"Trophozoite Cultures, Growth of ABZ-Resistant Clones and Obtention of H 2 O 2 -Resistant Trophozoites Giardia duodenalis trophozoites of the WB strain (ATCC#30957) and ABZ-resistant clones were maintained in TYI-S-33 medium supplemented with 10% adult bovine serum (HyClone) and antibiotic/antimycotic solution (Thermo, USA) at 37 • C (Keister, 1983) in 4.5 mL screw-capped vials. ABZ-resistant trophozoites were selected by continuous subculture under increasing sublethal concentrations of ABZ (Sigma cat. "
[Show abstract][Hide abstract] ABSTRACT: The control of Giardia duodenalis infections is carried out mainly by drugs, among these albendazole (ABZ) is commonly used. Although the cytotoxic effect of ABZ usually involves binding to β-tubulin, it has been suggested that oxidative stress may also play a role in its parasiticidal mechanism. In this work the effect of ABZ in Giardia clones that are susceptible or resistant to different concentrations (1.35, 8, and 250 μM) of this drug was analyzed. Reactive oxygen species (ROS) were induced by ABZ in susceptible clones and this was associated with a decrease in growth that was alleviated by cysteine supplementation. Remarkably, ABZ-resistant clones exhibited partial cross-resistance to H 2 O 2, whereas a Giardia H 2 O 2-resistant strain can grow in the presence of ABZ. Lipid oxidation and protein carbonylation in ABZ-treated parasites did not show significant differences as compared to untreated parasites; however, ABZ induced the formation of 8OHdG adducts and DNA degradation, indicating nucleic acid oxidative damage. This was supported by observations of histone H2AX phosphorylation in ABZ-susceptible trophozoites treated with 250 μM ABZ. Flow cytometry analysis showed that ABZ partially arrested cell cycle in drug-susceptible clones at G2/M phase at the expense of cells in G1 phase. Also, ABZ treatment resulted in phosphatidylserine exposure on the parasite surface, an event related to apoptosis. All together these data suggest that ROS induced by ABZ affect Giardia genetic material through oxidative stress mechanisms and subsequent induction of apoptotic-like events.
Frontiers in Microbiology 08/2015; 6(800). DOI:10.3389/fmicb.2015.00800 · 3.99 Impact Factor
"Giardia NF trophozoites were originally obtained from a water sample during an outbreak of giardiasis in Newfoundland, Canada , while Giardia GS/M clone H7 trophozoites were obtained from ATCC (50581) as previously described . Trophozoites were grown axenically in 15 mL polystyrene tubes (Becton-Dickinson Falcon) in Keister’s modified TYI-S-33 medium ,  supplemented with piperacillin (Sigma-Aldrich) and used at peak density culture. "
[Show abstract][Hide abstract] ABSTRACT: Giardia duodenalis (syn. G. intestinalis, G. lamblia) is a predominant cause of waterborne diarrheal disease that may lead to post-infectious functional gastrointestinal disorders. Although Giardia-infected individuals could carry as much as 106 trophozoites per centimetre of gut, their intestinal mucosa is devoid of overt signs of inflammation. Recent studies have shown that in endemic countries where bacterial infectious diseases are common, Giardia infections can protect against the development of diarrheal disease and fever. Conversely, separate observations have indicated Giardia infections may enhance the severity of diarrheal disease from a co-infecting pathogen. Polymorphonuclear leukocytes or neutrophils (PMNs) are granulocytic, innate immune cells characteristic of acute intestinal inflammatory responses against bacterial pathogens that contribute to the development of diarrheal disease following recruitment into intestinal tissues. Giardia cathepsin B cysteine proteases have been shown to attenuate PMN chemotaxis towards IL-8/CXCL8, suggesting Giardia targets PMN accumulation. However, the ability of Giardia infections to attenuate PMN accumulation in vivo and how in turn this effect may alter the host inflammatory response in the intestine has yet to be demonstrated. Herein, we report that Giardia infection attenuates granulocyte tissue infiltration induced by intra-rectal instillation of Clostridium difficile toxin A and B in an isolate-dependent manner. This attenuation of granulocyte infiltration into colonic tissues paralled decreased expression of several cytokines associated with the recruitment of PMNs. Giardia trophozoite isolates that attenuated granulocyte infiltration in vivo also decreased protein expression of cytokines released from inflamed mucosal biopsy tissues collected from patients with active Crohn's disease, including several cytokines associated with PMN recruitment. These results demonstrate for the first time that certain Giardia infections may attenuate PMN accumulation by decreasing the expression of the mediators responsible for their recruitment.
PLoS ONE 10/2014; 9(10):e109087. DOI:10.1371/journal.pone.0109087 · 3.23 Impact Factor
"Cell cultures G. intestinalis strain HP-1 (i.e., Prague line of Portland-1 isolate (ATCC 3088), assemblage A1), which was an original human isolate provided by E.A. Meyer (Oregon Health Science University, Portland, USA) and clone WBc6 (ATCC 50803, assemblage A1), provided by W.Z. Cande (UC- Berkeley, USA), was used. Axenic cultures were routinely maintained in TYI-S-33 medium (pH 6.8) supplemented with 10 % (v/v) heat-inactivated bovine serum (Gibco, Life Technologies) and 0.1 % (v/v) bovine bile (Keister 1983). Enrichment with mitotic cells was performed according to the protocol described by Sagolla et al. (2006). "
[Show abstract][Hide abstract] ABSTRACT: During mitotic prophase, chromosomes of the pathogenic unicellular eukaryote Giardia intestinalis condense in each of the cell's two nuclei. In this study, Giardia chromosomes were investigated using light microscopy, high-resolution field emission scanning electron microscopy, and in situ hybridization. For the first time, we describe the overall morphology, condensation stages, and mitotic segregation of these chromosomes. Despite the absence of several genes involved in the cohesion and condensation pathways in the Giardia genome, we observed chromatin organization similar to those found in eukaryotes, i.e., 10-nm nucleosomal fibrils, 30-nm fibrils coiled to chromomeres or in parallel arrangements, and closely aligned sister chromatids. DNA molecules of Giardia terminate with telomeric repeats that we visualized on each of the four chromatid endings of metaphase chromosomes. Giardia chromosomes lack primary and secondary constrictions, thus preventing their classification based on the position of the centromere. The anaphase poleward segregation of sister chromatids is atypical in orientation and tends to generate lagging chromatids between daughter nuclei. In the Giardia genome database, we identified two putative members of the kleisin family thought to be responsible for condensin ring establishment. Thus far, Giardia chromosomes (300 nm to 1.5 μm) are the smallest chromosomes that were analyzed at the ultrastructural level. This study complements the existing molecular and sequencing data on Giardia chromosomes with cytological and ultrastructural information.
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