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5) The electron cryo-tomography of the prepore reveals a structure of 34 protomers in the prepore, a number expected based on numerous other reports. However, the CryoEM structure of the pore reveals a larger 42 protomer pore. This is unexpected but interestingly the authors note a range of ring structures were present with this being the most stable. So the question arises whether the pore structure is an artifact. Can the authors model a 34 protomer pore based on their 42 protomer structure and convince that the details they discuss in the 42 protomer pore are likely relevant to the smaller pore? Is the 20º incline in the β-hairpin maintained in modeling the smaller pore? Like the authors, other workers have found that PLY form linear oligomers in their crystals. The authors should discuss how linear oligomers fit into their detailed mechanism of pore formation.
|
review
| 99.25 |
6) The authors have skirted around another controversial issue of whether arcs and incomplete rings observed by other workers are physiological relevant as they are not observed in this work. Some discussion of why PLY does not form such structures in their hands but do in others would be insightful.
|
other
| 99.9 |
7) Subsection “Three stages of PLY pore formation”: In the previous AFM study (van Pee et. Al, Nano Letters, 2016) only 13% of the lower height rings were pre-pores on supported lipid membranes. This could be mentioned to indicate that the occurrence of a "late prepore" is rare event even on membranes without curvature.
|
study
| 99.94 |
8) The literature review, particularly in the Discussion, has been narrowly focused to a large degree on the PLY literature and should be broadened out to other CDCs, particularly to PFO where there is an extensive literature on how it forms pores. Does the current study support the major findings in the PFO literature and does it disagree with any aspects?
|
review
| 99.9 |
9) Parts of the Discussion should be moderated. The limited resolution and a few static snapshots do not reveal all the details of membrane insertion and pore formation. Such details will come gradually from a host of biochemical and biophysical approaches. The suggestion that the CryoEM structure forms a good basis for drug discovery to treat numerous Gram-positive infections is not supported by any results in the manuscript so should be restricted to a speculative claim about S. pneumoniae.
|
other
| 95.25 |
Thank you for resubmitting your work entitled "CryoEM structures of membrane pore and prepore complex reveal cytolytic mechanism of Pneumolysin" for further consideration at eLife. Your revised article has been favorably evaluated by Richard Aldrich (Senior editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors.
|
other
| 99.94 |
1) Over-interpretation of lower-resolution parts of the map in terms of the atomic model. Although the new supplementary figures now reveal the limitations of the map more clearly, over-interpretation in the text still remains a problem. Specifically, W433 is mentioned to move wrt the crystal structure, yet there is no density visible for W433 in Video 1, Figure 2—figure supplement 2 or 3 to support this statement. The map in this region is ~6Å. The same can be said for H407 that does also not have clear density. The authors should stay away from over-interpretation in this region and simply state that this domain is likely to contribute to the inter-molecular interactions that stabilize the pore, as they have done in the Discussion section, paragraph three. Likewise, there is no obvious density that connects D168 and K271 (Figure 5 instead shows connecting density between β-strands that is expected at resolutions lower than 4.7A). The resolution in this area is again ~6-7Å according to the local resolution estimation. The authors should be more cautious and can suggest that a salt bridge may exist as evident by the point mutant D168A, which has an 80% reduction in hemolytic activity.
|
other
| 65.06 |
2) The comment in the Discussion section: "The 4.5 Å cryoEM structure of the PLY pore complex thus paves the way towards the design of new drugs that inhibit toxin oligomerization and pore formation as a promising approach towards combating infections by wide-spread and dangerous Gram-positive pathogens" jars with the reply "the different CDCs seem to follow different pathways of pore formation…. Any more discussion of PFO papers would distract from the main focus of our work, which is on PLY." Therefore, the authors should focus the therapeutic claim focus to diseases that PLY has shown to contribute.
|
other
| 91.4 |
We are aware, and do not approve of the general tendency to overstate the resolution and quality of cryoEM maps. In this case, however, we have a high-resolution x-ray structure of the soluble form of PLY, and several of its domains move as rigid bodies. We would therefore be justified to describe at least these parts as an atomic resolution structure. Nevertheless we have refrained from such a description, and instead describe the structure in terms of an atomic model, which it no doubt is.
|
study
| 98.8 |
Locally the resolution is probably even worse than 4.5A, as for example the β strands are not well separated in Figure 1A. Overall, too little information is given on the quality of the map and the corresponding atomic model. In fact, at 4.5A, the map alone will probably still leave considerable scope for registry or main chain tracing errors.
|
other
| 99.25 |
Register and chain-tracing errors in the β-strands of the barrel are actually not a problem, because the first and last stretches of each strand are fixed in the x-ray structure of domain 3. Together with the hydrophobic residues facing the lipid bilayer on the outside of the barrel and the polar and charged residues facing the inside, this leaves virtually no room for chain trace errors even at 4.5 Å. Moreover, we know that the β-strands in the barrel are well-resolved because of the sharp peak at 4.8 Å in the FSC curve (Figure 1—figure supplement 2B), which is the characteristic distance between β-strands. We can therefore be sure that our atomic model of the β-barrel, which is the major new part of the structure, is correct and accurate within the constraints of the map.
|
study
| 100.0 |
It is true that the local resolution of some parts of the map, especially the loops connecting the long β-strands and one loop in the periphery of domain 4, is worse than 4.5 Å, but the resolution of domains 1, 2 and parts of domain 4 is significantly better. The local resolution estimate (new Figure 1—figure supplement 3) shows this clearly for the whole pore complex and for an individual protomer seen from different directions. The new supplementary figures demonstrate the excellent quality of the map, which resolves many of the tryptophan and tyrosine sidechains (Figure 2—figure supplements 1–3). This would be expected at 4 Å rather than at 4.5 Å resolution.
|
study
| 100.0 |
Figure 2—figure supplement 4: stereo diagram of domain 3 (green) with the upper part of the β-barrel and the new helix-turn-helix motif (HTH) that forms the α-barrel inside the pore as a stick model in the cryoEM map. The local resolution estimate indicates 4 to 4.5 Å for the β-barrel outside the lipid bilayer and around 5 Å for the α-barrel. Also shown is the new helix α3a (red and yellow) of the neighboring monomer, indicating a potential ionic inter-monomer interaction of Lys196 with Asp59 that would stabilize the membrane-inserted pore complex.
|
study
| 100.0 |
The FSC curve between the model and the map is now included in Figure 1—figure supplement 2B (blue curve). Note that the model was used as built into the map, but not refined, for the reasons given below. Like the masked FSC, this curve shows a sharp peak at 4.8 Å, due to the strong contribution of the resolved long β hairpins in the barrel. The correlation factor at the steep drop beyond this peak at 4.5 Å is not 0.5 (which is an arbitrary value), but approximately 0.3. We draw the reviewers’ and editor’s attention to two papers, which will be familiar to the BRE of our study, that report similar thresholds for the map against model FSC of the 3.4 Å structure of the ribosome/Sec61 complex (Voorhees et al., Cell 2014) and the tri-snRNP spliceosome at 5.9 Å (Nguyen et al., Nature 2015).
|
review
| 95.44 |
We can rule out overfitting for three reasons: (1) Substantial portions of the model were not built from scratch, but they were rigid-body fitted domains from our 2.4 Å x-ray structure of soluble PLY. These map regions cannot be overfitted. (2) The major new part of the membrane-inserted form of the PLY protomer is a β-barrel with 84 long β-hairpins of rigid, pre-defined geometry, so the risk of overfitting in this region is minimal. (3) The comparison of the phase-randomized vs masked FSC (Figure 1—figure supplement 2B) indicates that there is no overfitted noise.
|
study
| 100.0 |
The individual domains of the PLY x-ray structure were fitted manually into the cryoEM map as rigid bodies with COOT. Where necessary, flexible or refolded protein regions were then refitted manually, followed by geometry regularization in COOT. We had described this procedure clearly in the methods of the original manuscript, but now describe it also in the main text, where it is less easily overlooked.
|
other
| 98.5 |
Fitting and refinement with Phenix or Refmac was tried but did not work, because these automatic procedures attempted to e.g. fit the membrane-inserted β-hairpins into the amphipol density around the hydrophobic part of the pore. The densities of amphipol and protein are comparable at 4-4.5 Å resolution, and only manual fitting can differentiate between them. Therefore, it was unfortunately not possible to employ automatic model fitting and refinement. It is well known that refinement of x-ray structures at similar resolutions also does not work, for the same reasons.
|
study
| 99.94 |
Even though this salt bridge is in the least well-defined map region near the tip of the long β-hairpins, there was significant density for it, as shown in the new panel G of Figure 5. We would like to point out that we did not look for this salt bridge, but deduced that there had to be one in this position from our chain trace, and then found the corresponding map density, which again shows there were no chain tracing errors in the β-barrel. Moreover, the interaction of adjacent β-hairpins that includes this salt bridge is in excellent agreement with the S-S crosslinking studies of Tweten and colleagues in the related PFO (Sato et al. 2013, NatChemBiol). This is now explained more fully in subsection “CryoEM structure of the PLY pore complex” paragraph four of the revised manuscript.
|
other
| 66.5 |
The reviewer did not read our manuscript carefully. The mutation of Asp168 to alanine reduces the hemolytic activity by 80%, not to 80%! This is exactly the strong effect that one would expect from the disruption of a salt bridge stabilizing the membrane-inserted form. We do not see the point of mutating Lys271 as well.
|
other
| 99.9 |
In addition the mutation seems to affect membrane binding as the authors mention (Figure 7 legend) and the ring attachment from the membranes (subsection “Determinants of lytic activity”). Does this imply that pores are formed but are then released? Previous AFM studies by the authors (van Pee et. Al, Nano Letters, 2016) suggest the process is irreversible. Some clarity and caution interpreting this salt bridge would improve the manuscript.
|
other
| 95.2 |
We apologize if this was not clear in the original manuscript. In the original Figure 7 legend we wrote “Many PLYD168A rings become detached from the liposomes, probably due to reduced binding affinity, and tend to break into fragments.” At this stage, the rings are of course prepores, and it is these, not the pores, that become detached and break into fragments. In the revised manuscript (subsection “Determinants of lytic activity” paragraph two) and Figure legend, we have clarified this point.
|
other
| 99.9 |
Note that the AFM study (van Pee et. al, Nano Letters, 2016, Figure 5) describes the transformation of membrane-attached rings into pores as irreversible, not the membrane attachment of the rings. Rings of this PLY mutant in the early prepore state detach from the membrane easily, but only before they (irreversibly) insert into the membrane to form the pore. This is now discussed in on paragraph two of the Discussion section.
|
other
| 63.56 |
3) Also related to the limited resolution: interactions of the Trp-rich loop are discussed (Results section) but the electron density map in that region looks poorly resolved. This is a very important part of a CDC and the motif defines the family so another panel is required to show the quality of the electron density map in that region. (A close up stereo showing side-chains would help). There is great interest in seeing what conformation this loop adopts in the prepore and pore states since it is thought to control prepore to pore conversion. In Results paragraph two, the authors state that the undecapeptide is interacting with Thr405. This sentence should be re-worded to provide more detail. What is the nature of this interaction and again is the resolution of this area sufficient to justify this comment? From Figure 2C the loop itself does not seem to be resolved even at the Cα backbone level.
|
other
| 97.94 |
The new Figure 2—figure supplement 2 shows domain 4 of PLY in the cryoEM (green) and x-ray structure from the inside of the ring. Differences between the undecapeptide structure in the soluble and pore forms of PLY are surprisingly small. The closest distance between Trp433 and Asp403 or Thr405 is 4-5 Å.
|
study
| 100.0 |
The stereo image of Figure 2—figure supplement 3 (see response to comment 1 above) shows the interactions of the D4 loops between two monomers. These interactions and their consequences are now discussed in more detail in the revised manuscript in paragraph three of the Discussion section.
|
other
| 99.9 |
4) A major finding of this work is that domain 2 collapses but does not lose its structure (Results section). Earlier EM studies suggested it does lose its structure but modeling and FRET studies suggested otherwise. More discussion of this controversy is required. Importantly, the electron density of this key region should be shown in a figure to satisfy the reader that the structure has been maintained. (Figure 2E is not convincing enough. A stereo with side-chains focused on D2 only would be easier to evaluate). Some new detailed insights are discussed; namely, the remodeled helix-turn-helix motif and helix alpha3a. Can stereo figures of the electron density with side-chains displayed of these regions be shown?
|
other
| 98.4 |
In the earlier cryoEM studies no intramolecular details were resolved and the rotation of domain 2 was interpreted as a “collapse”. To propose a rotation of domain 2 would have been more plausible but was not suggested. The FRET study (Ramachandran et al. 2005, PNAS) was interpreted in terms of a bending of domain 2 to account for the height change observed by AFM (Czajkowsky et al. 2004, PNAS). Our present cryoEM structure now shows that both interpretations were incorrect. We rewrote the corresponding text passage (Discussion section paragraph three) that now includes a reference to the FRET study. The AFM study had already been cited.
|
study
| 98.75 |
5) The electron cryo-tomography of the prepore reveals a structure of 34 protomers in the prepore, a number expected based on numerous other reports. However, the CryoEM structure of the pore reveals a larger 42 protomer pore. This is unexpected but interestingly the authors note a range of ring structures were present with this being the most stable. So the question arises whether the pore structure is an artifact.
|
study
| 99.94 |
We do not think that the numbers of subunits we found in the PLY prepore and pore are at all unexpected. Tilley et al. (Cell, 2005) reported 31 ± 3 monomers in the PLY prepore, and two classes with 38 or 44 monomers in the PLY pore, in excellent overall agreement with our findings. Both the prepore and pore of Tilley et al. were in liposomes, so there can be no question that the number of subunits in our detergent-solubilized PLY pores is artefactual.
|
study
| 100.0 |
Can the authors model a 34 protomer pore based on their 42 protomer structure and convince that the details they discuss in the 42 protomer pore are likely relevant to the smaller pore? Is the 20º incline in the β-hairpin maintained in modeling the smaller pore?
|
other
| 99.56 |
It must have escaped the reviewer’s notice that we already built a 34-protomer model into the smaller pore of the subtomogram average, and that this was shown in Figure 6E of the original manuscript. No significant modification of the pore subunit, in particular not of the 20° incline of the β-hairpins relative to the membrane normal, was necessary to fit 34 copies of the pore-forming subunit into the 27 Å-resolution cryoET map. This is now pointed out more clearly in subsection “Three stages of PLY pore formation” of the revised manuscript
|
other
| 99.7 |
A description of how the linear arrays of the various x-ray structures relate to the prepore and pore form of PLY, and how this fits our detailed mechanism of pore formation can be found in our recent AFM paper (van Pee et al., Nano Lett 2016; supporting figure S2 and text relating to it).
|
study
| 99.94 |
6) The authors have skirted around another controversial issue of whether arcs and incomplete rings observed by other workers are physiological relevant as they are not observed in this work. Some discussion of why PLY does not form such structures in their hands but do in others would be insightful.
|
other
| 99.9 |
We certainly have no intention of skirting around this issue. Arcs and slits of PLYwt are actually visible in Figure 7A. This figure was there in the original manuscript, but we now point them out specially with coloured arrows. However, in contrast to LLO, PLY forms mostly complete rings.
|
other
| 99.94 |
7) Subsection “Three stages of PLY pore formation”: In the previous AFM study (van Pee et. Al, Nano Letters, 2016) only 13% of the lower height rings were pre-pores on supported lipid membranes. This could be mentioned to indicate that the occurrence of a "late prepore" is rare event even on membranes without curvature.
|
study
| 99.94 |
8) The literature review, particularly in the Discussion, has been narrowly focused to a large degree on the PLY literature and should be broadened out to other CDCs, particularly to PFO where there is an extensive literature on how it forms pores. Does the current study support the major findings in the PFO literature and does it disagree with any aspects?
|
review
| 99.9 |
Notwithstanding their high degree of sequence identity and similar monomer and oligomer structures, the different CDCs seem to follow different pathways of pore formation. Of the 21 papers on pore-forming toxins that we cite in our manuscript, more than half deal with PFO. Any more discussion of PFO papers would distract from the main focus of our work, which is on PLY.
|
other
| 98.5 |
9) Parts of the Discussion should be moderated. The limited resolution and a few static snapshots do not reveal all the details of membrane insertion and pore formation. Such details will come gradually from a host of biochemical and biophysical approaches. The suggestion that the CryoEM structure forms a good basis for drug discovery to treat numerous Gram-positive infections is not supported by any results in the manuscript so should be restricted to a speculative claim about S. pneumoniae.
|
other
| 95.2 |
The cryoEM structure of the PLY pore complex shows clearly the interactions of the secondary structure elements of neighboring protomers. The structure further shows how elements of secondary structure present in the soluble form refold and take on a completely different structure in the membrane. While the formation of the long membrane-inserted β-hairpins was predicted as such, no one could know or predict the exact structure of the β-strands and how they interact to form the membrane-perforating barrel. Other structural elements, including the inner α-barrel and the newly formed helix in domain 1, are entirely new. These elements are involved in inter- and intramolecular interactions in the pore complex. Even at 4.5 to 5 Å resolution there can be no reasonable doubt of their existence and interaction. Targeting these regions with newly developed drugs would be an elegant way to inhibit pore formation and infection, and thus an effective means of putting these pathogen out of action. We think that our discussion of this aspect of the PLY structure is reasonable and adequate.
|
study
| 99.9 |
1) Over-interpretation of lower-resolution parts of the map in terms of the atomic model. Although the new supplementary figures now reveal the limitations of the map more clearly, over-interpretation in the text still remains a problem. Specifically, W433 is mentioned to move wrt the crystal structure, yet there is no density visible for W433 in Video-1, Figure 2—figure supplement 2 or 3 to support this statement. The map in this region is ~6Å. The same can be said for H407 that does also not have clear density. The authors should stay away from over-interpretation in this region and simply state that this domain is likely to contribute to the inter-molecular interactions that stabilize the pore, as they have done in the Discussion section, paragraph three.
|
other
| 98.2 |
As explained in our first response letter, the location of most residues in domain 4, which hardly changes its conformation upon membrane insertion, is predefined by the crystal structure of the soluble PLY monomer. Although the resolution of the cryoEM map in the loop regions is lower, there is little room for misinterpreting the loop conformations. Nevertheless, we changed the manuscript text as requested by the reviewers.
|
other
| 99.9 |
“The close proximity of loop β18/19 that contains Asp403, Thr405, and His407 of one monomer to loop β22/23 and the uncedapeptide containing Trp433 of the adjacent monomer suggests a critical role of these loops not only in receptor recognition, but also in oligomer formation.”
|
study
| 98.5 |
“The conserved Trp433 in the undecapeptide loop seems to form a polar interaction with Asp403 and Thr405 in the loop that connects β18/19 in the neighbouring monomer, and Try461 interacts with His407 of the next monomer, most likely by π-stacking (Figure 2—figure supplements 2, 3).”
|
study
| 99.9 |
Likewise, there is no obvious density that connects D168 and K271 (Figure 5 instead shows connecting density between β-strands that is expected at resolutions lower than 4.7A). The resolution in this area is again ~6-7Å according to the local resolution estimation. The authors should be more cautious and can suggest that a salt bridge may exist as evident by the point mutant D168A, which has an 80% reduction in hemolytic activity.
|
study
| 99.9 |
“[…]and (3) an ion bridge between Asp168 and Lys271 in adjacent b-strands of the pore barrel (Figure 5G). Even though the hairpins at the ends of the trans-membrane b-strands are the least well-ordered part of the ring (Figure 1—figure supplement 3), there is significant map density for the salt bridge connecting them (Figure 5G).”
|
study
| 99.94 |
“[…]and (3) ionic interactions between charged sidechains in adjacent β-strands of the pore barrel (Figure 5G). In particular, Asp168 and Glu170 in β–strand β7 are in a good position for forming a salt bridge with Lys271 in β–strand β10 of the next-door monomer (Figure 5G).”
|
study
| 99.94 |
2) The comment in the Discussion section: "The 4.5 Å cryoEM structure of the PLY pore complex thus paves the way towards the design of new drugs that inhibit toxin oligomerization and pore formation as a promising approach towards combating infections by wide-spread and dangerous Gram-positive pathogens" jars with the reply "the different CDCs seem to follow different pathways of pore formation…. Any more discussion of PFO papers would distract from the main focus of our work, which is on PLY." Therefore, the authors should focus the therapeutic claim focus to diseases that PLY has shown to contribute.
|
other
| 91.4 |
We apologize for not making this clear. As far as anyone knows, the mechanism of membrane insertion by rearrangement of the conserved domains 1 – 4 is the same for all CDCs. What we meant was that the exact pathways by which the membrane-inserted toxin monomers assemble into pores may differ between species. While most CDCs form circular pores, a small number of bacteria, for example Listeria monocytogenes or Streptococcus pyogenes, give rise to slit-like or arch-shaped pores. However, the structure of the membrane-inserted monomers in these slit-like or arch-shaped pores would be essentially the same as the membrane-inserted form of PLY. Therefore, compounds that interfere with refolding and membrane insertion of soluble monomers would prevent infection by Streptococcus pneumoniae and other CDC-producing Gram-positive bacteria. We have modified the last paragraph of the revised manuscript as follows:
|
other
| 52.25 |
“The mechanism of membrane insertion by rearrangement of the conserved domains 1 – 4 is likely to be the same for all CDCs. Therefore, compounds that interfere with refolding and membrane insertion of soluble monomers would prevent infection by Streptococcus pneumoniae and other CDC-producing Gram-positive bacteria that attack human cells with similar pore-forming toxins.
|
study
| 76.44 |
The curative treatment modalities for hepatocellular carcinoma (HCC) include liver transplantation, surgical resection (SR), and radiofrequency ablation (RFA). Although liver transplantation is the best treatment option for patients with HCC, SR and RFA are most commonly considered first-line treatments because of the graft shortage. Numerous studies suggest both SR and RFA are comparable in terms of long-term survival for patients with early stage HCC. The 5-year survival rates are 42%–56% for SR and 42%–70% for RFA [1–6]. However, even in patients who underwent liver transplantation, tumor recurrence is not uncommon with a 10%–15% risk of recurrence . The 5-year recurrence rate after SR is 42%–52% [8–11], which is higher up to 42%–70% after RFA [12, 13]. Therefore, how to manage recurrent HCC is important in improving the survival and merits further evaluation. Of all the recurrence patterns, intrahepatic recurrence is the most common and the size of the recurring HCC is usually smaller than that of the initial HCC because the surveillance interval is shorter . Accordingly, the treatment options do not particularly differ between recurrent HCC after SR and primary tumor. Liver transplantation is still recognized as the better choice for recurrent HCC , but its wide application has been limited by the shortage of donors. Repeated SR for recurrent HCC has been reported to be an effective treatment option with a comparable survival rate to that of primary SR [16–18], but its feasibility is limited by small liver remnants, poor liver function reserve, or technical difficulties owing to expected postoperative adhesion [19–21]. RFA, as a nonsurgical, less invasive, and repeatable therapeutic approach, is safer and causes less damage in treating recurrent HCC following primary resection [22–24]. As to the choice of repeated SR and RFA, although several studies have compared the clinical outcome of RFA vs. SR for recurrent HCC, no clear recommendation has yet been established. Many of the studies were limited by small case numbers or large tumor size [25–31] and there remain many controversies regarding the choice of repeated SR and RFA in treating recurrent tumor after primary resection. This study aimed to compare the efficacy, safety, and long-term survival of repeated SR and RFA for recurrent HCCs.
|
review
| 95.7 |
From January 2002 to September 2014, a total of 530 consecutive patients with early and intermediate stage HCC underwent hepatic resection at the Division of General Surgery, Department of Surgery, Kaohsiung Veteran General Hospital. At the time of the data collection and analysis, 271 patients were free of tumor recurrence, 235 patients had intrahepatic recurrence, and 24 patients had extrahepatic recurrence. For matching comparison between repeated surgical resection and RFA, patients with fewer or equal to 3 recurrent tumors and each tumor size less than or equal to 3cm were enrolled. The detailed inclusion and exclusion of patients were shown in Figure 1. Among the 235 patients with intrahepatic recurrence, 100 patients with fewer or equal to 3 recurrent tumors and each tumor size ≦ 3cm received either repeated SR or RFA as the secondary treatment, and these patients were enrolled in our study The data of patient characteristics, clinicopathologic features, and survival outcomes were reviewed.
|
study
| 100.0 |
In our hospital, all patients received computed tomography (CT) or magnetic resonance imaging (MRI) of the liver within one to two months after primary hepatectomy of the HCC to confirm complete tumor clearance. Thereafter, surveillance for recurrent HCC consisted of measurements of serum alpha-fetoprotein (AFP), liver biochemistry, and ultrasonography, CT scan, or MRI scans of the liver every three months. Intrahepatic recurrence was defined as a new lesion with arterial contrast enhancement and portal venous washout. If there was a new lesion less than 1cm and without typical HCC imaging pattern on contrast-enhanced CT or MRI scans, we arranged image studies every three months. If there was a new lesion more than 1cm and without a typical HCC imaging pattern on contrast-enhanced CT or MRI scans, we would arrange a needle biopsy for histological confirmation.
|
study
| 99.9 |
Neither SR and RFA were adopted for recurrent HCC treatment if a patient had any symptoms and signs of irreversible liver decompensation and severe portal hypertension such as jaundice, ascites, encephalopathy, PTINR >1.5x, the presence of severe varix, and thrombocytopenia with a platelet count < 50 x× 109/Cumm. Repeated hepatic resection was considered if a patient had a single tumor or tumors within a monosegment of liver with good liver function reserve. Repeated hepatic resection was generally avoided if patients had gross ascites, an indocyanine green (ICG) retention rate of more than 20% at 15 minutes and/or a serum total bilirubin level of more than 1.5 mg/dL, or the presence of moderate esophageal varix. RFA was generally selected in patients with Child-Pugh class A or B disease, prothrombin time ratio of more than 50%, and platelet count of more than 50 000/mm3 (50 × 109/L). RFA also was considered if the indocyanine green (ICG) retention rate was more than 20% at 15 minutes or recurrent tumors in a deep-seated intraparenchymal location where anatomical resection would remove more than one segment of liver, resulting in insufficient liver reservation. CT guided RFA was more preferred than sono-guided RFA if the tumor size was small and invisible by sonogram, difficult to approach due to altered anatomy, and tumors in high-risk locations, which was defined as tumors less than 5 mm adjacent to the hollow viscera, big bile duct, gallbladder, diaphragm, liver capsule, liver hilum, heart, major portal or hepatic vein.
|
other
| 99.7 |
Continuous variables were expressed as medians and interquartile ranges and were compared using the Mann–Whitney U-test. Categorical variables were compared with the χ2 test or Fisher’s exact test when appropriate. Overall survival, disease-free survival, and cumulative incidence of second recurrence of the two study groups were estimated by the Kaplan-Meier method. Comparison of survival between groups was performed with the log-rank test. Disease-free survival following treatment of the first recurrence of HCC was defined as the period from the date of treatment of the first recurrence of HCC to the date of the second tumor recurrence or death. Overall survival after treatment of the first recurrence of HCC was defined as the period from the date of treatment of the first recurrence to the date of death related to any cause. Univariate and multivariate analyses of prognostic factors with the overall survival were evaluated by step wise forwards Cox’s regression analysis. A p value ≤ 0.05 was considered significant. Statistical analysis was performed using the SPSS 19.0 computer software program.
|
study
| 100.0 |
Table 1 shows the patient and tumor characteristics of the initial HCC. The age, gender ratio, incidence of comorbidity, and positive rates of viral hepatitis, and laboratory data were comparable in both groups. In the repeated SR group, 35 (81.4%) patients had Child-Pugh class A cirrhosis and 1 (2.3%) had Child-Pugh class B cirrhosis, whereas 50 (87.7%) patients in the RFA group had Child-Pugh class A cirrhosis (p = 0.41). In each group, 7 patients did not have cirrhosis, which was based on radiological finding and Ishak score of non-tumor part in the surgical pathology. The two groups had similar clinico-pathological features of initial HCC in terms of tumor size, tumor number, distribution of tumor location, proportion of tumor BCLC staging, histological grade of tumor cell differentiation, and presence of microvascular invasion. Most patients had laparotomy segmentectomy or wedge resection for treatment of the initial HCC, and only four (7%) patients in the RFA group received a lobectomy.
|
study
| 99.94 |
With the regards to the recurrent HCC (Table 2), the median time from initial resection to first recurrence was longer in the repeated SR group, but the there was no statistically significant difference (26 vs. 14 months, p = 0.06). The clinical characteristics of patients and the recurrent tumor were not significantly different in the two groups including laboratory data, tumor size, tumor number, and tumor staging. In the repeated SR group, 41 (90.7%) patients underwent segmentectomy and four (9.3%) wedge resections. In the RFA group, most (81%) procedures were performed via a CT guided approach, and 46 (81%) patients received one session of RFA, whereas 11 (19%) needed two sessions of RFA for complete treatment of recurrent tumors.
|
study
| 100.0 |
Table 3 showed the postoperative and long term outcomes of patients receiving repeated SR and RFA for the treatment of the first recurrence of HCC. Seven (16%) patients developed a total of 10 operation related complications in the repeated SR group, and one (2%) patient experienced a major complication: death due to bile duct injury with bile leakage and septic shock. In the RFA group, four (7%) patients developed a total of six procedure related complications, and there was no complication related mortality. The incidences of treatment related morbidity (p = 0.14) and mortality (p = 0.25) did not differ in the two groups. The total hospital day was longer in the repeated SR group (13 vs. 5 days, p < 0.05). The overall treatment response rates were similar (repeated SR 100% vs. RFA 98%, p = 0.67). At the time of analysis, 30.2% of patients over the median follow up time of 53 months in the repeated SR group and 28.1% patients over the median follow up time of 54 months in the RFA group remained free of a second recurrence. The median time from the treatment of recurrent HCC to developing a second recurrence was 11 months in the repeated SR group and 10 months in the RFA group (p = 0.74). Intrahepatic recurrence was the most common recurrence pattern in both groups. For the treatment of the second recurrence of HCC, 55% of patients received curative treatment in the repeated SR group, whereas 67% of patients received curative treatment in the RFA group.
|
study
| 99.94 |
The 1-, 3-, 5-year overall survival rates after treatment of the first recurrence of HCC were 97.6%, 82.7%, 56.4% in the repeated SR group and 98.2%, 77.2%, 52.6% in the RFA group (P = 0.69) (Figure 2). The 1-, 3-, 5-year disease-free survival rates were 57.0%, 32.1%, 28.6% in the repeated SR group and 60.8%, 26.6%, 16.6% in the RFA group (P = 0.89) (Figure 3). The 1-, 3-, 5-year cumulative incidences of the second recurrence after treatment of the first recurrence of HCC were 41.7%, 67.1%, 70.8% in the repeated SR and 28.1%, 72.9%, 79.7% in the RFA group (P = 0.94) (Figure 4). In the Cox’s regression analysis, with regard to overall survival, the histologic grade of the initial tumor (I vs non-I), AFP level at the time of tumor recurrence, time from initial resection to 1st recurrence, and time from treatment of 1st recurrent HCC to 2nd recurrence were significant prognostic factors at univariate analysis; HBsAg positive, age at the time of tumor recurrence, the histologic grade of the initial tumor (I vs non-I), AFP level at the time of tumor recurrence, time from initial resection to 1st recurrence, and time from treatment of 1st recurrent HCC to 2nd recurrence were significant prognostic factors at multivariate analysis (Table 4).
|
study
| 99.94 |
Our study suggested RFA achieves long-term survival outcomes similar to repeated SR in small recurring HCCs less than 3cm after primary resection. Previous studies comparing the efficacy of repeated RFA vs. resection after recurrence of HCCs usually also enrolled recurring tumors of more than 3cm in size (25, 28, 30, 31). Actually, following surgical resection of the primary tumors, intensive screening was usually applied and the recurring tumors were usually smaller than 3cm and the recurrent HCCs in our study were relatively small: 46% were smaller than 2cm, which was more like the real situation in the recurrence of tumors. A recent meta-analysis reported RFA was associated with lower disease-free survival rates, however, our study we found both SR and RFA achieved a similar outcome for recurrent HCCs.. This might be explained by the inclusion criteria whereby the recurrent HCCs in our study were relatively small. As expected, a smaller tumor size is closely related to a higher rate of complete tumor elimination after RFA and a greater safety margin with fewer non-tumorous liver parenchyma resections in repeated SR, so the outcomes will be better. Besides, most RFA were performed under sonogram guidance in previous reports, but most (81%) patients received RFA under CT guidance in our study because the tumor size was usually small and some were not visible by sonogram. Altered anatomy after resection also contributed to difficulty with US guided RFA. Some experts suggested CT-guided RFA provides better detection of RFA lesions, margin discrimination, immediate ablation zone evaluation, and few artifacts , which may achieve better complete tumor ablation and better outcomes. Indeed, the RFA treated group in our study had a better 5-year overall survival rate and disease free survival rates than several previous studies [25–28]. Although in a recent study, Lee et al. found that either US or CT guided RFA was comparable for treatment naïve HCC , there was no study that compared the efficacy and safety of US or CT in the guidance of RFA for recurrent HCC after primary resection. This interesting problem merits further investigation in the future.
|
study
| 99.9 |
In our study, both repeated SR and RFA completely eliminated recurrent HCC and achieved near 100% response rates. However, a second recurrence of HCC is not uncommon. Chan et al showed 72.4% of patients in the repeated SR group and 84.4% of patients in the RFA group developed a second recurrence with a similar median time from treatment of the first recurrence to second recurrence (6.3 vs. 9.5 months, p = 0.25). Our study showed 79.8% of patients in the repeated SR group and 71.9% of patients in the RFA group developed a second recurrence with a similar median time from treatment of the first recurrence to second recurrence (11 vs. 10 months, p = 0.74).
|
study
| 99.94 |
The improvement in liver function evaluation, surgical technique and perioperative care and decrease in postoperative morbidity and mortality make it possible for more patients to receive surgical resection [16, 35, 36]. However, surgical resection of recurrent HCC is still challenging due to small liver remnants, poor liver function reserve, intra-abdominal adhesion from previous surgery, and the tumor location being adjacent to major vascular or biliary structures. Although the latest literatures suggest repeated SR is considered a favorable and important curative treatment for recurrent HCC, postoperative complications, and in particular hepatic failure, are not uncommon and can not be overlooked . A meta-analysis study showed repeated SR for recurrent HCC was associated with a higher procedure related morbidity rate compared with RFA . Our study showed the procedure-related complication rates were higher in the repeated SR group than that in the RFA group (16% vs. 7% p = 0.14). One patient with repeated SR had major complications and died of bile duct injury with bile leakage and septic shock. RFA is a minimally invasive procedure and can be performed percutaneously either by ultrasound or CT guided approach. Compared with repeated SR via the open or laparoscopic approach, RFA is a highly target-selective thermal treatment technique to conserve non-tumorous liver parenchyma and minimize the degree of surgical insult to the limited liver reserve. Apart from surgery, RFA can be performed under conscious sedation and has a shorter hospital day, making it more cost-effective than surgical resection [2, 38]. In our study, median total hospital stay for patients who underwent RFA was significantly shorter than for those who underwent repeated SR (5 vs. 13 days, p < 0.05). Further, the characteristics and benefits of less invasiveness and highly-targeted tumor treatment improved the feasibility of patients and repeatability of RFA for recurrent HCC. Our study showed more than one third of patients in both groups underwent loco-regional treatment for a second recurrence of HCC, whereas 8% of patients in the RFA group and 27% of patients in the repeated SR group were amenable to surgical resection of a second recurrence of HCC. The aforementioned beneficial factors of RFA make it more safe and feasible in treating recurrent HCC after hepatectomy.
|
study
| 98.8 |
Previous studies reported the serum albumin level, serum alpha fetoprotein level, recurrent tumor size, time interval from primary hepatectomy to first recurrence, and time interval from treatment of recurrent HCC to second recurrence were significant prognostic factors to overall survival [23, 25, 28]. A shorter time interval from treatment to recurrence, is associated with poor prognosis. The finding of our study is consistent with the result of previous report by Albert C.Y. Chan, et al in 2012 . The univariate and multivariate analyses in our study also showed similar findings, except recurrent tumor size was not related to overall survival. Our finding can be explained by the relatively small recurrent tumor size in our study and complete elimination rate of recurrent tumor increase with a decreased possibility of the presence of satellite nodules which decrease early recurrence and improve overall survival. Moreover, the presence of HBV infection and younger age at the time of tumor recurrence were significant factors of better overall survival at univariate analysis. Although an acceptable long term survival is reachable in carefully selected elderly patients [40, 41], patient age is theoretically related to the post-treatment complication and disease morbidity and is always more relevant for the prognosis than many tumor and treatment factors. However, the reason why the presence of HBV infection was also a favorable factor was unknown. It was probably because all HBV patients with indication (45.3%) in our study had anti-viral agent treatment, which had been proved to reduce HBV related long-term complications and lead to better prognosis .
|
study
| 99.94 |
The limitation of our study included non-randomization. Although this was not a randomized study, the baseline characteristics including the ICG retention rate at 15 minutes at initial diagnosis of HCC and at recurrence of HCC after surgical resection between the two groups of patients were similar. A prospective randomized controlled trial to compare the efficacy or repeated RFA and surgical resection for recurrent HCCs after surgical resection is required.
|
other
| 82.94 |
Blood donor screening practices for the hepatitis B virus (HBV) infections vary from country to country both in terms of the manufacturers of the reagents and the testing methods. Over the past 30 years, the risk of TT-HBV has markedly decreased due to the development of more sensitive hepatitis B surface antigen (HBsAg) tests, the introduction of screening for antibodies against the hepatitis B core antigen (anti-HBc) in some countries, the use of nucleic acid tests (NAT), and improved volunteer donor recruitment processes [1–3]. In recent years, the government of China has taken several measures to improve the safety of the blood supply, which included promulgating a new blood donation law in 1998 and revising standard protocols for donor screening and donation screening processes in 2012 . However, in China, the risk of TT-HBV still remains higher than that of other routinely screened viruses, such as HCV and HIV. When using only HBsAg tests for HBV infection screening, the risk of TT-HBV is high because donors may appear HBsAg-negative but are actually HBV infected, such as those currently in the window period (WP) or at the late stage of infection. This is especially true for the areas with both a high prevalence of HBV and a lack of NAT screening . Also, there is an additional risk associated with chronic OBI. OBI is usually defined (in the blood donor screening context) as an HBV infection without detectable HBsAg, usually presenting itself as anti-HBc positive and, typically, with low levels of HBV DNA. Liang et al. reported that the prevalence of HBsAg in the general population in China fell to 7.2% from 9.8% after the implementation of vaccination against hepatitis B , which has played an important role in decreasing the rate of HBV infection.
|
review
| 99.5 |
The residual risk (RR) is different with different blood screening strategies. Using NAT for donor screening can shorten the window period and identify occult HBV infections (OBI) which cannot be detected by HBsAg tests . In terms of the risk for TT-HBV, it is a problem particularly in the countries and/or areas with both a high prevalence of HBV and where NAT for HBV is not used routinely for donor screening . Despite the implementation of donor screening by NAT, there are still a number of countries where there is a residual risk for TT-HBV [9–11].
|
review
| 99.3 |
China still is a developing country with most areas undeveloped economically. At present, most blood centers cannot afford NAT, thus most donations are routinely screened with two ELISA tests for HBsAg in China. This issue has been brought to the attention of the relevant authorities of the Chinese Government, which have decided to pilot NAT testing for HBV, HCV, and HIV in all provincial blood centers in 2015 during blood donor screening . This study aims to evaluate the residual risk (RR) of HBV infections in China before the implementation of NAT. This study will provide helpful data to assess the effectiveness of the implementation of NAT in China. Available data about the prevalence, incidence, and RR of HBV infections among Chinese blood donors is limited. In 2013, Wuping Li and his colleagues reported their findings on the prevalence, incidence, and residual risk of HBV infections in the Anhui Blood Center from 2009 to 2011 . However, that study was based on the data from one single blood center, which is the limitation of that study.
|
study
| 99.94 |
The present study involves six blood centers located in different regions of China (Fig. 1).Hopefully this study was therefore more representative. The aims of the present study were to evaluate the current prevalence, incidences and RR of HBV infections of blood donors among the six blood centers, and to provide guidance for developing and monitoring evidence-based blood donor management strategies to improve the safety of the blood supplies regarding the RR of TT-HBV infections.Fig. 1Geographic Distribution of the Six Blood Centers and the Institute of Blood Transfusion
|
study
| 99.94 |
This study was approved by the Ethics Committee of the Institute of Blood Transfusion, of the Chinese Academy of Medical Sciences & Peking Union Medical College. Written informed consent was obtained from each study participant before the interview, sample collection and testing.
|
other
| 99.94 |
This study was a collaborative effort between the Institute of Blood Transfusion (IBT) of the Chinese Academy of Medical Sciences and six blood centers located in different regions of China. The six Chinese regional blood centers were the Anhui Blood Center (Hefei, Anhui, located in the east), Fujian Blood Center (Fuzhou, Fujian, located in the south), Dalian Blood Center (Dalian, Liaoning, located in the north), Changzhi Blood Center (Changzhi, Shanxi, located centrally), Kaifeng Blood Center (Kaifeng, Henan, located centrally) and Mianyang Blood Center (Mianyang, Sichuan, located in the west). Figure 1 shows the geographic distribution of the six blood centers. The study population consisted of all blood donors who donated at one of the six participating blood centers or at one of their mobile blood collection vehicles between July 1, 2014 and June 30, 2015. All blood donations were screened with the serological tests for HIV, HBV, HCV and syphilis. All samples of HBsAg reactive screening were sent to the IBT national reference laboratory for blood donor testing, and then were subjected to the HBsAg neutralization test and anti-HBc tests. Residual risks could be calculated by the HBV yield approach as Li et al. described .
|
study
| 99.94 |
Following the “Technical and Operational Guidelines and Procedures for Blood Centers” issued by the Chinese Ministry of Health on December 31, 2011 , all six blood centers had the same approach for blood donor screening, requiring all blood donors to pass a routine pre-donation screening process that consisted of a medical history questionnaire, a brief physical examination, and pre-donation rapid screening. The medical history questionnaire included questions about their histories of sexually transmitted diseases, hepatitis, illegal parenteral drug use, sex with multiple partners, and men who had sex with men (MSM). If any of the above items screened positive, the donors were permanently deferred. The physical examination included body temperature, body weight, and blood pressure. Before blood collection, all donors underwent rapid testing at the collection sites for the hepatitis B surface antigen (HBsAg, Rapid Test Kit, Aikang Bio-technology Co., Ltd., Hangzhou, China) as well as rapid testing for Alanine Aminotransferase (ALT) (ALT Rapid Test Kit, Rongsheng Biological Pharmaceutical Co., Ltd., Shanghai, China) and hemoglobin (Hb) (Hemoglobin Assay Kit, Amyjet Scientific Inc., Wuhan, China). The donors with increased ALT levels or a reactive HBsAg result would be temporarily deferred, the samples and information were saved if donors tested HBsAg positive on the rapid test.
|
study
| 99.9 |
All successful donations were subjected to tests on two different HBsAg assays by an enzyme-linked immuno sorbent assay (ELISA). If both testing on the two different HBsAg assays were reactive, the screening test was defined as reactive. If the results from either one of two tests was reactive, the sample was retested in duplicate on the same assay and/or another appropriate kit. The screening test was defined as reactive if either one or two positive reactive results were obtained during retesting of the HBsAg ELISA tests. If both tests showed non-reactive results, the screening test was defined as non-reactive, and the corresponding donation was qualified for transfusion.
|
study
| 99.94 |
All test kits were approved and licensed by the Chinese State Food and Drug Administration (or Food and Drug Administration, FDA). The reagents used for donor screening tests are listed in Table 1, and the assays were performed following the manufacturer’s instructions.Table 1Screening Test Kits for HBsAg Used at Each Blood CenterBlood CenterFirst AssaySecond AssayTest KitSensitivity (%) / Specificity (%) / Limit (ng/ml)Test KitSensitivity (%) /Specificity (%) / Limit (ng/ml)AnhuiWANTAI (Beijing)99.5 / 98.5 / 0.03BIO-RAD (United States)100 / 97.55 / NDFujianBIO-RAD (United States)100 / 97.55 / NDWANTAI (Beijing)99.5 / 98.5 / 0.03DalianInTec (Xiamen, China)100 / 98.6 / NDBIO-RAD (United States)100 / 97.55 / NDChangzhiBIO-RAD (United States)100 / 97.55 / NDInTec (Xiamen, China)100 / 98.6 / NDKaifengBIO-RAD (United States)100 / 97.55 / NDWANTAI (Beijing)99.5 / 98.5 / 0.03MianyangWANTAI (Beijing)99.5 / 98.5 / 0.03BIO-RAD (United States)100 / 97.55 / ND ND no data WANTAI Beijing WANTAI Biological Pharmacy Enterprise Co., Ltd. BIO-RAD BIO-RAD Clinical Diagnostics InTec Xiamen InTec Biological Pharmacy Enterprise Co., Ltd.
|
other
| 99.06 |
All HBsAg screening reactive samples, including pre-donation rapid screening and after-donation reactive testing by ELISA, were sent to the IBT national reference laboratory for donor testing where they were tested for HBsAg, via the ELISA test (MONOLISA TM HBsAg ULTRA, BIO-RAD, California, USA), and anti-HBc antibodies (HBcAb ELISA Kit, Beijing WANTAI Biological Pharmacy Enterprise Co. Ltd., Beijing, China). When the signal to cutoff ratio (S/CO) for the HBsAg test was greater than or equal to 1.0, the ELISA for HBsAg tests were considered as reactive, after which they were confirmed with a neutralization assay (Reagent Kit for the Confirmation of the HBV Surface Antigen, ZHUHAI LIVZON DIAGNOSTICS INC., Zhuhai, China). If there was a positive confirmed result by neutralisation, the sample was confirmed as HBsAg-positive; if a negative neutralisation result was obtained, the samples were considered HBsAg-negative. The reagents used for donor screening tests in IBT are listed in Table 2, and the testing algorithm is listed in Fig. 2 . Table 2Screening Test Kits Used for IBTKit NameCompanySensitivity / Specificity (%)Diagnostic Kit for HBVBIO-RAD (United States)100 / 97.55Surface Antigen (ELISA)HBsAg Neutralization,Livzon (Zhuhai)no dataAntibody to Hepatitis BSurface Antigen (Human)Diagnostic Kit forWANTAI (Beijing)99.4 / 99.1Antibody to HepatitisCore Antigen (ELISA) Fig. 2Testing Algorithm. Note: §: We do not take into account the risk from OBI. HBsAg: Hepatitis B surface Antigen. HBc antibody: Hepatitis B core antibody. ELISA: Enzyme-Linked Immuno Sorbent Assay. IBT: Institute of Blood Transfusion. S/CO: the signal to cutoff ratio
|
study
| 100.0 |
Testing Algorithm. Note: §: We do not take into account the risk from OBI. HBsAg: Hepatitis B surface Antigen. HBc antibody: Hepatitis B core antibody. ELISA: Enzyme-Linked Immuno Sorbent Assay. IBT: Institute of Blood Transfusion. S/CO: the signal to cutoff ratio
|
other
| 99.94 |
The demographic characteristics of all donors among the six blood centers were collected and analyzed (Table 3) according to and including donor status (first time donors vs repeat donors), gender, age, occupation and education. First-time donors were defined as donors who had no record on file according to the blood center databases. Repeat donors were donors who had a previous record in the databases of the blood centers.Table 3Demographic Characteristics of all Donors Across Six Blood Centers from June, 2014 to July, 2015Blood CentersAnhuiDalianChangzhiKaifengMianyangFujianDonors Status First-Time60,812(62.26%)38,503(49.75%)10,643(32.43%)40,227(67.11%)18,889(41.52%)43,384(51.92%) Repeat36,856(37.74%)38,892(50.25%)22,174(67.57%)19,711(32.89%)26,605(58.48%)40,170(48.08%)Gender Male65,105(66.66%)48,496(62.66%)20,790(63.35%)38,942(64.97%)27,742(60.98%)50,124(59.99%) Female32,563(33.34%)28,899(37.34%)12,027(36.65%)20,996(35.03%)17,752(39.02%)33,430(40.01%)Age Group 18~2542,212(43.22%)32,597(42.12%)2701(8.23%)11,688(19.50%)9313(20.47%)28,450(34.05%) 25~3527,328(27.98%)21,421(27.68%)7269(22.15%)15,740(26.26%)10,150(22.31%)21,933(26.25%) 35~4518,762(19.21%)15,439(19.95%)13,199(40.22%)19,234(32.09%)16,005(35.18%)20,989(25.12%) 45~558907(9.12%)7536(9.74%)8765(26.71%)11,976(19.98%)9276(20.39%)10,854(12.99%) 55~60459(0.47%)402(0.52%)883(2.69%)1301(2.17%)751(1.65%)1329(1.58%)Occupation Farmers5801(5.94%)3243(4.19%)12,270(37.39%)16,405(27.37%)5687(12.50%)2565(3.07%) Workers10,206(10.45%)12,027(15.54%)5907(18.00%)11,286(18.83%)4841(10.64%)2423(2.90%) Students23,020(23.57%)18,103(23.39%)1414(4.31%)5580(9.31%)4599(10.11%)14,647(17.53%) Soldiers1845(1.89%)859(1.11%)98(0.30%)623(1.04%)641(1.41%)1504(1.80%) Teachers2724(2.79%)410(0.53%)755(2.30%)468(0.78%)1210(2.66%)/ Civil Servants3437(3.52%)186(0.24%)1195(3.64%)336(0.56%)1843(4.05%)4278(5.12%) Doctors4189(4.29%)573(0.74%)597(1.82%)2242(3.74%)3071(6.75%)3175(3.81%) Staff24,026(24.60%)2748(3.55%)2110(6.44%)22,914(38.23%)3662(8.05%)19,518(23.36%) Other22,414(22.95%)39,247(50.71%)8470(25.81%)84(0.14%)19,940(43.83%)35,444(42.42%)Education Below High School18,645(19.09%)22,174(28.65%)15,765(48.04%)21,799(36.37%)14,185(31.18%)31,458(37.65%) High School and42,662(43.68%)21,733(28.08%)14,407(43.90%)30,784(51.36%)22,893(50.32%)23,111(27.66%)Associate Degree Bachelor’s Degree22,044(22.57%)20,726(26.78%)2543(7.75%)5556(9.27%)7670(16.86%)28,525(34.14%) Master’s Degree3340(3.42%)805(1.04%)30(0.09%)1091(1.82%)669(1.47%)460(0.55%) Others10,977(11.24%)11,958(15.45%)72(0.22%)707(1.19%)77(0.17%)/ Sum up97,66877,39532,81759,93845,49483,554 Total396,866
|
study
| 100.0 |
The incidence rate of HBV was determined by using the HBsAg yield approach as previously described . In the process of the HBV infection, HBsAg becomes detectable earlier than anti-HBc. HBsAg may be transient, while anti-HBc can persist for a long time. Therefore, donations confirmed as HBsAg-positive but non-reactive for anti-HBc can be regarded as newly infected cases that are considered to be HBsAg yield cases. The number of HBsAg yield cases divided by the total number of donations is equal to the HBsAg yield rate. The quotient divided by the length of time a patient has been HBsAg-positive prior to anti-HBc seroconversion (termed the HBsAg yield window, calculated as 44 days) gives an incidence estimate for HBV infections among blood donors .
|
study
| 100.0 |
The RR attributable to WP donations was calculated using the following equation:\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \mathrm{HBsAg}\ \mathrm{new}\ \mathrm{infection}\ \mathrm{rate}=\mathrm{Number}\ \mathrm{of}\ \mathrm{HBsAg}\ \mathrm{positive}\ \mathrm{but}\ \mathrm{anti}-\mathrm{HBc}-\mathrm{negative}/\mathrm{The}\ \mathrm{total}\ \mathrm{number}\ \mathrm{of}\ \mathrm{donations}; $$\end{document}HBsAgnewinfection rate=Number of HBsAg positivebutanti−HBc−negative/The total number of donations;the ratio of HBsAg new infection rate = HBsAg new infection rate of first − time donors over HBsAg new infection rate of repeat donors;\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \mathrm{Incidence}\ \mathrm{rate}\ \mathrm{for}\ \mathrm{HBV}\ \mathrm{infection}=\mathrm{HBsAg}\ \mathrm{new}\ \mathrm{infection}\ \mathrm{rate}/\mathrm{HBsAg}\ \mathrm{yield}\ \mathrm{window}\ \left(44\ \mathrm{days}\right); $$\end{document}Incidence rate forHBVinfection=HBsAgnewinfection rate/HBsAg yield window44days; \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \mathrm{RR}=\mathrm{incidence}\ \mathrm{rate}\ \mathrm{for}\ \mathrm{HBV}\ \mathrm{infection}\times \mathrm{window}\ \mathrm{period}\ \left(0.16\ \mathrm{years}\ \mathrm{or}\ 59\ \mathrm{days}\right); $$\end{document}RR=incidence rate forHBVinfection×window period0.16years or59days;
|
study
| 99.94 |
Data were statistically analyzed using computer software (SPSS 17.0, SPSS, Chicago, IL). An approximate 95% confidence interval (95% CI) was obtained using the Poisson distribution model. The Chi-square test was performed to assess the association between the categorical variants. A P-value of <0.05 was used as the cut-off level for significance.
|
study
| 99.94 |
From July 1, 2014 to June 30, 2015, a total of 558,089 blood donations were collected at six blood centers located in different areas of China (see Figure 1). The demographic characteristics of all of the donors at the six blood center were collected, and the distributions of all demographic characteristics were examined (see Table 3). Among the Anhui, Dalian and Fujian Blood Centers, almost 60% of all donations came from donors aged between 18 and 35 years old, while almost 40% at Changzhi, Kaifeng and Mianyang blood centers were of this age group. Across all of the blood centers, the proportions of male and female donors were 63% and 37%, respectively. Donors with a high school education or less contributed to 70% of all donations. Overall, more than half of the donors were employees and students. There were significant differences in the constitutions of donors in terms of their demographic characteristics across the six blood centers in this study.
|
study
| 100.0 |
In this study there were 558,089 donor samples, of which there were 1664 donor samples found to be HBsAg reactive. All of the 1664 samples were tested for anti-HBc and HBsAg and confirmed by neutralisation, and those that were HBsAg positive and anti-HBc positive totaled 484, while those that were HBsAg positive and anti-HBc negative totaled 94; those that were HBsAg negative and anti-HBc positive totaled 326, and those that were HBsAg negative and anti-HBc negative totaled 760. 746 of the 1664 were repeat donors, the mean of interdonation interval for repeat donors was about 11.9 months, and the interdonation interval data for all 746 repeat donors is shown in Table 4 . Table 4The interdonation interval data for all 746 repeat donorsThe interdonation interval (month)6-9(including 9)9-1212-1515-1818-2121-24>24 TotalDonors3391946234415719,746Mean(month)11.9
|
study
| 100.0 |
Overall, the serologic prevalence of confirmed HBV infections from first-time and repeat donations is shown in Tables 5 and 6. In all, 578 samples were HBsAg positive, confirmed by neutralization assay. The overall prevalence was 0.15%. The seroprevalence of HBsAg was estimated to be 0.13%, 0.078%, 0.16%, 0.07%, 0.20%, and 0.25% at the Hefei, Dalian, Changzhi, Kaifeng, Mianyang and Fujian Blood Centers, respectively. The prevalence of HBsAg varied significantly among the six blood centers, and the prevalence in Fujian was much higher (0.25%) than all of the other blood centers. By contrast, the prevalence in Kaifeng was the lowest (0.07%) among all of the blood centers. Of all 578 reactive samples, 520 (98.5%, 520/528) were from first-time donors.Table 5HBsAg Prevalence by Blood Center / Bank Among all Donors (First-Time and Repeat)Blood CenterDonations(Total)First-TimeRepeatPrevalence(%)(Among all Donors)DonationsNumber of HBsAgConfirmed PositivePrevalence(%) (95% Confidence Intervals)DonationsNumber of HBsAgConfirmed PositivePrevalence(%) (95% Confidence Intervals)Hefei97,66860,8121150.19 (0.15-2.23)36,856100.027 (0.01-0.04)0.13 (0.11-0.15)Dalian77,39538,503520.14 (0.09-0.17)38,89280.02 (0.01-0.04)0.08 (0.06-0.09)Changzhi32,81710,643400.38 (0.26-0.49)22,174120.05 (0.02-0.08)0.16 (0.12-0.20)Kaifeng59,93840,227360.09 (0.06-0.12)19,71140.02 (0.004-0.04)0.07 (0.05-0.09)Mianyang45,49418,889770.41 (0.32-0.50)26,605120.05 (0.02-0.07)0.20 (0.16-0.24)Fujian83,55443,3842000.46 (0.40-0.52)40,170120.03 (0.013-0.05)0.25 (0.22-0.29)Total396,866212,4585200.24 (0.22-0.27)184,408580.03 (0.02-0.04)0.15 (0.13-0.16) Table 6HBV Prevalence for Blood Donors Above and Below 35 Years OldBlood CenterDonationsBelow 35Over 35 P-valueTotal PrevalenceDonationsNumber of HBsAg confirmed positivePrevalenceDonationsNumber of HBsAg Confirmed PositivePrevalenceHefei97,66869,540380.06%28,128870.31%<0.0010.13%Dalian77,39554,018160.03%23,377440.19%<0.0010.08%Changzhi32,81712,671110.09%20,146410.20%0.010.17%Kaifeng59,93827,428120.04%32,510280.09%0.0450.07%Mianyang45,49419,463210.11%26,031680.26%<0.0010.20%Fujian83,55450,383550.11%33,1711570.47%<0.0010.25%Total396,866233,5031530.07%163,3636250.38%<0.0010.20%
|
study
| 100.0 |
In order to estimate HBV incidence accurately, an HBsAg yield window of 44 days was used as described in a previous study . The ratio of HBsAg new infection rate of donations from first-time donors to repeat donors was 6.97, 6.57, 6.94, 4.41, 9.04 and 15.43, at the Hefei, Dalian, Changzhi, Kaifeng, Mianyang and Fujian Blood Centers, respectively. Overall, the estimated incidence rate was 213.44, 161.59, 989.80, 278.05, 125.31 and 352.19 (per 100,000 person-years) in Hefei, Dalian, Changzhi, Changzhi, Kaifeng, Mianyang and Fujian, respectively (see Table 7).Table 7HBsAg Incidence by Blood Center/ Bank Among First-Time and Repeat DonorsBlood CenterFirst-TimeRepeatRatio ofa HBsAg new infection rateYield Window(year)Incidence(per 105 py)Number of HBsAgConfirmed Positive, Anti-HBc NegativeIncidence(per 105 py)Number of HBsAgConfirmed Positive, Anti-HBc NegativeYield Rate(per 105 py)Incidence(per 105 py)Hefei13315.3925.4345.256.970.12213.3Dalian36281.5225.1442.856.570.12161.59Changzhi52347.34940.59338.236.940.12989.80Kaifeng10372.89210.1584.564.410.12278.05Mianyang2261.1453.4728.899.040.12125.31Fujian6640.1924.9841.1915.430.12352.19Total72773.492211.9399.427.780.12197.38 aThe HBsAg new infection rate in repeat donors is the HBsAg+/HBc antibody- prevalence, and the incidence for HBV infections in first-time donors is equal to the incidence for HBV infections in repeat donors multiplied by the ratio of HBsAg new infection rate (the ratio of HBsAg new infection rate = HBsAg new infection rate of first-time donors over HBsAg new infection rate of repeat donors). The overall incidence for HBV infections in all donors is the percentage of incidence in first-time donors plus the percentage of incidence in repeat donors. For Hefei, 315.18 × 10−5 × 0.6226 (Number of first-times / number of first-times + number of repeats = 60,812 / 97,668) + 45.22 × 10−5 × 0.3774 = 196.23 × 10−5 + 17.07 × 10−5 = 213.3 × 10−5)The residual risk calculation model used in this study is one of the more suitable computational models in the absence of nucleic acid detection. Based on the results of the last two blood tests, it is possible to extrapolate whether the recent donors have a new infection. However, for the first-time donors, the new infection ratio cannot be determined or calculated directly. Therefore, this model is adopted to indirectly establish the incidence and residual risk of the first-time donors
|
study
| 100.0 |
aThe HBsAg new infection rate in repeat donors is the HBsAg+/HBc antibody- prevalence, and the incidence for HBV infections in first-time donors is equal to the incidence for HBV infections in repeat donors multiplied by the ratio of HBsAg new infection rate (the ratio of HBsAg new infection rate = HBsAg new infection rate of first-time donors over HBsAg new infection rate of repeat donors). The overall incidence for HBV infections in all donors is the percentage of incidence in first-time donors plus the percentage of incidence in repeat donors. For Hefei, 315.18 × 10−5 × 0.6226 (Number of first-times / number of first-times + number of repeats = 60,812 / 97,668) + 45.22 × 10−5 × 0.3774 = 196.23 × 10−5 + 17.07 × 10−5 = 213.3 × 10−5)
|
study
| 99.94 |
The residual risk calculation model used in this study is one of the more suitable computational models in the absence of nucleic acid detection. Based on the results of the last two blood tests, it is possible to extrapolate whether the recent donors have a new infection. However, for the first-time donors, the new infection ratio cannot be determined or calculated directly. Therefore, this model is adopted to indirectly establish the incidence and residual risk of the first-time donors
|
study
| 100.0 |
The residual risk of HBV infection was estimated by applying the refined infectious window-period estimate of 0.16 years (or 59 days) to the derived incidence estimates. The residual risk of HBV infection among blood donations from the participating blood centers is shown in Table 8 . The estimated residual risks were 34.14, 25.85, 158.35, 44.48, 20.04 and 56.35 (per 100,000 person-years) in Hefei, Dalian, Changzhi, Kaifeng, Mianyang and Fujian, respectively. The residual risk of Dalian was the highest, approximately 6 times that of Fujian, which had the lowest residual risk.Table 8HBsAg Residual Risk by Blood Center/ Banks Among First-Time and Repeat DonorsBlood CenterFirst-timeRepeatWindow Periodsin YearOverall(per 105 py)Incidence(per 105 py)Residual risk(per 105 py)Incidence(per 105 py)Residual risk(per 105 py)Hefei315.3950.46 (6.13-182.04)45.257.24 (0.88-26.12)0.1634.14Dalian281.5245.04 (5.46-162.62)42.856.85 (0.83-24.75)0.1625.85Changzhi2347.34375.57 (64.40-721.85)338.2354.12 (9.28-104.01)0.16158.35Kaifeng372.8959.66 (7.22-215.38)84.5613.53 (1.64-48.84)0.1644.48Mianyang261.1441.78 (3.98-65.31)28.894.62 (0.44-7.22)0.1620.04Fujian640.19102.43 (12.39-369.78)41.196.59 (0.80-23.96)0.1656.35Average703.08112.49 (15.04-187.96)96.8315.49 (1.93-24.16)0.1656.53
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study
| 100.0 |
In China, most donations were routinely subjected to screening for HBsAg by ELISA twice. The remaining few blood centers screen HBV infection by ELISA(×1) plus NAT. However, due to the transient nature of the HBsAg detectable period during the HBV infection process, and also due to the lack of routine screening testing for anti-HBc antibodies, the residual risk of HBV infection has remained high in China [8, 13, 16]. In order to develop an evidence-based, efficient and safe blood donor screening strategy and/or policies to decrease the RR of HBV infections, it is essential to have epidemiological information regarding the prevalence, incidence and RR of HBV infections and the associated demographic characteristics of both high-risk and low-risk populations of voluntary blood donors. The demographic characteristics of volunteer blood donors in different blood centers in China were quite different. For example, the percent of the first time donors among the six blood centers ranged from 32.43% to 67.11% of all donations. The proportion of young donors 18 to 25 years old was 43.22% of all donations at the Anhui Blood Center, but only 8.23% at the Changzhi Blood Center. Meanwhile, the proportion of middle aged donors 35 to 55 years old was 28.33% of all donations at the Anhui Blood Center, but was 66.93% at the Changzhi Blood Center. There were also some differences in the occupations and education levels at the different blood centers. For instance, 37.39% of donors were farmers, 18.00% were workers and 4.31% were students at the Changzhi Blood Center, while these proportions were 3.07%, 2.9% and 17.53%, respectively, at the Fujian Blood Center. 91.94% of donors had either received an Associate’s degree, a high school diploma or below at the Changzhi Blood Center, while this figure was 56.68% at the Dalian Blood Center. However, some similarities were noted among different blood centers regarding the gender of the donors; namely male donors comprised the majority of donors (with a mean of 61%) at all of the blood centers. In addition, more than half of all donors at all of the blood centers were under 35 years old in this study except for those at the Changzhi Blood Center (see Table 3).
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study
| 100.0 |
The differences found in the demographic characteristics at different blood centers may have contributed to the differences of estimated residual risks at the blood centers. A higher RR was found at Changzhi (158.35 per 100,000 donations per year), while the lowest was found at Mianyang (20.04 per 100,000 donations per year) (see Table 7). According to the characteristics of the blood donors, most of the blood donors were first-time donors, and only Changzhi had the most repeat blood donors, reaching 67.57%. Theoretically, and according to published data, repeat donors have a lower RR for infectious diseases [12, 14]. However, the results of this study showed that the HBV incidence and residual risks at Changzhi were the highest. With regards to the age of the blood donors, most of them were under the age of 35, but most (70%) donors at Changzhi were over 35 years old. China began nationwide hepatitis B vaccinations in 1992, so the residual risk of donors under 35 years old is lower than that of donors above 35 years old. Perhaps this is thus a major cause of Changzhi having the highest residual risk. This data also proved that the HBV vaccine in China has been successful, and it shows that hepatitis B vaccination can effectively reduce residual risks. Second, the proportion of farmers at the Changzhi Blood Center was 37.39%, much higher than the proportions at the other five blood centers. (These proportions ranged from 3.07% at the Fujian Blood Center to 27.37% at the Kaifeng Blood Center. See Table 3). Due to economic barriers and other multivariate factors, farmers had the lowest vaccination rate, which again could have led to their higher susceptibility to HBV infections. Third, the proportion of donors without a high school education was relatively high at the Changzhi Blood Center. These donors may have had a relatively low level of health knowledge, which also could have accounted for their elevated rates of HBV infection. The findings in this study are very interesting, because the evidence above indicates that the evaluation of residual risks should take into consideration not only the proportions of first time and repeat donors, but also their ages, occupations and education backgrounds.
|
study
| 99.94 |
TT-HBV can still occur after transfusion, even though the carrier blood has tested negative for HBsAg. Apart from occult HBV infections, there remains a limitation in the model that was used to estimate the residual risk of HBV, because neither the window period nor the incidence of donor HBV infections is precisely known. Thus, theoretically, the actual infection risk may be underestimated, which might not accurately reflect infectivity. However, the estimated result of Li’s model can still be used to evaluate the safety of blood supplies.
|
study
| 99.94 |
Some countries and/or regions have low prevalence of HBV infections, such as Canada and Hong Kong. These two locations exhibit low residual risks of 1:1,700,000 and 1:22,000 , respectively, whereas China has a moderate-to-high residual risk for HBV infections (about 1:13,670 in Shanghai) . Although the prevalence of HBsAg in the Chinese population has dropped to 7.2% in 2006 from 9.8% since the implementation of the nationwide HBV vaccination program in 1992, the prevalence of HBV infections in China is still high [7, 8, 13, 18]. Continued efforts are still needed in donor education, improving donor recruitment and screening strategies. The incidence window period model used in this study can also be used to evaluate the potential impact of HBV NAT implementation by calculating the expected percentage residual risk reduction and yield of a particular assay system . Per the request of the Chinese Ministry of Health, NAT testing for HBV, HCV, and HIV will have been piloted in all provincial blood centers by 2015 . This may decrease the residual risk of TT-HBV markedly.
|
study
| 100.0 |
This study may underestimate the incidence of HBV infections due to its definition of an infection incident and because its risk estimates do not take into account the risk from OBI. The numbers of HBsAg positive and anti-HBc negative tests in each region are low, leading to a general lack of precision in RR calculations (see the wide 95% CIs). In terms of limitations, the incidence calculations are sensitive to assumptions around the window period. In addition, the study may have a number of other limitations around window period estimates. Although, this study included six different locations of blood centers from a diversity of geographical areas, it still may not be representative of all of China.
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study
| 100.0 |
Despite the introduction of more sensitive assays in blood donor screening, our data revealed that the current residual risk of transfusion-transmitted HBV infection is still high (overall 56.53 per 105 py). To improve the safety of blood supplies, we need to continue to educate blood donors and improve donor recruitment and screening strategies. NAT for blood donor screening is needed in China, which may markedly decrease the RR for HBV infections and improve the safety of blood supplies. It is worth considering testing for anti-HBc during blood donor screening in China.
|
study
| 88.5 |
In 2012, breast cancer (BC) accounted for 25% of all cancer cases worldwide and 15% of all cancer deaths among females.1 Late-stage BC is often associated with an immunosuppressive microenvironment and lacks of antitumor immune responses. Natural killer (NK) cells mediate direct cytotoxic activity against tumor cells and provide an early host defense against the transformed cells.2 NKG2D is one of the most important NK cell-activating receptors, which is also expressed on a subset of CD8+ T cells, γ/δ T cells, NK1.1+ T cells and lymphokine-activated killer (LAK) cells.3, 4, 5 Ligands for NKG2D receptors (NKG2DLs) comprise major histocompatibility complex class I chain-related proteins A and B (MICA/B) and unique long 16 (UL16) binding proteins 1–6 (ULBP1–6).6 NKG2D–NKG2DL stimulation of NK cells leads to strong activation and tumor cell rejection.7, 8, 9 However, malignant cells decrease their surface expression of NKG2DLs through downregulation and/or internalization10 as well as the shedding of NKG2DL extracellular domains.11 The downregulation of NKG2DL prevents the detection of malignant cells by immune cells, although the underlying mechanisms remain unclear.
|
study
| 99.6 |
MicroRNAs (miRNAs) are short non-coding RNA molecules that usually repress gene expression by binding to the 3'-untranslated region (3'-UTR) of their target mRNAs. Increasing evidence indicates that miRNAs have important roles in tumor formation and immunogenicity.12, 13, 14 A group of miRNAs was predicted to target the mRNA of the NKG2DLs by the TargetScan database.15 Previous study found that miR-20a, miR-93, miR-106b, miR-373 and miR-520d could repress MICA and MICB expression by binding to the mRNA 3'-UTRs in human cancer cells (mainly HeLa, 293T, DU145 cells) and normal cells (human foreskin fibroblasts and human umbilical vein endothelial cells).16 Paula Codo et al.17 found that miR-20a, miR-93 and miR-106b contributed to the immune evasion of glioma cells by inhibiting MICA/B. These data led us to question whether miRNAs contribute to the post-transcriptional regulation of NKG2DL expression and how they influence the immunogenicity in the context of BC.
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study
| 99.94 |
Histone deacetylase inhibitors (HDACis), as important reagents in epigenetic therapy, have shown promising effect in clinical trials for the treatment of human malignancies.18 Suberoylanilide hydroxamic acid (SAHA) and valproic acid (VPA) are potent HDACi drugs that have been approved by the United States Food and Drug Administration to treat cutaneous T-cell lymphoma and epilepsy, respectively. Both SAHA and VPA display antitumor activity in vivo with a favorable pharmacological profile and well-tolerated side effects.19, 20 In addition, HDACis might sensitize malignant cells to NK cell recognition depending on NKG2D–NKG2DL signaling.21, 22 These data suggest that HDACis may serve as a new and tumor-selective drug class by enhancing immune surveillance in the treatment of BC.
|
review
| 95.1 |
In the present study, we found that the high expression of MICB, which is an important NKG2DL, was an indicator of good prognosis in BC. Next, we characterized the important role of the miR-17–92 cluster in MICA/B and ULBP2 regulation and the functional impact of the miR-17–92 cluster on the BC immunogenicity. Furthermore, HDACis were found to enhance NK cell recognition in a miRNA-dependent manner.
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study
| 100.0 |
MICA/B protein was rarely detected in the normal breast tissues of BC patients (84.4% showed negative MICA/B expression). However, in BC tissues, 92.2% showed positive MICA/B expression (Figures 1a and b). The MICA/B mRNA expression level was detected less in normal breast tissues than in paired BC tissues (Supplementary Figure 1a). Together, these results showed that the expression of MICA/B was higher in BC tissues than in normal breast tissues.
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study
| 100.0 |
We found that the protein expression of MICA/B was significantly higher in BC cells at early stages (Tumor Node Metastasis (TNM) stages I and II) than in BC cells at advanced stages (TNM stage III) (Figures 1a and b). In addition, this inverse correlation was confirmed between MICA/B mRNA expression and the TNM stages by quantitative PCR analysis (Figure 1c). However, no correction was found between MICA/B expression and the WHO grade of BC (Supplementary Figure 1b). For ULBP1/2/3, no specific expression patterns were found for different TNM stages or WHO grades of BC (data not shown).
|
study
| 100.0 |
To determine the prognostic value of MICA/B expression in BC, 80 BC patients at the early stages of disease (TNM stages I and II) were divided into two groups according to the median value of MICA or MICB mRNA expression. Kaplan–Meier survival analysis was performed to evaluate the relationship between MICA or MICB expression and patient survival. The results indicated that patients with high expression of MICB had a longer overall survival compared with those with low expression of MICB. However, the results showed no significant difference in the overall survival between the two groups with different MICA expression (Figure 1d). Importantly, we found similar results in the early-stage BC cohort from The Cancer Genome Atlas (TCGA, TNM stages I and II) (N=855) with a long-term follow-up period (up to 20 years) (Figure 1e). Similar results were obtained if we included advanced-stage BC patients in the calculation above (Supplementary Figure 1c and 1d). Taken together, these results suggest that MICB might act as an important predictive factor in BC.
|
study
| 99.94 |
The TargetScan database predicted numerous miRNAs with binding sites in the 3'-UTR of different NKG2DL members. Among them, miR-20a, miR-20b, miR-93 and miR-106b belong to the miR-17-92 cluster and its paralogs, which share a similar seed sequence. Members of the miR-17-92 cluster were reported to downregulate MICA/B in other carcinomas.16, 17, 23 Thus, we examined miR-20a, miR-20b, miR-93 and miR-106b as NKG2DL-targeting miRNAs in BC.
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study
| 100.0 |
To assess the influence of endogenous miR-17-92 on NKG2DL expression in BC cells, seven human BC cell lines (BCap37, Hs 578T, MDA-MB-231, MDA-MB-468, BT-474, SK-BR-3 and MCF-7) and two human normal breast cell lines (HBL-100 and Hs 578Bst) were studied in parallel for their expression levels of NKG2DL surface proteins and the examined miRNAs. All the examined cell lines expressed NKG2DLs and the tested miRNAs at different levels (Figures 2a and b). In BC cell lines, statistical analysis revealed an inverse correlation between the expression levels of endogenous miR-20a and MICA/B protein expression (Figure 2c). Importantly, in the BC tissues from TCGA's cohort (N=1042), statistical analysis revealed an inverse correlation between the expression levels of endogenous miR-20a and MICA/B mRNA expression, which again pointed toward the involvement of the tested miRNAs in NKG2DL regulation (Figure 2d).
|
study
| 100.0 |
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