ABSTRACT
@#Cryptococcus neoformans is an encapsulated fungal pathogen that causes severe disease primarily in immunocompromised patients. Adherence and internalisation of microbial pathogens into host cells often begin with engagement of microbes to the surface receptors of host. However, the mechanisms involved remain poorly understood. In this study, we investigated the association of cell surface determinants of C. neoformans with mammalian cells. Our results showed that treatment with trypsin, but not paraformaldehyde or heat killing, could reduce host-cryptococci interaction, suggesting the involvement of cell surface proteins (CSPs) of C. neoformans in the interaction. We extended our investigations to determine the roles of CSPs during cryptococci-host cells interaction by extracting and conjugating CSPs of C. neoformans to latex beads. Conjugation of CSPs with both encapsulated and acapsular C. neoformans increased the association of latex beads with mammalian alveolar epithelial cells, alveolar macrophages and monocyte-derived macrophages. Further examination on the actin organisation of the host cells implied the involvement of actin-dependent phagocytosis in the internalisation of C. neoformans in CSP-conjugated latex beads. We hypothesised that CSPs present on the cell wall of C. neoformans mediate the adherence and actin-dependent phagocytosis of cryptococci by mammalian cells. Our results warrant further studies on the exact role of CSPs in the pathogenesis of cryptococcosis
ABSTRACT
Background Endothelin-1 (ET-1) is an active regulator of intraocular pressure.The ET-1 level in aqueous humor is elevated in primary open-angle glaucoma,normal intraocular tension glaucoma and the animal model of glaucoma.There is now accumulating evidence for a role of ET-1 in the pathogenesis of glaucoma.However,the effect of ET-1 on the phagocytic function in trabecular meshwork cells (TMCs) is unclear.ObjectiveThis study is to observe the effect of ET-1 on the phagocytic function in cultured human TMCs.Methods Human trabecular meshwork tissue was obtained from healthy donator and cultured and subcultured in vitro by the explant culture method.The third passage of human TMCs were incubated with fluoresent red-labeled latex beads for 0,4,8,12,24,48 and 72 hours.The phagocytic kinetics of human TMCs were continuously evaluated by counting the number of latex beads in TMCs using a fluorescence microscope.Depending on the concentrations of ET-1 in culture medium,the TMCs were divided into control group (without ET-1),low-dose ET-1 (10~(-9)mol/L) treatment group,middle-dose ET-1 (10~(-8)mol/L) treatment group and high-dose ET-1 (10~(-7) mol/L) treatment group.In addition,based on the addition of endothelin receptor (ETAR) antagonist,the TMCs were divided into control group (without ETAR antagonist),ET-1 (10~(-8)mol/L) treatment group,ETAR antagonist (1×10~(-7)mol/L BQ123+10~(-8)mol/L ET-1) treatment group and ETBR antagonist(1×10~(-7) mol/L BQ788+10~(-8) mol/L ET-1)treatment group.TMCs of each group were incubated with latex beads,and the numbers of latex beads in TMCs were counted under a fluorescent microscope.Results Cultured HTM cells showed positive reactions for FN,LN,NSE and negative response for FⅧRag.The phagocytic kinetics test revealed that the latex beads were detected 4 hours after incubation.The density of latex beads was gradually increased with the delay of incubation duration and peaked at 24 hours.The number of the latex beads saturated after 48 hours of incubation.However,the number of latex beads in TMCs was significantly reduced after the addition of ET-1 in a dose-dependent manner (F=28.91,P＜0.05).The number of latex beads in the ET-1 group was less than that in the control group and the ETAR receptor antagonist group (q=13.7228,q=9.4312,P＜0.05).No significant difference was found in latex beads number between the ET-1 group and the ETBR antagonist group (q=1.1600,P＞0.05).Conclusion ET-1 inhibits the phagocytic function of human TMCs and ETAR plays a partial role in the phagocytic function of human TMCs.
ABSTRACT
The aims of this study were to describe a reproducible method for the isolation, purification and primary culture of rat Kupffer cells, and were to develop in vitro system which could provide a tool for the study of ischemia-reperfusion injury. Kupffer cells were isolated following sequential collagenase digestion of the liver by perfusion and enrichment of a nonparenchymal cell fraction by a double-densities gradient centrifugation step using Percoll and were selected by allowing them to adhere to culture vessel for 2 h at 37 degrees C under 5% CO2. The purity of obtained Kupffer cell was about 90% assessed by the phagocytosis of 3 micrometer latex beads. This method for Kupffer cell isolation resulted in yields of 1~5 x10(7) Kupffer cells per liver and Kupffer cells were preserved in maintenance cultures for 10 days. The phagocytic capacity of cultured Kupffer cells was measured according to the amount of latex beads incorporated into the cytoplasm. Larger round Kupffer cells in the culture had higher phagocytic capacity compared with smaller round or irregular shaped Kupffer cells. The different phagocytic capacity of Kupffer cells which was dependent on size and shape in vivo was well preserved during culture. The experimental group of Kupffer cells in culture were sequentially treated with ischemia and reperfusion at 1h and 30 min. The ratio of Kupffer cells having latex beads in their cytoplasm was significantly increased compared with control (p<0.01). This result was able to explain the Kupffer cells' activation after ischemia-reperfusion injury in vivo. In conclusion, Kupffer cells in this culture well resembled the cells in vivo and this in vitro model could provide a valuable tool for the study of Kupffer cells with a key role in pathophysiology of ischemia-reperfusion injury.