletal muscle were noted. In subsequent studies, mice were immunized with the same regimen of glucan--T. cruzi and challenged with 500 or 5000 T. cruzi. Glucan significantly (P less than 0.05) increased survival as denoted by 60% and 50% survival in the glucan-T. cruzi group vs 0% in controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of B-Lymphocyte and NK Cell Activities by Glycoinositolphospholipid Purified from Trypanosoma cruzi

Glycoinositolphospholipids (GIPLs) are some of the major glycolipids of the Trypanosoma cruzi surface that were previously shown to activate B cells. In the present study, we investigated whether (i) T. cruzi GIPLs could induce immunoglobulin secretion from B cells in the absence of T cells and NK cells and whether (ii) NK cells are also stimulated by the GIPLs. B cells purified from mice deficient in both T and NK cells (CD3epsilon transgenic mice) secreted immunoglobulin in response to the GIPL. This response was increased by coculture with a murine NK cell line. The T. cruzi GIPL also increased the NK cell (interleukin-2 induced) proliferative response. Our data indicate that the T. cruzi GIPL has a direct stimulatory effect on NK cells and induces immunoglobulin secretion in the absence of T lymphocytes and NK cells. These findings suggest that this T. cruzi-derived molecule may be one of the stimulators that lead to NK cell activation during T. cruzi infection.     

Evaluation of the efficacy of miconazole nitrate shampoo for prevention against T. tonsurans infection]

We report the efficacy of miconazole nitrate shampoo (Furfur Shampoo) to prevent T. tonsurans infection. METHOD: Experimental models were made from stratum corneum of healthy human heel or guinea pig skin. Before and/or after T. tonsurans was applied, samples were washed with miconazole nitrate shampoo, then inoculated on a Sabouraud culture plate. RESULT: Though miconazole nitrate shampoo did not eliminate T. tonsurans on the sample completely, the time required to develop the colony (9.9 days) was obviously extended (7.7 days by control). DISCUSSION: We concluded that miconazole nitrate shampoo is a useful method of preventing T. tonsurans infection, and should be used every day (before/after the chance to infect).     

Combined use of enzyme-linked immunosorbent assay and flow cytometry to detect antibodies to Trypanosoma cruzi in domestic canines in Texas

Canines may be sentinels and/or reservoirs for human Trypanosoma cruzi exposures. This study adapted a method originally designed for human diagnostics to detect serum immunoglobulin G to T. cruzi in canines. The method combined an enzyme-linked immunosorbent assay (ELISA) for screening and flow cytometry detection of anti-live trypomastigote antibodies (ALTA) for confirmation. The assays were optimized by using known positive and negative control canine sera, and cutoff values were established. The ELISA and ALTA assay easily distinguished between reactive (positive controls) and nonreactive (negative controls) sera and were used to test sera collected in a cross-sectional seroprevalence survey of 356 domestic canines from Harris County, Tex., and the surrounding area. Fifty-three (14.9%) of 356 asymptomatic canines in the survey were positive by ELISA, and 5 (1.4%) were confirmed positive with the ALTA assay, with an additional 4 (1.1%) canines classified as "suspect positive." Thus, the overall prevalence of T. cruzi antibodies in this population was 2.6%. This is the first U.S. study to use the combination of ELISA and ALTA to detect serum antibodies to T. cruzi and the first report of the prevalence of T. cruzi infection in domestic canines in the Houston, Tex. (Harris County), region. Our results demonstrate that the combination of ELISA and ALTA has been successfully adapted for use in testing canines for serological evidence of T. cruzi infection. Seroprevalence survey results suggest that T. cruzi antibody-positive domestic canines in the peridomestic setting are present in the Houston, Tex., region and further suggest that T. cruzi is enzootic in the region.