Impairment of Interleukin-17A Expression in Canine Visceral Leishmaniosis is Correlated with Reduced Interferon-γ and Inducible Nitric Oxide Synthase Expression.

Dogs are the primary urban reservoir of Leishmania infantum and play a crucial role in the transmission of this parasite to man via sandflies. The spleen and liver are the main target organs of L. infantum infection, but few studies have evaluated the immune response to this infection in the canine liver. To identify the immunological mediators involved in resistance and/or susceptibility to canine visceral leishmaniosis (CVL), we selected 21 dogs naturally infected by L. infantum and classified as asymptomatic or symptomatic. Immunological parameters were analysed and correlations with clinical signs were determined. Symptomatic dogs showed higher numbers of parasites and less leucocyte infiltration in the liver compared with asymptomatic dogs. The progression of this disease was characterized not only by the down regulation of T helper (Th) 1-related cytokines, such as interferon (IFN)-γ and tumour necrosis factor (TNF)-α, but also by the down regulation of genes encoding interleukin (IL)-17A, inducible nitric oxide synthase (iNOS) and IL-10 in the spleen and liver in symptomatic dogs compared with asymptomatic dogs. Importantly, IL-17A gene transcription level was positively correlated with mRNA expression for iNOS and IFN-γ. Th1- and Th17-related cytokines therefore appear to play a role in restricting parasite growth via iNOS activation and decrease susceptibility of dogs to CVL.

Novel target genes of the yeast regulator Pdr1p: a contribution of the TPO1 gene in resistance to quinidine and other drugs.

The yeast transcription factor Pdr1p regulates the expression of a number of genes, several of which encode ATP-driven transport proteins involved in multiple drug resistance. Among 20 genes containing binding consensus sequences for the transcription factor Pdr1p in their promoter, we studied more particularly the regulation and function of PDR16 (involved in phospholipid synthesis), TPO1 (involved in vacuolar transport of polyamines), YAL061W (homologous to polyol dehydrogenases) and YLR346C (unknown function). We found that the regulation of these four genes depends on Pdr1p, since promoter activities studied by lacZ fusion analysis and mRNA levels studied by Northern blotting analysis changed upon deletion or hyperactivation by the pdr1-3 mutant of this transcription factor. The drug sensitivity of the strains deleted for these genes revealed that TPO1, a gene previously found to be involved in spermidine resistance and vacuolar polyamine transport, is a determinant of multidrug transporter since it also mediates growth resistance to cycloheximide and quinidine. This resistance pattern overlapped with that of YOR273C, a homolog of TPO1. These two homologous transporters are thus bona fide members of the phylogenetic subfamily DHA1 (drug/proton antiport TC 2.A.1. 2) of the major facilitator superfamily. Both YOR273C and TPO1 as well as at least one other determinant involved in the yeast pleiotropic drug resistance network contribute to resistance to a quinoline-containing antimalarial drug.

Cell-surface sialoglycoconjugate structures in wild-type and mutant Crithidia fasciculata.

The cell-surface expression of sialoglycoconjugate structures in wild-type Crithidia fasciculata and its TFR(R1) drug-resistant mutant was analyzed with the aid of an influenza C virus strain, lectin, enzymatic treatment, and flow cytofluorimetry analysis probed with fluorescein isothiocyanate-labeled (FITC) lectins. 9-O-Acetyl-N-acetyl neuraminic acid (Neu5,9Ac2) structures mediate influenza C virus cell-binding. The SAalpha2,3Gal and SAalpha2,6Gal sequences are specifically recognized by Maackia amurensis (MAA) and Sambucus nigra (SNA) lectins, respectively. On the basis of these parameters the TFR(R1) mutant strain of C. fasciculata was found to contain exposed sialoglycoconjugates bearing Neu5,9Ac2 surface structures. After the removal of sialic acid residues by neuraminidase activity the marked increases in PNA (peanut agglutinin)-mediated agglutinating activity showed that those acidic units on C. fasciculata cells were glycosidically linked to D-galactose. The bond involves SAalpha2,6Gal and SAalpha2,3Gal linkages as suggested by the use of FITC-SNA and FITC-MAA lectins, respectively. Both SAalpha2,3Gal and SAalpha2,6Gal sequences were preferentially expressed by the TFR(R1) mutant. The SAalpha2,6 linkage markedly predominated. In the TFR(R1) mutant, but not in wild-type cells, two distinct populations of cells were distinguished by reactivity with FITC-SNA, one of which was enriched with surface SAalpha2,6Gal sequences. These diverse findings suggest that sialoglycoconjugate structures present on the flagellate surface may be associated with mutation and the cell growth cycle in C. fasciculata.

PDR16 and PDR17, two homologous genes of Saccharomyces cerevisiae, affect lipid biosynthesis and resistance to multiple drugs.

The Saccharomyces cerevisiae open reading frame YNL231C was recently found to be controlled by the multiple drug resistance regulator Pdr1p. Here we characterize YNL231C (PDR16) and its homologue YNL264C (PDR17). Deletion of PDR16 resulted in hypersensitivity of yeast to azole inhibitors of ergosterol biosynthesis. While no increase in drug sensitivity was found upon deletion of PDR17 alone, a Deltapdr16,Deltapdr17 double mutant was hypersensitive to a broad range of drugs. Both mutations caused significant changes of the lipid composition of plasma membrane and total cell extracts. Deletion of PDR16 had pronounced effects on the sterol composition, whereas PDR17 deletion mainly affected the phospholipid composition. Thus, Pdr16p and Pdr17p may regulate yeast lipid synthesis like their distant homologue, Sec14p. The azole sensitivity of the PDR16-deleted strain may be the result of imbalanced ergosterol synthesis. Impaired plasma membrane barrier function resulting from a change in the lipid composition appears to cause the increased drug sensitivity of the double mutant strain Deltapdr16,Deltapdr17. The uptake rate of rhodamine-6-G into de-energized cells was shown to be almost 2-fold increased in a Deltapdr16,Deltapdr17 strain as compared with wild-type and Deltapdr5 strains. Collectively, our results indicate that PDR16 and PDR17 control levels of various lipids in various compartments of the cell and thereby provide a mechanism for multidrug resistance unrecognized so far.

Occurrence of N-acetyl- and N-O-diacetyl-neuraminic acid derivatives in wild and mutant Crithidia fasciculata.

The cell-surface expression of sialic acids in wild-type Crithidia fasciculata and three drug-resistant mutants (FU(R)11, TR3, and TFRR1) was analyzed using fluorescein-labeled Limulus polyphemus agglutinin (LPA) binding, glycosidase of known sugar specificity, and thin-layer chromatography (TLC). Gas-liquid chromatography-mass spectrometry (GC-MS) analysis using both electron-impact (EI-MS) and chemical ionization (CI-MS) by isobutane with selected ion monitoring (SIM) was also used. The surface location of sialic acid was inferred from LPA binding to whole cells abrogated by previous treatment with neuraminidase. An exception occurred with the TFRR1 strain, which after incubation with neuraminidase showed increased reactivity with the fluorescent lectin. Both N-acetyl- and N-O-diacetyl-neuraminic acids were identified in the flagellates by TLC, with a clear predominance being noted for the former derivative. However, the content of N-O-diacetyl-neuraminic acid was preferentially found in the TFRR1 strain. The GC-MS analysis of the acidic component of the TFRR1 mutant strain confirmed the occurrence of N-acetyl-neuraminic acid (Neu5Ac) by the presence of the diagnostic ions (m/z values: 684 and 594 for CI-MS and 478, 298, and 317 for EI-MS) and also by comparison with the standard Neu5Ac retention time. GC-MS analysis also showed fragments (m/z values: 654 and 564 for CI-MS and 594, 478, 298, and 317 for EI-MS) expected for the 7-O- and 9-O-acetyl-N-acetyl-neuraminic acids (Neu5,7Ac2 and Neu 5,9Ac2, respectively).

Surface charge and hydrophobicity of wild and mutant Crithidia fasciculata.

Surface charge of wild-type Crithidia fasciculata and three drug-resistant mutants (TR3, TFRR1, and FUR11) was studied by direct zeta-potential determination and ultrastructural cytochemistry. Surface tension was also investigated by measurements of the advancing contact angle formed by the protozoa monolayers with drops of liquids of different polarities. The individual zeta potential varies markedly among the C. fasciculata cells. The wild and FUR11 mutant strains displayed lower negative surface charge (-12.5 and -9.5 mV, respectively) as compared with the TR3 (-14.8 mV) and TFRR1 (-14.7 mV) mutant strains. Binding of cationized ferritin (CF) was observed at the cell surface of wild and mutant strains of C. fasciculata. Neuraminidase treatment reduced the negative surface charge in the TFRR1 and TR3 mutants in about 37 and 29%, respectively, whereas no significant change was observed with the wild and FUR11 mutant strains. These findings suggest that sialic acid residues are the major anionogenic groups on the surface of C. fasciculata. The density of sialic acid residues per cell in wild and mutant strains of C. fasciculata falls in a range of 1.4 x 10(4) to 3.6 x 10(4). Marked differences of hydrophobicity were also observed. For example, the TFRR1, FUR11, and TR3 drug-resistant mutant strains showed higher contact angle values (55.4, 54.2, and 49.3, respectively) than the wild-type (35.6), as assessed by alpha-bromonaphtalene.

Epidural injection of xylazine for perineal analgesia in horses.

Local anesthetics given in the epidural space of a horse may cause hind limb weakness in addition to analgesia. Because alpha 2 agonists given by epidural injection cause sensory blockade without motor effects in human beings and other species, their use in veterinary anesthesia is appealing. This study was designed to examine the effectiveness of xylazine HCl, an alpha 2 agonist commonly used in horses. Xylazine, 0.9% NaCl, and lidocaine were given by epidural injection to horses subjected to perineal electrical stimulation. Administration of xylazine (0.17 mg/kg of body weight, diluted to a 10-ml volume, using 0.9% NaCl) induced approximately 2.5 hours of local analgesia without apparent side effects. Higher doses of xylazine caused mild hind limb ataxia. Administration of lidocaine induced a similar duration of analgesia, with severe hind limb ataxia (100% incidence). We concluded that xylazine given by epidural injection results in safe, effective perineal analgesia in horses.