Secreted Giardia intestinalis cysteine proteases disrupt intestinal epithelial cell junctional complexes and degrade chemokines.

Giardiasis is a common diarrheal disease caused by the protozoan parasite Giardia intestinalis. Cysteine proteases (CPs) are acknowledged as virulence factors in Giardia but their specific role in the molecular pathogenesis of disease is not known. Herein, we aimed to characterize the three main secreted CPs (CP14019, CP16160 and CP16779), which were identified by mass spectrometry in the medium during interaction with intestinal epithelial cells (IECs) in vitro. First, the CPs were epitope-tagged and localized to the endoplasmic reticulum and cytoplasmic vesicle-like structures. Second, we showed that recombinant CPs, expressed in Pichia pastoris, are more active in acidic environment (pH 5.5-6) and we determined the kinetic parameters using fluorogenic substrates. Third, excretory-secretory proteins (ESPs) from Giardia trophozoites affect the localization of apical junctional complex (AJC) proteins and recombinant CPs cleave or re-localize the AJC proteins (claudin-1 and -4, occludin, JAM-1, ß-catenin and E-cadherin) of IECs. Finally, we showed that the ESPs and recombinant CPs can degrade several chemokines, including CXCL1, CXCL2, CXCL3, IL-8, CCL2, and CCL20, which are up-regulated in IECs during Giardia-host cell interactions. This is the first study that characterizes the role of specific CPs secreted from Giardia and our results collectively indicate their roles in the disruption of the intestinal epithelial barrier and modulating immune responses during Giardia infections.

Acanthamoeba culbertsoni isolated from a clinical case with intraocular dissemination: Structure and in vitro analysis of the interaction with hamster cornea and MDCK epithelial cell monolayers.

Acanthamoeba culbertsoni trophozoites, previously isolated from a human keratitis case with severe intraocular damage, were maintained in axenic culture. Co-incubation of amoebae with MDCK cell monolayers demonstrated an apparent preference of the amoebae to introduce themselves between the cells. The trophozoites appeared to cross the cell monolayer through the tight junctions, which resulted in decreased trans-epithelial resistance (TER) measurements. Unexpectedly, after co-incubation of amoebae with hamster corneas, we observed that the trophozoites were able to cross the different cell layers and reach the corneal stroma after only 12 h of interaction, in contrast to other Acanthamoeba species. These observations suggest that this A. culbertsoni isolate is particularly pathogenic. Further research with diverse methodologies needs to be performed to explain the unique behavior of this Acanthamoeba strain.

Protective effects of hydrogen sulfide on portal hypertensive vasculopathy in rabbits by activating AKT-NF-κB pathway.

The role of hydrogen sulfide (H2S) in portal hypertension (PH)-induced esophagus-gastric junction vascular lesions in rabbits was observed. The rabbit PH models were established. The animals were randomly divided into the following groups: normal, PH, PH+sodium hydrosulfide (PH+S), PH+propargylglycine (PH+PPG). The plasma H2S levels, apoptosis of esophageal-gastric junction vascular smooth muscle cells, and the expression of nuclear transcription factor-κB (NF-κB), p-AKT, IκBa and Bcl-2 were detected. The cystathionine γ lyase (cystathionine-gamma-splitting enzyme, CSE) in the junction vascular tissue was measured. The results showed that the plasma H2S levels and the CSE expression levels had statistically significant difference among different groups (P<0.05). As compared with PH group, plasma H2S levels were declined obviously (11.9±4.2 vs. 20.6±4.5, P<0.05), and CSE expression levels in the junction vascular tissue were notably reduced (1.7±0.6 vs. 2.8±0.8, P<0.05), apoptosis rate of vascular smooth muscle cells per unit area was significantly decreased (0.10±0.15 vs. 0.24±0.07, P<0.05), and the expression levels of p-AKT and NF-κB were significantly decreased (2.31±0.33 vs. 3.04±0.38, P<0.05; 0.33±0.17 vs. 0.51±0.23, P<0.05), however, IκBa and Bcl-2 expression increased obviously (5.57±0.17 vs. 3.67±0.13, P<0.05; 0.79±0.29 vs. 0.44±0.36, P<0.05) in PH+PPG group. As compared with PH group, H2S levels were notably increased (32.7±7.3 vs. 20.6±4.5, P<0.05), the CSE levels in the junction vascular tissue were significantly increased (6.3±0.7 vs. 2.8±0.8, P<0.05), apoptosis rate of vascular smooth muscle cells per unit area was significantly increased (0.35±0.14 vs. 0.24±0.07, P<0.05), and the expression levels of p-AKT and NF-κB were significantly increased (4.29±0.49 vs. 3.04±0.38, P<0.05; 0.77±0.27 vs. 0.51±0.23, P<0.05), yet IκBa and Bcl-2 expression decreased significantly (3.23±0.24 vs. 3.67±0.13, P<0.05; 0.31±0.23 vs. 0.48±0.34, P<0.05) in PH+S group. It is concluded that esophagus-gastric junction vascular lesions happen under PH, and apoptosis of smooth muscle cells is declined. H2S can activate NF-κB by the p-AKT pathway, leading to the down-regulation of Bcl-2, eventually stimulating apoptosis of vascular smooth muscle cells, easing PH. H2S/CSE system may play an important role in remission of PH via the AKT-NF-κB pathway.

Transepithelial migration of Toxoplasma gondii involves an interaction of intercellular adhesion molecule 1 (ICAM-1) with the parasite adhesin MIC2.

Toxoplasma gondii crosses non-permissive biological barriers such as the intestine, the blood-brain barrier and the placenta thereby gaining access to tissues where it most commonly causes severe pathology. Herein we show that in the process of migration Toxoplasma initially concentrates around intercellular junctions and probably uses a paracellular pathway to transmigrate across biological barriers. Parasite transmigration required viable and actively motile parasites. Interestingly, the integrity of host cell barriers was not altered during parasite transmigration. As intercellular adhesion molecule 1 (ICAM-1) is upregulated on cellular barriers during Toxoplasma infection, we investigated the role of this receptor in parasite transmigration. Soluble human ICAM-1 and ICAM-1 antibodies inhibited transmigration of parasites across cellular barriers implicating this receptor in the process of transmigration. Furthermore, human ICAM-1 immunoprecipitated the mature form of the parasite adhesin MIC2 present on the parasite surface, indicating that this interaction may contribute to cellular migration. These findings reveal that Toxoplasma exploits the natural cell trafficking pathways in the host to cross cellular barriers and disseminate to deep tissues.

Tight junctional inhibition of entry of Toxoplasma gondii into MDCK cells.

Various conditions of cultures were performed to investigate the role of tight junctions formed between adjacent MDCK cells on the entry of Toxoplasma. When MDCK cells were cocultured with excess number of Toxoplasma at the seeding density of 1 x 10(5), 3 x 10(5), and 5 x 10(5) cells/ml for 4 days, the number of intracellular parasites decreased rapidly as the host cells reached saturation density, i.e., the formation of tight junctions. When the concentration of calcium in the media (1.8 mM in general) was shifted to 5 microM that resulted in the elimination of tight junction, the penetration of Toxoplasma increased about 2-fold (p less than 0.05) in the saturated culture, while that of non-saturated culture decreased by half. Trypsin-EDTA which was treated to conquer the tight junctions of saturated culture favored the entry of Toxoplasma about 2.5-fold (p less than 0.05) compared to the non-treated, while that of non-saturated culture decreased to about one fifth. It was suggested that the tight junctions of epithelial cells play a role as a barrier for the entry of Toxoplasma and Toxoplasma penetrate into host cells through membrane structure-specific, i.e., certain kind of receptors present on the basolateral rather than apical surface of MDCK cells.