Month: February 2019

Resolution of acute hepatitis C correlates with the induction of strong and broad CD4+ and CD8+ T cell responses. However, the majority of patients fail to eliminate HCV and develop chronic infection. The high genetic variability of HCV significantly contributes to the escape from the immune system and complicates the development of an efficient vaccine. Nevertheless, more recent data indicate that there is protective immunity against HCV. A critical step for the understanding of the immunopathogenesis of HCV infection and HCV clearance is the presentation of viral epitopes on MHC class I molecules from infected cells. Most of the currently available experimental systems are limited, since an a priori defined set of synthetic peptides is used to either externally load target cells or to expand epitope-specific CD8+ T cells which are then used in downstream readout applications. Therefore, the aim of this study was to identify specific Ginsenoside-Rd ligands which are naturally processed and presented by cells expressing HCV proteins. To this end, we engineered continuous human cell lines to inducibly express HCV proteins and to constitutively express high levels of functional HLA-A2. MHC class I molecules were isolated from large-scale cultures of these cell lines, followed by elution and identification of naturally processed CTL epitopes. This proof-of-concept study allowed the identification of two naturally processed HCV-derived MHC class I ligands. Although both epitopes have been described previously by conventional T-cell dependent methods, this novel approach has the potential to identify novel and unconventional epitopes. It has been estimated that approx. 2,000–10,000 molecules of a protein are required to allow the presentation of one antigen. In fact, antigen processing involves a highly complex interplay of multiple steps and factors. Degradation by the proteasome, interaction with chaperones such as calnexin, incorporation into the peptide loading complex, involving other chaperones such as tapasin, peptide trimming by aminopeptidases, loading onto empty MHC I molecules, and, finally,(S)Ginsenoside-Rh2 transport across the secretory pathway to the cell surface are pivotal steps that are tightly coordinated during this process. The identification of two known HCV HLA-A2 ligands that are localized in NS3 and NS5B demonstrates their authentic processing and presentation in vivo. Furthermore, a more elaborate approach, using tracer substances such as isotope-labeled peptides, might hold promise for the quantification of an epitope relative to the complete repertoire of presented ligands. Few reports on Epstein-Barr virus encoded proteins investigated how the amount of presented MHC I antigen complex could influence the efficiency of recognition by CD8+ T cells. Therefore, in addition to the proof-of principle of this particular experimental setting, the identification of the two epitopes by this novel approach underlines their importance as natural targets for HCV-specific T cells. CTL responses against these epitopes could be of particular importance to control viral infection and may be included as targets in future vaccination strategies. In principle, the direct sequencing of MHC I ligands should allow to identify epitopes after posttranslational protein modifications such as glycosylation, phosphorylation and proteolytic processing as well as unconventional epitopes derived from alternative reading frames or RNA splicing that are not detected by the current conventional methods. Future studies aimed at identifying naturally processed HCV-derived MHC class I ligands may provide novel insights into epitope processing and presentation as well as recognition, thereby contributing to the understanding of HCV pathogenesis.

The critical role of maintaining ATP levels in the survival of the heart during cold storage has long been recognized. Even if glycolysis is enhanced, the supply of adenine nucleotides could limit energy production. In addition to conversion of ATP to ADP and AMP, the total adenine nucleotide pool can be depleted under ischemic conditions due to the further degradation to adenosine, inosine, and hypoxanthine, all of which can penetrate the plasma membrane and be lost to cardiac myocytes. One strategy for overcoming this problem is to provide the precursors for de novo adenine nucleotide synthesis, adenine and ribose. In various studies, these two compounds have been shown to be beneficial for the heart, either alone or in combination. However, these experiments have focused on recovery of function and of ATP during reperfusion, rather than preservation during a period of cold storage. We tested whether adenine and ribose, when present during hypothermic incubation of cardiac myocytes,Ginsenoside-Rg2 could reduce their death rates. Neither adenine nor ribose showed significant effects when added individually. Moreover, the combination of the two also was not beneficial. While it is possible that the treatments would aid in the resynthesis of adenine nucleotides following return to normoxia and normothermia, it appeared that any synthesis of adenine nucleotides was insufficient to improve survival while the cells remained under ischemic, hypothermic conditions. Recent studies have often focused on diverse pathways that are altered in response to FBP, such as inflammation and apoptosis. However, the underlying connection between FBP and such effects are unclear. Our results suggest two linked mechanisms for FBP effects: calcium and energy. If FBP acts to chelate calcium, it will spare ATP that would otherwise be used in calcium pumping. Alternatively, If FBP is used to provide glycolytic ATP, the increased cellular energy can help control intracellular Ca2+ levels. Together, effects of FBP on ATP and Ca2+ levels will influence many regulatory pathways,Ginsenoside-Rb3 and these pathways deserve further exploration. Also, in the course of our studies we observed that albumin can contribute significant amounts of calcium to cell culture media, and that different lots of the same commercial albumin product appear to differ considerably in their calcium content. This may be important to researchers employing albumin in situations where calcium concentration is critical. BDM also has strong protective effects in our experimental system. Thus, FBP and BDM may be useful in hypothermic preservation of hearts for transplantation. Because calcium levels are normally well controlled in vivo, especially in clinical situations, calcium chelation might be less relevant compared to other beneficial effects of these two agents. However, during ex vivo preservation experiments, this could be a major factor in cardiomyocyte survival. Pyruvate, adenine, and ribose had little or no beneficial effects during the ischemic, hypothermic incubation. However, it remains possible that these compounds could be protective upon return to physiological temperature and oxygen levels. With an estimated 120–180 million chronically infected individuals, HCV is a leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide. Antiviral therapy has improved considerably with the introduction of pegylated interferon-a and ribavirin as well as, more recently, the first generation of directly acting antivirals. However, many patients still do not respond to or cannot tolerate antiviral therapy. In addition, HCV continues to be transmitted in certain areas of the world. Therefore, the development of preventive and therapeutic vaccines against hepatitis C is of major public health importance. Innate and adaptive immune responses to HCV have been studied in great detail.

It should be noted that Hassinen et al. reported metabolic effects of FBP in perfused hearts that were not produced by EGTA and thus did not appear to be due to calcium chelation. Effects on basal levels were not reported. While the authors suggested that preservation of ATP was responsible for the FBP effect, they also suggested that Ca2+ chelation could be involved. Cavallini et al. showed that FBP inhibited the thrombin-induced increase in cytosolic Ca2+ in platelets, though it did not appear that there was any effect on the resting Ca2+ level. The authors proposed effects on ‘‘the transmission of signal at the level of the receptor-G-protein-phospholipase C system.’’ Tamaki et al. reported that FBP inhibited the increase in cytosolic Ca2+ in response to phorbol ester treatment of Kupffer cells. The authors proposed that the effects came about via both chelation of extracellular Ca2+ and by provision of glycolytic ATP, allowing greater Ca2+-ATPase activity. Two of the studies that looked at intracellular Ca2+ concerned neurons, and found increases in cytosolic Ca2+ in response to FBP treatment, in contrast to our observations. However, in Ref., FBP, while it increased basal Ca2+ levels, prevented the increase in Ca2+ in response to hypoxia. In another study, using synaptosomes rather than intact cells, Zeng et al. showed that FBP reduced Ginsenoside-Rb2 level during ischemic conditions. They proposed metabolic effects of FBP and did not address the possible role of chelation. Thus, our studies add to the weight of evidence concerning a role for Ca2+ in protective effects of FBP, and provide the first experimental evidence related to an effect of FBP on Pseudoginsenoside-F11 homeostasis in heart preservation. Possible further experiments that would help confirm our hypothesis would include determining the effect of FBP on survival under conditions in which extracellular Ca2+ is fixed, as well as measurements of intracellular Ca2+ for EGTA-treated myocytes. Determining the combined effect of EGTA and FBP on intracellular Ca2+ would help establish whether or not FBP acts by multiple mechanisms. These include effects on calcium fluxes at low concentrations and inhibition of myosin ATPase at somewhat higher concentrations. Beneficial effects of BDM in the preparation of cardiac myocytes have been characterized, and the compound is used in the procedure we employed. Several studies have found benefits in preservation of the intact heart. However, at high concentrations, BDM can also have deleterious effects, possibly through action as a phosphatase. However, the effect was much lower than that of FBP. Possibly this is because the effect of BDM on calcium is indirect; by inhibiting myosin ATPase and preserving ATP, it enables the cells to maintain calcium pumping activity better than untreated cells. Because they have different modes of action, the combination of FBP and BDM may have benefits beyond either alone, as indicated by the furthest right hatched bar in Fig. 5. Pyruvate is employed during the procedure we used for the preparation of cardiac myocytes. The general metabolic benefits of pyruvate in the heart have been reviewed by Mallet. Most studies of pyruvate’s effects on the heart have examined periods of reperfusion after ischemia, rather than effects during cold storage. We found that pyruvate produced, at best, relatively small decreases in the death rate of myocytes incubated at 3uC. When combined with 5 mM FBP, effects were no greater than those of FBP alone. While we hypothesized that there might be sufficient residual oxygen in the ischemic cell suspensions to support pyruvate oxidation, the results suggest that either such metabolism is relatively small, or it provides little survival advantage to the myocytes. This is consistent with our previous finding that dichloroacetate, which stimulates pyruvate dehydrogenase, produced no beneficial effects under these conditions.

By reducing the extracellular Ca2+ level, FBP would make it easier for cells to maintain low intracellular levels and prevent such damage. In the experiments shown in Fig. 3, the calcium-chelating agent EGTA reduced the death rate by 60–70%, similar to the effects of FBP. Thus, chelation of calcium is protective in our experimental system, even though the final medium used to wash and incubate the myocytes is nominally calcium-free. It is difficult to measure the levels of calcium in this medium. However, the results indicated that the medium likely contained micromolar levels of calcium, and that most of this probably came from the albumin. Thus, the idea of a chelating effect of extracellular FBP is reasonable. Using a dissociation constant of Mogroside-III would bind approximately 60% of the calcium in the medium. We found that there was considerable variation in the quality of myocytes prepared using different lots of albumin having the same product number. Possibly this was the result of differences in calcium content of the different batches. Evidence for effects of FBP on calcium homeostasis is shown in Fig. 4. For freshly-prepared myocytes, cytosolic calcium was an average of 33% lower in FBP-treated cells compared to control cells. For freshly-prepared cells there is expected to be little effect of FBP on the cellular ATP levels; in Ref. we observed only a 16% higher ATP level for cells treated with 5 mM FBP compared to control cells after 2 hours of hypothermic incubation. Thus, the differences for the fresh myocytes in Fig. 4 are likely direct effects of calcium chelation rather than due to increased availability of ATP for Ca2+ pumping. Our earlier experiments found that ATP levels were about 30% higher at 6 hours and 50% higher at 24 hours in FBP-treated cells. Therefore the larger reduction in calcium with 24 hours of FBP treatment may be due to both chelation of Ca2+ by FBP and the cumulative effects of maintaining higher ATP levels in the cells, which should allow them to reduce the calcium levels through Ca2+-ATPase activity. In addition to producing extracellular effects, it is possible that FBP could be taken up by the cells and chelate intracellular Ca2+. We previously showed that label from radiolabeled FBP at 5 mM could be taken up by myocytes, both at room temperature and at 3uC. However, it is likely that much of this label was converted to other metabolites, and it seems unlikely that intracellular FBP would rise to levels high enough to provide a significant chelating effect. Additional support for a chelating effect of extracellular FBP,Mogroside-IIA2 rather than an effect via glycolytic ATP production, comes from a comparison of the effects of BDM and FBP. These compounds at 5 mM had similar effects on the hypothermic survival of myocytes. However, in myocytes incubated for 24 hours, BDM produced a much smaller reduction in free calcium than did FBP. Nevertheless, the data in Fig. 3 indicate that FBP may have protective effects beyond those due to calcium chelation. For both 0.3 and 1.0 mM EGTA, it appeared that the combination of FBP and EGTA produced greater reductions in the death rate than EGTA alone, although the differences were not statistically significant at the p,0.05 level by paired ttests. Because of the much greater affinity of EGTA for Ca2+ compared to the affinity of FBP, 5 mM FBP would not lower the Ca2+ level significantly in the presence of these levels of EGTA. We previously showed that FBP could be taken up by cardiac myocytes at 21uC and 3uC. It is possible that FBP taken up during or after the transition to hypothermia could be used to provide glycolytic ATP even at the reduced temperature. Since energyconsuming processes would also be slowed by the hypothermia, this ATP could provide significant protection against cell death. As described above, we previously demonstrated that FBP helped maintain higher levels of ATP during hypothermic incubation.

FVs have been described, depending on mammalian species, as being either fusiform or discoidal in cross-section. According to Staehelin et al., they have a form of biconvex discs with a diameter 0.5–1 mm. Minsky and Chlapowsky proposed that FVs are pancake-like flattened spheres, but this has never been confirmed by ultrastructural 3D analyses. FVs are lined by an asymmetric unit membrane, which contains four major integral proteins, uroplakins Ia, Ib, II and IIIa. Uroplakins form 16-nm intramembranous uroplakin particles, which are hexagonally arranged in urothelial plaques. Plaques measure between 0.3 and 1 mm in diameter, and they are connected by a non-thickened membrane, called hinge region. UPs are synthesized in the endoplasmic reticulum, where UPIa and UPIb form heterodimers with UPII and UPIIIa, respectively. Conformational changes in the Golgi apparatus enable the formation of 16-nm intramembranous particles, which are hexagonally arranged into 2D crystalline plaques in the post-Golgi compartments. While the structure of the 16-nm particles is largely known, the information on the 3D structure of mature FVs is missing. The plaque composition of mature FVs is identical to that of the apical plasma membrane of umbrella cells, therefore it has been proposed that FVs are transported from the Golgi apparatus towards the apical cell surface where they fuse with the plasma membrane. According to one hypothesis,Timosaponin-BII FVs are inserted into the apical plasma membrane during bladder distension and retrieved during bladder contraction. This membrane recycling therefore provides a mechanism to adjust surface area of umbrella cells during distension-contraction cycles of the urinary bladder. Alternative hypothesis says that FVs are not retrieved during contraction of the bladder; instead the apical surface area is accommodated only by the apical plasma membrane infolding. The analyses of morpho-functional organization of FVs are therefore essential for understanding their role in the intracellular membrane traffic and in the turn-over of the apical plasma membrane. Electron tomography, which allows 3D reconstructions of objects with the resolution below 10 nm, has greatly contributed to the understanding of subcellular structures and compartments. In order to analyse subcellular structures by ET in the state ‘close to native’, samples should be fixed by high pressure freezing, which allows immobilization within milliseconds,Theaflavin followed by freeze substitution. Because FVs are relatively large compartments, their 3D reconstruction requires serial sectioning and joining of tomograms. Here we demonstrate that high pressure freezing and ET of serial sections, supported by freeze-fracture and immunocytochemistry, give a new insight into the structure and organization of FVs in umbrella cells. We also compared the arrangement of FVs during distension-contraction cycle of the urinary bladder, and analysed intermediate cells with respect to the occurrence of FVs. FVs are highly specialized compartments that transport urothelial plaques in umbrella cells of the urothelium. Here we employed ET, freeze-fracturing and immuno-electron microscopy of the mouse urothelium in order to resolve the 3D ultrastructure and organization of FVs in their ‘close to native’ state. Our results indicate that rapid cryo-fixation is the superior method for ultrastructural studies of FVs and their morphofunctional organization in umbrella cells. Until now, conclusions about the nature of FVs were based on observations of ultrathin sections and freeze-fracturing whereas 3D models were not done yet. To investigate cellular structures in 3D rather than their 2D projections, ET is the method of choice.