8 research outputs found
Responses of the Differentiated Intestinal Epithelial Cell Line Caco-2 to Infection With the Giardia intestinalis GS Isolate
Giardia intestinalis is a parasitic protist that causes diarrhea in humans, affecting mainly children of the developing world, elderly and immunocompromised individuals. Humans are infected by two major Giardia assemblages (i.e. genetic subtypes), A and B, with the latter being the most common. So far, there is little information on molecular or cellular changes during infections with assemblage B. Here, we used RNA sequencing to study transcriptional changes in Caco-2 intestinal epithelial cells (IECs) co-incubated with assemblage B (GS isolate) trophozoites for 1.5, 3, and 4.5 h. We aimed to identify early molecular events associated with the establishment of infection and followed cellular protein changes up to 10 h. IEC transcriptomes showed a dominance of immediate early response genes which was sustained across all time points. Transcription of inflammatory cytokines (e.g., cxcl1-3, ccl2, 1l1a, and il1b) peaked at 1.5 and 3 h of infection. Compared to co-incubation with assemblage A Giardia, we identified the induction of novel cytokines (cxcl8, cxcl10, csf1, cx3cl1, il12a, il11) and showed that inflammatory signaling is mediated by Erk1/2 phosphorylation (mitogen activated protein kinase, MAPK), nuclear factor kappa B (NFκB) and adaptor protein-1 (AP-1). We also showed that GS trophozoites attenuate P38 (MAPK) phosphorylation in IECs. Low amounts of IL-8, CXCL1 and CCL20 proteins were measured in the interaction medium, which was attributed to cytokine degradation by trophozoite secreted proteases. Based on the transcriptome, the decay of cytokines mRNA mediated by zinc finger protein 36 might be another mechanism controlling cytokine levels at later time points. IEC transcriptomes suggested homeostatic responses to counter oxidative stress, glucose starvation, and disturbances in amino acid and lipid metabolism. A large group of differentially transcribed genes were associated with cell cycle arrest and induction of apoptosis, which was validated at protein level. IEC transcriptomes also suggested changes in tight junction's integrity, microvilli structure and the extracellular mucin layer. This is the first study to illuminate transcriptional and protein regulatory events underlying IECs responses and pathogenesis during Giardia assemblage B infection. It highlights differences compared to assemblage A infections which might account for the differences observed in human infections with the two assemblages
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Early Molecular Mechanisms Involved Post Cochlear Implant Electrode Insertion Trauma
Objectives: Cochlear implant electrode insertion trauma (EIT), not only causes direct tissue trauma and cell losses by necrosis, but also generates programmed cell death (PCD) and can lead to a loss of residual hearing. We aimed to: 1) Analyze the molecular mechanisms involved in PCD of hair cell (HCs) and support cells (SCs). 2) Compare the time of onset of these events in HCs and SCs. Methods: Guinea pigs were categorized in three groups: EIT; pre-treated with AM-111 at round window ½ hr before EIT, and unoperated contralateral ears as controls. Immunostaining for phospho-c-Jun, activated Caspase-3, CellROX, HNE and Reactive nitrogen species (RNS) were performed at 6hrs, 12hrs and 24hrs post-EIT. Results: At 6 hrs Post-EIT immunostaining SCs is positive for phosphorylation of c-Jun and activation of caspase-3. p-c-Jun labeling was observed at 12 hrs in both HCs and SCs of middle and basal turn. Caspase-3 activation was not observed in HCs of any turn. Lipid peroxidation starts 12hrs post EIT in both HCs and SCs of basal turn, and reaches up to apex turn at 24hrs post EIT. Conclusions: PCD starts initially in the SCs post EIT, followed by PCD in HCs. Caspase 3 activation is observed only in SCs. Protecting SCs with selective otoprotective drugs at early stage post implantation may help to preserve HCs and residual hearing