Using selection for BFV variants with high cell-free infectivity in bovine and non-bovine cells, infectivity dramatically increased from almost no infectious units to about 105–106 FFU (fluorescent focus forming units)/mL in both cell types. Importantly, the selected BFV variants with high titer (HT) cell-free infectivity could still transmit via cell-cell contacts and were neutralized by serum from naturally infected cows. These selected HT–BFV variants will shed light into virus transmission and potential routes of intervention in the spread of viral infections. It will also allow the improvement or development of new promising approaches for antiretroviral therapies.Virus transmission is essential for spreading viral infections and is a highly coordinated process which occurs by cell-free transmission or cell–cell contact. The transmission of Bovine Foamy Virus (BFV) is highly cell-associated, with undetectable cell-free transmission. However, BFV particle budding can be induced by overexpression of wild-type (wt) BFV Gag and Env or artificial retargeting of Gag to the plasma membrane via myristoylation membrane targeting signals, closely resembling observations in other foamy viruses. Thus, the particle release machinery of wt BFV appears to be an excellent model system to study viral adaption to cell-free transmission by  They also contain additional open reading frames under the control of the 5′-long terminal repeat (LTR) and an internal promoter located in the 3′-end of the env gene [pol gene is encoded by a spliced mRNA. Furthermore, FVs release non-infectious Env-only subviral particles and there is a strict dependence on capsid-glycoprotein interactions for virion release from the cells [Spumaretroviruses, also known as Foamy Viruses (FVs), were initially discovered in 1954 by Enders and Peebles in primary monkey kidney cell cultures . Similarhe cells ,7. Thesehe cells ,8. FVs ahe cells . Due to Bovine foamy virus  is a member of the understudied non-human Spumaretrovirus subfamily and was first isolated from cattle in 1983 . Though in vitro, leading to Env-independent SVP release [Unlike orthoretroviruses, FV particle budding requires the co-expression of Gag and Env and depends on specific interactions between the capsid and the N-terminal Env leader protein Elp ,7. Known release ,27,28. A release .in vitro, eliminating any effects arising from co-adaptation to heterologous host cells [Viral transmission from infected to non-infected cells occurs by cell-free transmission, involving the release of particles into the extracellular space, or cell-cell contact ,31. Virust cells .First, BFV-Riems replication in primary calf trachea cells was examined for BFV gene expression, cyto-pathogenicity, particle release, cell-cell transmission, and neutralization by serum factors. Next, we showed that the BFV-Riems budding machinery is similar to that of other FVs. This led us to select for BFV variants with enhanced cell-free transmission in bovine and non-bovine cultures by serial passaging. Interestingly, cell-free high titer (HT) BFV still transmits by cell–cell transmission and is neutralized by serum from naturally infected cows. The experimental system described here will extend our basic knowledge on FV particle release and viral transmission. This knowledge may contribute to the optimization of vectors for applications in cancer gene- or immunotherapy. A better understanding of virus transmission may also enhance our ability to intervene with the spread of viral infections and enable improvement or development of promising novel approaches for antiretroviral therapies.The BFV strain BFV-Riems  was obtaThe baby hamster kidney cell line BHK-21 (ATCC CCL-10) and Madin-Darby bovine kidney (MDBK) cells were grown in DMEM supplemented with 10% fetal horse serum (FHS) and 1% penicillin-streptomycin solution and used for cell-free BFV serial passaging and selection. The BFV indicator cell line (BICL) and MDBK indicator cell line (MICL) for BFV were derived from BHK and MDBK cells, respectively, and contained a green fluorescent protein (Gfp) expression cassette under the control of the BFV long-terminal repeat (LTR) promoter . Both weThe original BFV-Riems isolate was initially propagated by co-cultivation of infected and uninfected KTR cells. Non-adherent cells were removed after 24 h and the cultures were passaged at 1:3 ratios until massive CPE appeared. Once CPE was evident, the cells were harvested or, for propagation of the virus, fresh cells were added.To test the influence of serum on BFV produced in bovine KTR cells, infected cells were propagated in DMEM supplemented with 0%, 1%, and 10% of FCS, FHS, Serum Replacement Solutions 1, 2, and 3 , and Serum Replacement Solution , according to the manufacturer’s instructions, or in OptiMEM .6 BFV-infected KTR cells and supernatant were frozen in liquid nitrogen and thawed on ice for 30 min. This process was repeated three times. After removal of cell debris by centrifugation at 3000 rpm for 10 min, the resulting virus supernatants were filtered through 0.45 μm filters and used to infect new MDBK or BHK-21 cells. Supernatants from 80%–100% infected cells were filtered and added to new target cells during serial BFV passaging and in vitro evolution.BFV was adapted to use cell-free transmission in MDBK and BHK-21 cells by serial passaging using cell-free culture supernatants rather than cell co-cultivation. Aliquots of 6 × 104 cells/well). After 2 h, BFV-containing supernatants were serially diluted 1:6 on the cells. After five days, the supernatants were replaced by phosphate-buffered saline (PBS). BFV titers were determined by life cell fluorescence microscopy for Gfp signals .BFV titers were determined using MICL or BICL cells. Indicator cells were seeded in 96-well plates (1 × 104 to 101 fluorescent focus forming units (FFU)/mL and incubated with serially diluted cow sera 11 and 33  for 30 min and subsequently inoculated on sub-confluent MICL and BICL cells. The inoculum was replaced after 4 h and the cultures were supplemented with normal 10% FHS-DMEM. Viral titers were determined after five days by fluorescence microscopy.For serum neutralization assays, late passage HT–BFV derived from BHK-21 or MDBK cells was serially diluted from 104) method. In a 10 cm dish, a total amount of 10 μg plasmid DNA was diluted in a total volume of 450 µL double-distilled water (ddH2O). A mixture of plasmid DNA in water and 45 µL of 2.5 M CaCl2 was prepared and added dropwise to 450 µL of 2 x HEPES-buffered saline  under constant gentle shaking [2, the medium was replaced with fresh growth medium and the cells incubated for an additional 36 h.The day before transfection, 293T cells were seeded at a density of 20%–30%. The next day, cells were transfected with plasmid DNA by using the calcium phosphate  and separated by ultracentrifugation in a SW41Ti rotor  at 4 °C for 2 h at 28,000 rpm. After removing the medium and sucrose, the non-visible pellet was resuspended in 30 μL 1% SDS in PBS containing complete protease inhibitor cocktail  and stored at −20 °C before immunoblotting.For indirect immunofluorescence (IIF), BFV-infected KTR cells were seeded on glass coverslips. Five days post-infection (p.i.), the cells were washed with PBS and fixed in ice-cold 100% methanol/0.02% ethylene glycol tetraacetic acid (EGTA). Polyclonal BFV Gag-MA (matrix domain of Gag) specific hyper-immune serum was used as the primary detection serum ). As the secondary antibody, Alexa-594 or Alexa-488-coupled anti-rabbit immunoglobulin IgG antibodies (diluted 1:2000 in PBS with 3% BSA) were used. The respective antibody was incubated for 60 min on the coverslip followed by three 10-min PBS wash steps. For nuclear staining, Hoechst 33342 was used (1:1000 dilution) concurrently with the secondary antibody. Coverslips were finally washed in double-distilled water and ethanol before mounting on glass slides.BFV-infected cells were harvested and processed for transmission electron microscopy (TEM) as described previously .® software .BFV-infected MICL cells were harvested at different time points. The cells were washed three times in PBS  and strained before measurement by flow cytometry. Acquisition was performed using a Becton Dickinson FACSCalibur  and data analyzed using FlowJoTo extract viral DNA, BFV-infected cells were washed with PBS, transferred into an Eppendorf tube using a cell scraper, pelleted in a micro-centrifuge, and resuspended in an appropriate volume of PBS. DNA was extracted using a DNeasy Blood and Tissue Kit  following the manufacturer's instructions.gag and env genes were amplified using the high-fidelity Phusion® DNA polymerase  using the primers listed in To construct eukaryotic BFV expression vectors, BFV gag was replaced by PCR-amplified BFV gag using AgeI and BamHI resulting in clone pBC-BFV-Gag-oPRE. The plasmid pBC-Gag-oPRE was subsequently used as the backbone to construct the Gag myr-sub and myr-added clones using AgeI and BamHI. BFV env was cloned to the pBC12 backbone using HindIII and SmaI.The cytomegalovirus immediate-early (CMV-IE) promoter-based FFV Gag expression construct pBC-Gag-oPRE was used as backbone for the BFV Gag expression construct, utilizing an optimized post-transcriptional regulatory element (oPRE) from the woodchuck hepatitis virus and a non-coding heterologous sequence providing the splice-donor (SD) and splice-acceptor (SA) sites . FFV gag® Nuclease . Protein samples were boiled at 95 °C for 1–3 min in SDS loading buffer with containing reducing agent . Samples were separated by SDS-PAGE followed by transfer to a nitrocellulose membrane. BFV proteins were detected using polyclonal hyper-immune sera specific against Gag-MA (matrix domain of Gag) or Bet (full protein) (1:5000 dilutions in PBS with 3% BSA). Anti-rabbit IgG conjugated with horseradish peroxidase was used as secondary antibody. Signals were visualized using the enhanced chemiluminescence (ECL) detection system . Likewise, BFV-Riems Gag and Bet were detected using bovine reference sera of BFV100-infected cattle (provided by Jacek Kuzmak) and protein G conjugated to horseradish peroxidase as a secondary antibody.Cells were washed with PBS, lysed by adding an appropriate amount of lysis buffer  and transferred into an Eppendorf tube using a cell scraper. Genomic DNA was digested with 250 units of BenzonaseThe original BFV–Riems isolate from KTR cells was characterized for gene expression, particle release, serum neutralization, and transmission. Since the BFV isolates are known to be highly cell-associated and virus propagation is best performed by co-cultivation of uninfected cells with BFV-infected cells , BFV-RieTo further explore the tight cell association of BFV, TEM was performed to check for aberrant budding, a tetherin-like phenotype with particle trapping, or other hints of a mechanism hindering efficient infectious particle release. We did not find any evidence for altered particle assembly, release and budding, since both intact and budding particles were observed in almost all images .gfp reporter gene cassette were generated using standard methods. A single cell clone, designated MICL, with no basal Gfp expression but strong green fluorescence upon BFV infection, was selected and used for BFV detection and quantification. Titers were determined by measuring fluorescence five days post-infection/titration.To sensitively and conveniently monitor BFV infectivity, a new bovine cell-based reporter cell line was constructed. Similar to the BHK-21-based BICL BFV titration cells , bovine Although fetal sera should not contain substantial amounts of maternal or fetal pathogen-specific antibodies, we analysed whether BFV particles were neutralized by antibodies in the bovine serum used for KTR cell culture. For this purpose, BFV-Riems-infected KTR cells were grown in FCS-containing medium. Cells displaying an obvious BFV-specific CPE were washed and further incubated in medium containing 0%, 1% or 10% of either FCS or FHS, four different serum replacement reagents, or OptiMEM. Infected KTR cells and supernatants were harvested three days after the medium change. Serial dilutions of both cells and supernatants were used to infect sub-confluent BICL and MICL titration cells grown in serum-free OptiMEM medium. After two days, the inoculum was replaced, the cells were washed once and grown for five days in standard cell culture medium before titration by Gfp auto-fluorescence. Data from both titration cell lines show that the different media supplements had variable effects on BFV production and viability of BFV-infected KTR target cells while the supernatants mostly reflected BFV titers in the cell-bound fraction (data not shown). In summary, growth in serum-free cultures did not increase cell-free BFV titers, which strongly implies a lack of BFV neutralization by antibodies in FCS/FHS. Therefore, BFV titers are mainly determined by the overall influence of media on BFV production and cell viability but not by antibodies present in the medium/serum.gag and env genes were cloned and expressed from sub-genomic expression plasmids and functionally analyzed. BFV gag and env were amplified via PCR by taking A3 8000 and genomic DNA from BFV-infected KTR as templates, respectively. A3 8000 is a plasmid containing an approximately 8 kb fragment of the BFV genome, from the 5’-end to the middle of BFV genome. This fragment was amplified by long-template PCR taking genomic DNA from BFV-infected KTR as template [gag gene was replaced by the corresponding BFV sequences using standard cloning procedures  and non-bovine, BFV-permissive hamster cells (BHK-21) to compare differences in the adaptive process and potentially distinguish between co-adaptations to the different host cell species and the HT cell-free transmission phenotype. Selection in the two cell lines was performed in duplicate by two persons to examine the reliability and applicability of the selection procedure.Since the particle budding machinery of the original BFV-Riems isolate showed similarity to that of other FVs, we aimed to select HT cell-free infectious BFV variants (HT–BFV) As starting material, KTR-associated BFV particles were harvested by repeated cycles of freezing and thawing of cells and culture supernatants. Lysates were cleared by low-speed centrifugation, filtered through a 0.45 µm filter, and applied to MDBK and BHK-21 cells. Under these conditions, infected cells were initially split several times before BFV-specific CPE appeared. From cell cultures with enhanced CPE in more than half the cells or more than 80% Gag positivity, as determined by IIF, supernatants were harvested, cleared by low speed centrifugation, and filtered through a 0.45 µm filter before inoculation of new MDBK and BHK21 target cells. During serial passaging, cell-free infectivity was determined using the BFV indicator cell lines MICL and BICL cells, respectively.6 FFU/mL for BHK-21 cells and 105 FFU/mL for MDBK cells , and BP10, BP62, and BP230 from BHK-21 cells  were analyzed by TEM. Capsids embedded in electron-dense material were frequently observed in the cytoplasm in early and middle passage HT–BFV. However, extracellular virions were rarely detected in the early samples, while moderate numbers of particles were observed in the middle passages. In the late passages, budding and bursts of viral particles were readily observed seen at the plasma membrane of HT–BFV-infected cells. The results demonstrated an increase in particle release with BFV selection A. To valFormation of multinuclear syncytia is a common feature of FVs in many infected cultures. In BFV-permissive KTR cell, FV-specific syncytia are easily detectable by phase contrast microscopy  and Since we detected a loss of syncytia formation of selected HT–BFV at passage 18 in MDBK and passage 24 in BHK-21 cells, we examined whether loss of syncytia induction was also detectable in KTR cells. Antigen expression, subcellular distribution, and syncytia formation in KTR cells infected with the original BFV-Riems isolated by co-cultivation and KTR cells infected by cell-free BFV particles from passage 30 in BHK-21 cells were analyzed by IIF using Gag MA antiserum. While Gag expression and sub-cellular localization did not change dramatically, BFV-specific CPE of large multinucleated giant cells was completely lost at higher passage numbers C.4 to 101 FFU/mL, incubated with serially diluted cow sera 11 and 33 for 30 min, and inoculated on sub-confluent MICL and BICL cells. The inoculum was replaced after 4 h and the cultures supplemented with normal 10% FHS-DMEM. Titers were determined after five days by fluorescence microscopy. Both HT-BFV were neutralized by sera from BFV infected cows 11 and 33  and were selected for the neutralization assays. Late-passage HT–BFV derived from BHK-21 and MDBK were serially diluted from 101 and 33  using HTTo determine whether HT–BFV can still spread via cell–cell contact, the efficiencies of cell-free and cell-cell transmission were measured. It was important to establish experimental conditions that allowed an approximate comparison of both modes of transmission. Cell-free transmission is more effort- and time-intensive than cell-cell transmission. Early after a new infection, viral infection of target cells is mediated mainly by cell-to-cell spreading, with little or no contribution from cell-free viral transfer, which is significantly less efficient ,36,42. Bin vitro selected BFV while the original BFV–Riems only spread via cell–cell transmission whereas cell-free transmission was almost undetectable throughout the whole time frame. As expected, BFV from late passages had better cell-free infectivity at 48 h compared to those from passage 22. Moreover, more time was required for complete infection of the culture by cell-free spread (70 h) than by cell-cell transfer (48 h). Overall, the data confirm a lack of cell-free transmission for the original BFV–Riems, while BFV with increased cell-free infectivity retained its ability to spread via cell–cell transmission.At 24 h post-infection, BFV infection by cell-free and cell-cell transmission was barely visible B. At 36 in vitro. Unlike the simian and feline FVs, BFV spreads pre-dominantly by cell-cell transmission and “filopodia-like” structures  may be utilized for BFV transmission. By using a rigorous in vitro evolution procedure, HT cell-free transmitted BFV was selected and characterized with special emphasis on gene expression, gross antigenic changes, and mode of transmission.In the current study, we set out to characterize and functionally analyse the BFV–Riems isolate in infected cultures with special emphasis on the low or almost non-existing cell-free virus transmission of all known BFV isolates in vitro, the inefficient particle release, the consequence of antibody-mediated immune responses, or anti-viral restriction factors [in vitro cell cultures cannot be formally excluded at present. In the current study, bovine non-transformed, mortal KTR , hamster BHK-21 (Baby hamster kidney fibroblast) and MDBK (Madin-Darby bovine kidney) cells were analyzed. The wt BFV-Riems isolate showed almost exclusively cell-to-cell transmission in all these cells, making a major effect of low level permissiveness of the target cells used here unlikely. In addition, FVs are considered to have a very broad range of permissive target cells, at least in vitro [in vitro evolution screen used here did not select host cells with a higher permissiveness towards BFV but only the serially passaged BFV-Riems isolates grown in the two unselected, parental cell lines. The selection was performed in bovine and hamster cells in parallel. It is especially noteworthy that comparisons between the cell lines showed that the titer of MDBK-selected BFV accelerated more rapidly in the initial period compared to the BHK-21-derived BFV. Similarly, HT cell-free BFV selection required more time and passages to reach a plateau in BHK-21 cells than in MDBK cells. This finding supports the view that BFV required additional genetic alterations to adapt to the heterologous hamster target cells. However, the absolute titers of HT-BFV from BHK-21 cells were higher than those from MDBK cells. The inefficient cell-free transmission of all known BFV isolates in cell cultures may be due to the non-permissiveness or reduced permissiveness of the target cells used  factors ,44,45. B factors ,27,28, c factors . However factors ,14,15,20in vitro . FinallyFurther virological work will be needed to address questions related to cellular restriction factors. Molecular virus genetics and evolutionary studies are required to address all issues related to alterations in viral functions involved in efficient cell-free transmission . Here, vversus free Env occurred and that therefore fusion-competent Env is simply no longer present on the surface of HT-BFV-infected cells.The viral proteins detected by immunoblotting in the cellular lysates show similar steady-state expression levels throughout the selection procedure, indicating that a simple increase in viral protein synthesis did not play a role when selecting for HT-BFV variants. In addition, we did not detect any gross differences in Gag protein sub-cellular localization, although TEM studies indicate increased budding at the plasma membrane in MDBK and BHK-21 cells. Here, the question emerges whether cytosolic trafficking of preassembled or fully assembled capsids described for PFV is affected by any of the induced adaptive changes . While GBFV is highly cell-associated in cell culture with no or only miniscule transmission via cell-free virus . The modin vitro molecular evolution of a specific viral function. In this case, we studied HT cell-free virus release and infectivity, though close relatives and likely also the ancestors of BFV carried this trait. The fact that the HT cell-free transmission phenotype was reproducibly acquired twice in two different cell lines indicates that the required molecular building blocks are still present in the original BFV-Riems isolate for further adaptation [in vitro evolution screen performed here. Instead, alternative or variant solutions to overcome the selection hurdle (cell-free transmission) have likely evolved. Corresponding molecular evolution studies are currently under way to address all these questions.The data presented here confirm that BFV is an excellent model system to study aptation . It is, versus cell-free virus transmission in pathogenic human and animal viruses, the BFV system, with the option to study virus spread and transmission in cattle and sheep, offers an excellent, manageable, reliable and robust animal model to investigate these issues [versus HT cell-free transmission for virus persistence, spread, molecular evolution, and immune detection or clearance within experimentally infected animals would be an excellent model.While there are many unknowns and speculations concerning the advantages of cell–cell e issues  while, fe issues . Compariin vitro evolution. The HT-BFV variants were characterized with respect to different parameters, such as gene expression, replication features and immunogenicity. Comparative genetic studies at different stages of the adaptive process conducted here should allow identification of the molecular mechanisms required for efficient cell-free retrovirus particle release, a process essential for translational studies in the area of retrovirus vector development. It is also of prime importance in understanding virus transmission and pathogenicity. In the current study, we established BFV variants that gained the ability of HT cell-free transmission using


As therapeutic vaccination approaches are explored for cancer therapy, there is a need to identify milestones that can predict the successful induction of sustained immunity. We examined whether the magnitude of immune responses seen prior to vaccination or at the completion of a vaccine series to a tumor-associated self antigen can predict long-term immunity.6 peripheral blood mononuclear cells. Responses were evaluated at baseline study entry, at one month after completion of vaccinations and at long-term follow-up, the last time point available at week 24-36. Correlative statistical analysis was performed using Pearson product-moment correlation coefficients of least-squares regression.Interferon gamma Elispot data was abstracted from patients enrolled in 3 clinical trials (n = 86) utilizing therapeutic vaccination targeting HER2/neu with long-term follow-up. A positive response/immunity was defined as a precursor frequency of greater than 1:20,000 or 50 interferon gamma corrected spots per well/10Although the magnitude of immunity at one month post vaccination and at baseline both correlated with immunity at long-term follow-up, immunity at one month post-vaccination has the stronger correlation:  versus . Immunity at one month post-vaccination predicts positivity at long-term follow-up (AUC = 0.7872). Defining a threshold of greater than 400 corrected spots per well at one month post-vaccination yields a 97% certainty that long-term follow-up responses will be positive. The increases from baseline interferon gamma Elispot responses against HER2/neu were also significantly higher in patients that achieved persistent immunity at long-term follow-up time points (p < 0.05).The magnitude of interferon gamma Elispot responses from peripheral blood mononuclear cells one month after completion of vaccination correlates best with persistent immunity at long-term follow-up time points. Pre-existing levels of immunity also influence the ability of patients to achieve persistent immunity at long-term follow-up with therapeutic vaccination. This knowledge may be exploited to identify the success of targeted cancer vaccines early and allow the implementation of boosters or the employment of vaccines against alternate targets.


A growing body of experimental evidence supports the diagnostic relevance of circulating microRNAs in various diseases including cancer. The biological relevance of circulating microRNAs is, however, largely unknown, particularly in healthy individuals. Here, we propose a hypothesis based on the relative abundance of microRNAs with predominant tumor suppressor activity in the blood of healthy individuals. According to our hypothesis, certain sets of circulating microRNAs might function as a tumor surveillance mechanism exerting continuous inhibition on tumor formation. The microRNA-mediated tumor surveillance might complement cancer immune surveillance. MicroRNAs are encoded by separate genes that are often clustered and undergo a complex maturation process giving rise to 22–25 nucleotide long single-stranded mature microRNA molecules targeting the 3′ non-coding region of messenger RNAs (mRNA) . MicroRNressors) –7.Numerous reports underline the relevance of tissue microRNAs both in normal and pathological conditions. Recent data appear to add a further layer of complexity to the biological relevance of microRNAs, as secreted microRNAs have been found in body fluids  , 9. MicrThe extracellular microRNAs are surprisingly stable and their analysis is possible even from archived body fluid samples. Circulating blood-borne microRNAs are being investigated as potent minimally invasive biomarkers of several diseases including tumors , 14.Despite their clear relevance as disease markers, the biological relevance of blood-borne microRNAs is largely unknown. Circulating microRNAs can be regarded as hormones that convey epigenetic information and might affect gene expression in cells distant to the cellular microRNA source . MembranThe relevance of circulating microRNAs in healthy individuals is, however, entirely unknown. Here, we present a hypothesis on the potential relevance of circulating blood-borne microRNAs.hsa-miR-451 (106-fold higher expression than that of the 20th microRNA in the ranking) [hsa-miR-223 (11 to 338-fold) [hsa-miR-16 (11 to 20-fold) [hsa-let-7f (16-fold) [hsa-miR-335 and hsa-miR-377, in the study by Weber et al. [The circulating microRNA profiles of healthy individuals have been analyzed in some studies and over 270 different microRNAs have been detected with highly variable expression in different studies on serum or plasma samples . By compranking) , hsa-miR38-fold) , 20, hsa20-fold) , 20 and 16-fold)  can be hr et al.  are alsowww.ncbi.nlm.nih.gov/geo). Data of 61 samples from 5 studies have been retrieved (four of these five studies include data from Asian populations). By rank ordering these microRNAs relative to the 20th most abundant microRNA, hsa-miR-451 has emerged among the top-ranked microRNAs in four of the five studies (up to 59-fold overrepresented). Hsa-miR-223 and hsa-miR-16 are also commonly found in three studies among the 20 most abundant microRNAs. Two further microRNAs worth mentioning: hsa-miR-486-5p that is abundant in three independent studies (up to 28.8-fold overrepresented), and hsa-miR-923 that is almost 200-fold overrepresented in one study   Two further microRNAs found to be abundant in our in silico analysis also have tumor suppressor activity: hsa-miR-486-5p in breast [hsa-miR-923 in chronic lymphocytic leukemia [Among these microRNAs, several act predominantly as tumor suppressors. leukemia . hsa-miRicroRNAs . The hsag cancer . Beside icroRNAs , other mculation . We haven et al.  and alson et al. –28. The  context . In glioppressor , 31. Theppressor , hsa-miRc cancer . The moslorectal , gastriclorectal  cancers lorectal . The thin breast  and lungn breast ; hsa-miRleukemia .hsa-miR-21 [It must be noted, however, that among the relatively most abundant microRNAs, microRNAs with predominant oncogenic properties can also be found like a-miR-21  [with nia-miR-21  and alsoThe predominant tumor suppressor activity of the most highly overexpressed microRNAs in the blood is intriguing. Is it possible that these microRNAs could inhibit malignant transformation? By entering transformed cells, tumor suppressor microRNAs targeting genes involved in the regulation of cell cycle, cell proliferation, etc., might lead to decreased proliferation, cell cycle arrest or apoptosis induction Fig. . It is hAs tissue microRNA dysregulation is considered to be an early event in tumorigenesis , alteratAnyway, an extensive research for identifying the mRNA targets of microRNAs isolated from healthy persons and cancerous patients might provide ideas on the tentative tumor suppressive or oncogenic mechanisms of circulating microRNAs, respectively.Apart from this hypothetical reprogramming of nascent tumor cells in the very early phase of tumorigenesis, blood-borne microRNAs might also influence other, more advanced phases  of tumor formation including angiogenesis, invasion, etc. . CirculaAn interesting similar hypothesis on the relevance of circulating microRNAs in tumors has been put forward by Chen et al. , who obshsa-miR-21. The relative abundance of microRNAs with predominantly tumor suppressor activity in the over tenfold overrepresented group is, however, noteworthy.A major counter argument against this hypothesis could be the cellular context-dependent activity of microRNAs. Even microRNAs with predominant tumor suppressor activity could have oncogenic relevance in certain tissues. Our tenfold boundary (tenfold overrepresented to the 20th microRNA in ranking) for discussing the most relevant circulating microRNAs is certainly arbitrary, and among the microRNAs below this threshold, several others can be found having both oncogenic and tumor suppressor activity or even predominantly oncogenic like Another major problem is related to the very low quantity of circulating microRNAs. Even the concentration of the most overrepresented circulating microRNAs is very low, and it is unknown whether microRNAs in such low concentration are potent enough to result in significant gene expression alterations in cells internalizing them. There are in vitro data supporting the gene expression modulating capacity of both membrane vesicle and lipoprotein-associated microRNAs , 46, butA further issue is related to the affected cell populations. Apart from the endothelial and blood cells getting into direct contact with circulating microRNAs, what other cells could be affected and how? Most probably, microRNAs can traverse the capillary endothelium, as there are data that even exogenous microRNAs might pass the gastrointestinal epithelium and enter the circulation , 48. TheThere are certainly interindividual and ethnic variations in microRNA profiles of healthy individuals as exemplified by the findings of different research groups Table . Habits,hsa-miR-223 and hsa-miR-451, however, no increase in spontaneous tumor frequency has been reported [It will be rather difficult to test this hypothesis experimentally. In vitro experiments could be conceived to test whether microRNAs could halt tumorigenesis or induce apoptosis in nascent tumor cells generated by, e.g., viral or chemical transformation induction processes in cell cultures. As our hypothesis relates to the inhibition of nascent tumor formation in vivo, animal tumor models should be examined. microRNAs with predominant tumor suppressor action could be administered to mouse tumor models  and the frequency of tumor formation could be tested. Several problems need to be addressed in these studies, e.g., form of administration, the concentration of the microRNA administered, potential off-target microRNA actions, etc. Alternatively, knockout or transgenic mouse models of selected microRNAs abundant in the circulation could be examined for spontaneous or induced tumor frequency. In the available knockout mouse models for reported , 53. LonHere, we have presented a hypothesis on the potential relevance of circulating, blood-borne microRNAs in healthy individuals. Our hypothesis is based on the relative abundance of microRNAs with predominant tumor suppressor activity among the most highly overrepresented microRNAs in serum or plasma. Their potential activity as tumor suppressors might be intriguing. It must be noted, however, that we have highlighted only one potential activity of circulating microRNAs in healthy individuals. Circulating microRNAs might affect other major homeostatic mechanisms, i.e., immune response, hematopoiesis, etc. Further studies will be necessary to gain deeper insight into this fascinating field and to be able to explore the biological relevance of circulating microRNAs.


Ependymal tumors comprise a heterogeneous group of neuroepithelial malignancies of the CNS with variable prognosis that can occur in children and adults along the entire neuroaxis, including the spine (SP), posterior fossa (PF), and supratentorial brain regions (ST). Currently, these tumors are classified and graded solely by morphological patterns, but it is commonly accepted that grading does not accurately predict their clinical behavior . A powerC11orf95- RELA gene fusions, recently identified by Parker et al. [RELA gene fusions were not found in any of the other subgroups. In a second ST subgroup (ST-EPN-YAP1) with good outcome we identified highly recurrent YAP1 fusions as the probably most important driver event, again not present in any of the other subgroups. We found no other recurrent gene fusions in the other subgroups, confirming previous sequencing studies [DNA methylation patterns in tumors have been shown to represent a very stable molecular memory of the respective cell of origin throughout disease course, making them particularly suitable for tumor classification purposes. We generated genome-wide DNA methylation profiles for 500 ependymal tumors using the Illumina 450k methylation array and identified nine distinct molecular subgroups, three within each CNS compartment. One of the subgroups within each compartment was enriched with grade I subependymomas (SE), named SP-SE, PF-SE and ST-SE. Other molecular subgroups within the spine showed a relatively good concordance with the histopathological subtypes myxopapillary ependymoma (SP-MPE) and (classic) ependymoma (SP-EPN). The remaining molecular subgroups within the posterior fossa were the previously described PFA and PFB ependymomas ,4, renam studies . These sThe molecular subgroups identified in our study were closely associated with specific age groups. Tumors of the PF-EPN-A subgroup were almost exclusively found in young children and also ST subgroups, ST-EPN-YAP1 and ST-EPN-RELA, were much more common in children. Tumors in the other subgroups were more common or exclusively found in adults. Patients with PF-EPN-A and ST-EPN-RELA tumors, together comprising two-thirds of all cases, have the worst outcome. In contrast, patients in the other subgroups have an excellent prognosis with 5-year overall survival rates close to 100%. Multivariable analysis identified molecular subgroup, level of resection and gain of chromosome arm 1q to be of independent prognostic value for both overall and progression free survival but did not show any predictive impact of WHO-grading .YAP1 locus, which is also on chromosome 11, while the remainder of the genome appeared to be relatively stable. Predominantly stable genomes were also found for all three molecular subependymoma subgroups  as well as for PF-EPN-A. The most frequent event in the latter subgroup was gain of chromosome 1q, which is an established marker for poor outcome in ependymoma [NF2. The PF-EPN-B subgroup showed by far the highest degree of genomic instability, with many gains and losses of entire chromosomes or chromosomal arms in each tumor. Taking into account that CNAs are not occurring randomly and therefore presumably contain drivers of cancer, Mohankumar and colleagues recently reported a cross-species in vivo screen of 84 candidate oncogenes and 39 candidate tumor suppressor genes (TSG), located within 28 recurrent CNAs in ependymoma [Genome-wide DNA copy number profiles showed strong differences of DNA copy number alterations (CNAs) between the nine molecular subgroups. Most CNAs across all subgroups involved gains or losses of whole chromosomes compatible with aneuploidy. In accordance with findings by Parker et al. , we freqendymoma . Spinal In summary, we have described a new, comprehensive and robust classification of ependymal tumors based on DNA methylation profiling. Importantly, we have also shown that subgroup classification remains stable at the time of recurrence . Future


ELISPOT is the only technique currently available that permits to enumerate antigen-specific B cells providing insights into humoral immune defense. Resting memory B cells can be detected in blood samples after polyclonal stimulation. In contrast, the spontaneous production of immunoglobulins (Ig) by B cells in freshly-isolated peripheral blood mononuclear cells (PBMC) signifies recent and ongoing antigen stimulation in vivo. ELISPOT has thus far been restricted to single color analysis of Ig classes or subclass for detection of Antibody-Secreting Cells. Consequently, a comprehensive study of all four Ig classes or IgG subclasses required fourfold amounts of PBMC, materials and labor. We have developed a 4-color B cell ImmunoSpot assay where all four Ig classes or IgG subclasses are detected simultaneously. This novel technique was applied to the characterization of Human Cytomegalus Virus (HCMV)-specific Ig responses in human subjects with previous exposure to the virus.® S6 Analyzer.PBMC were cultured for 4 days with polyclonal B cell activators. On day 4, cells were washed, counted and plated in a low autofluorescence PVDF plate, pre-coated with either anti-human Ig capture antibody for detection of total Ig secretion, or HCMV antigen for detection of HCMV-specific ASC. During a 4h culture, the Ig secreted by the individual B cells was captured on the membrane. The plate-bound human Ig "spots" were visualized using IgG subclass- or Ig class-specific detection reagents, whereby each detection reagent is distinguished from the other 3 reagents through its unique fluorescence. The four-color assays were analyzed using an ImmunoSpotThe four-color ImmunoSpot assay has identical sensitivity for detecting individual B cells secreting IgG subclasses as the respective single-color assays, and the four fluorochromes can be discerned unambiguously, without spectral overlap. Therefore, four color B cell ImmonoSpot assays are equally suitable for monitoring B cell-mediated immunity as four single-color assays. Applying the test to characterize the HCMV-specific ig response suggest that the same Ig classes and sub-classes are involved in different donors, yet at highly variable ratios. Morover, the spot morphologies showed striking differences amongs the test subjects suggesting the involvement of different B cell affinities: diffuse, "fuzzy" spots indicate relatively low affinity, while tight spots with well-defined borders indicate Ab with high affinity for the antigen. Four color B cell ImmunoSpot assays therefore pemit comprehensively measuring frequencies and affinities of antigen-specific B cells secreting Ig of all four Ig classes and IgG subclasses.


Hypoxic hepatitis (HH) is a frequent cause of acute hepatocellular damage in critically ill patients associated with high mortality. Indocyanine green, a medical dye, is removed solely by the liver without entering enterohepatic circulation. Therefore its plasma disappearance rate (ICG-PDR) is an effective clinical tool for assessment of liver function in acute and chronic hepatic disease.Aim of this study was to evaluate the prognostic impact of ICG-PDR in comparison to laboratory parameters and established scores in critically ill patients with HH and other liver disease entities.The impact of ICG-PDR was prospectively evaluated in 119 critically ill patients with different liver disease entities : n = 10, control group without signs of acute liver injury, viral or drug induced hepatitis or cirrhosis: n = 22). Plasma disappearance rate of ICG was measured non-invasively by using a finger pulse densitometry system.ICG-PDR on admission was significantly lower in patients with liver diseases  than in the control group without hepatic impairment .ICG-PDR predicted 28-day mortality independently of SOFA score and serum lactate in patients with underlying liver disease . AUROC of ICG-PDR predicting 28-day mortality in patients with HH was significantly higher than AUROC of SOFA, arterial serum lactate, INR and AST 48 hours after admission (p < 0.05). ICG-PDR increased significantly in 28-day survivors over course of time (p < 0.001). 28-day survivors could be identified using a cut-off ICG-PDR ≥ 9.0 %/min 48 hours after admission with sensitivity of 77% and specificity 100%.Diagnostic test accuracy of ICG-PDR was superior to standard liver function parameters and well established scoring systems in patients with HH. Therefore ICG-PDR is a feasible non-invasive tool for early risk stratification in patients with HH.


Right ventricular (RV) pressure overload results in interventricular septal displacement (IVSD) towards left ventricle in patients with pulmonary arterial hypertension (PAH). There is however scarce data on the duration of IVSD during cardiac cycle as expressed by curvature duration index (CDi) and its potential role in the evaluation of PAH patients. The aim of our study is to reveal the potential value of CDi as a marker of RV function and pressure overload.All patients underwent cardiac magnetic resonance . A routine set of LV and RV short-axis cines of 6 mm slice thickness, were acquired from base to apex using a breath-hold retrospective ECG-gated balanced steady state free precession (SSFP) sequence. CDi , left ventricular eccentricity index in end-systole (LVSei) and end-diastole (LVDei), left ventricular end-systolic (LVESarea) and end-diastolic area (LVEDarea), RV end-systolic (RVESarea) and end diastolic area (RVEDarea) were defined in the short-axis view in the level of papillary muscles. Interventricular septal curvature ratio (CR) was defined in the same level, at end-systole. The right ventricular wall thickness (RVWT) was assessed in the anterior segment of RV. Tricuspid annular plane systolic excursion (CMR-TAPSE) was defined in the 4-chamber view. Right ventricular ejection fraction (RVEF) and RV end-systolic volume (RVESV) and end-diastolic volume (RVEDV) were obtained with the use of serial short axis cine-MRI views from base to the apex, according to Simpson's rule.Our study included 41 consecutive patients  with precapillary pulmonary hypertension . A direct linear correlation between CDi and CMR-TAPSE , CR , LVSei  and LVDei , LVESarea , LVEDarea , RVEF , RVESV , RVESarea , RVEDarea , RVWT  was observed.CDi is a potential non invasive simple marker for the evaluation of RV pressure overload and function in patients with precapillary pulmonary hypertension. Its prognostic significance remains to be established in further studies.Hellenic Cardiological Society.


Although avoidance of death is a key goal of critical care, so too is provision of high-quality end-of-life care when life-prolonging therapy is not desired. One often-used measure of ICU performance is risk-adjusted mortality, yet this measure treats all deaths equally and thus is flawed. In this work, we identify different death typologies using cluster analysis, with the ultimate goal of more narrowly targeted ICU quality improvement measures and efforts.We performed k-means cluster analysis in 2,047 ICU decedents admitted to a university medical center in 2011/12. Variables for the analysis included ICU length of stay (LOS), mechanical ventilation, tracheostomy, gastrostomy tube insertion, dialysis, enteral or parenteral nutrition, and cardiopulmonary resuscitation (CPR).Four clusters were identified. Short ICU LOS and low life- sustaining therapy utilization but relatively frequent CPR characterized Cluster 1. Intermediate ICU LOS and moderate life-sustaining therapy utilization characterized Clusters 2 and 3. Prolonged ICU LOS and high life-sustaining therapy utilization (except CPR) characterized Cluster 4. Age and severity of illness decreased across clusters with the oldest, sickest patients in Cluster 1 and the youngest, least sick patients in Cluster 4. See Table Cluster analysis can identify unique typologies of death within ICUs. This approach may be a novel method to more specifically target ICU efforts to reduce mortality and improve the quality of death.


Photodynamic therapy (PDT) of cancer is an approved therapeutic procedure that generates oxidative stress leading to cell death of tumor and stromal cells. Cell death resulting from oxidative damage to intracellular components leads to the release of damage-associated molecular patterns (DAMPs) that trigger robust inflammatory response and creates local conditions for effective sampling of tumor-associated antigens (TAA) by antigen-presenting cells. The latter can trigger development of TAA-specific adaptive immune response. However, due to a number of mechanisms, including epigenetic regulation of TAA expression, tumor cells evade immune recognition. Therefore, numerous approaches are being developed to combine PDT with immunotherapies to allow development of systemic immunity. In this review, we describe immunoregulatory mechanisms of epigenetic treatments that were shown to restore the expression of epigenetically silenced or down-regulated major histocompatibility complex molecules as well as TAA. We also discuss the results of our recent studies showing that epigenetic treatments based on administration of methyltransferase inhibitors in combination with PDT can release effective mechanisms leading to development of antitumor immunity and potentiated antitumor effects. Photodynamic therapy (PDT) is a light-based therapeutic approach used for the treatment of various solid tumors and non-malignant diseases. Its mechanism of action involves three non-toxic and harmless components: photosensitizer (PS), light, and oxygen. Their spatiotemporal encounter triggers photochemical reaction leading to formation of singlet oxygen and ensuing photodamage in tumor tissue . PS can Direct cytotoxic effects induced by PDT do not explain strong antitumor activity of this treatment observed in experimental animals. For example, cells from the tumors excised immediately after curative PDT are still clonogenic indicating that there must be other, indirect mechanisms triggered by PDT that contribute to the efficacy of the treatment . These iMassive PDT-induced photooxidative damage occurs mainly in the membranes and cytoplasm of tumor cells, tumor vasculature, and other stromal elements. This substantial PDT-mediated local injury threatens the host tissue integrity and homeostasis. Therefore, a host response develops as acute local inflammation in order to eliminate dead and injured cells, heal lesion, and restore tissue function as well as maintain its homeostasis . This reA number of cytokines have been detected both within the tumor and in the plasma of mice undergoing PDT. Among a wide range of cytokines  and chemokines (KC and MIP2) induced after PDT, a very important role is assigned to IL-1β and IL-6 . NeutralNeutrophils are the first cells invading PDT-treated tumor sites showing remarkable impact on PDT-mediated tumor damage. It was shown that neutrophils depletion in tumor bearing mice and rats attenuates the efficacy of antitumor PDT , 17. MonInnate immune cells encounter released tumor antigens (including oxidatively modified ones) together with molecules known as damage-associated molecular patterns (DAMPs) or cell death-associated molecular patterns (CDAMPs) . DAMPs p+ cytotoxic T cells (CTLs) in PDT outcome. Long-term tumor control after PDT treatment is possible only in immunocompetent mice, whereas in immunodeficient SCID or nude mice the long-term effects are abrogated. However, adoptive transfer of T cells from normal mice that underwent successful PDT restores antitumor PDT efficacy in immunodeficient animals. Importantly, T-cell depletion studies revealed that the CD8+ T-cell population is critical for a successful PDT response whereas CD4+ T cell population plays only a supportive role . It consists of four major steps: (1) peptide processing, (2) peptide transport, (3) MHC class I assembly, and (4) antigen presentation . A funda+ T lymphocytes isolated from patients with low expression of MUC1 do not react to autologous tumors  can also induce cell death in both T and NK cells. Another related mechanism is associated with surface expression of non-classical MHC class I molecules HLA-G and HLA-E that inhibit cytotoxic activity of effector CTLs and NK cells –46. Circin vivo and the efficacy of cancer immunotherapies  that down-regulate immune response , responsible for tryptophan catabolism. Enhanced expression of IDO in some types of tumors causes local shortage of tryptophan, leading to disturbances in proliferation of alloreactive T lymphocytes and their cell cycle arrest . Additioresponse , 54.L  and PD-L1, secretion of immunosuppressive cytokines such as IL-10 and TGF-β, and by consuming IL-2 , which under normal conditions serve as safeguards against overwhelming inflammation. In this way, tumors turn the immune system against itself, and exercise a powerful arsenal of mechanisms unavailable to tumor cells themselves to mitigate anti-tumor immune activity. Tregs inhibit activation and expansion of antigen-specific CD8ing IL-2 . There aing IL-2 . MDSCs aing IL-2 . MDSCs e T cells , 63. The T cells , 63, 64. T cells . Additio T cells .Epigenetic mechanisms include post-translational modifications of histone proteins affecting chromatin remodeling, DNA methylation, and regulation of gene expression by non-coding RNAs. A number of epigenetic events seem to play a pivotal role both in tumor progression and in avoiding immune recognition , 68. TheMethylation occurs predominantly in CpG-rich regions called “CpG islands.” A characteristic feature of tumor cells is global hypomethylation of their genome, and hypermethylation of CpG island in promoter regions of various genes . MethylaHistones acetylation is a reversible process of adding an acetyl moiety to lysine residues on histone proteins resulting in neutralization of their positive charge and impairing their interaction with negatively charged DNA. Therefore, acetylation increases the accessibility of regulatory proteins to DNA, enabling activation of various genes expression . This prDNA methylation is one of the most important epigenetic mechanism regulating expression of genes responsible for recognition of tumor cells by host immune system. Particularly, this is relevant to methylation of gene promoters, which leads to silencing of TAA and APM proteins, enabling escape from tumor immune-surveillance .The presence of TAA in cancer cells is a mandatory requirement for activation of effector CTLs. TAA can be divided into four different groups: (i) differentiation antigens, which are lineage-specific and expressed in tumor as well as in normal cells from which the tumors arise; (ii) overexpressed antigens, which are broadly expressed in many normal tissues, but present in tumor cells at higher levels; (iii) tumor-specific antigens, usually typical for individual tumors, resulting from genetic alterations; (iv) cancer–testis antigens (CTA) that are expressed in various types of malignant human tumors and are restricted in normal tissues to germ cells of the testis, with occasional expression in female reproductive organs . CTA are+ T cells  to radiation-induced fibrosarcoma cells was observed to induce strong tumor-specific immune response allowing for long-term tumor control. Re-challenge experiments revealed that survived mice developed resistance to GFP-positive cells . These d+ CTLs as their depletion with monoclonal antibodies almost completely abrogated antitumor effects. On the other hand, CD4+ T cells played only a supportive role. We have also observed that the combined treatment led to expansion of IL-17-producing CD4+ T cells, which are known to stimulate CD8+. Intriguingly, pentamer staining for P1A-specific CD8+ T cell population revealed no significant changes in draining LNs and spleens between experimental groups. Moreover, all mice treated with PDT and 5-aza-dC that remained long-term tumor-free have rejected re-inoculated tumor cells even if the cells were P1A-negative. These findings suggest that the presence of P1A is not essential for the maintenance of long-lasting antitumor immunity. It is possible that 5-aza-dC combined with PDT may lead to increased expression and release of TAA in the PDT-treated microenvironment. Together with PDT-induced inflammation and the release of DAMPs, the combination treatment would confer better antigen presentation of P1A. Improved tumor recognition by immune cells can further expand the repertoire of antigen-specific T cells thereby increasing the immunogenic outcome of PDT. Thus, it can be hypothesized that PDT of tumors with TAAs up-regulated by 5-aza-dC can induce concomitant immunity to other subdominant and possibly weakly immunogenic antigens and facilitate epitope spreading. Immunity to the latter can sustain antitumor activity in mice. To summarize, our findings demonstrate that treatment combining 5-aza-dC with PDT leads to local cytotoxic effects accompanied by the release of induced TAAs allowing for development of antitumor immune response and long-term survival (Figure Considering that 5-aza-dC restores expression of CTA including P1A as well as MHC class I molecules , we sougl Figure . Based oMW, AM, and JG reviewed relevant literature. MW, AM, and JG drafted the manuscript. JG revised the manuscript and supervised MW and AM. MW and AM designed the figure. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Operation therapy is more invasive than medication therapy and then operation-related medical errors (MEs) might be of more significant impact than medication errors. We assessed the incidence and characteristics of operation-related MEs to improve patient safety in such patients.The Japan Adverse Event (JET) study was a prospective cohort study which had evaluated AEs and MEs at two tertiary care hospitals. We included all adult patients aged ≥15 years old who had operations over a 2-month period. The primary outcome of this study was the operation-related MEs, defined as any deviation from appropriate process of an operation or perioperative care. Trained nurses placed at each participating hospital reviewed all charts daily on weekdays, along with laboratories, incident reports, and prescription queries to collect any potential event. They also collected the characteristics of the patients in the cohort. Some operation-related MEs are associated with operation-related AEs, which are operation-related preventable AEs. After those suspected events were collected, physician reviewers independently evaluated them and classified them as operation-related MEs, AEs, or rule violations. Physician reviewers assessed and rated operation-related AEs according to the symptom and the severity of injury.This study included 389 patients with 6,624 patient-days. The median age of patients was 69 years and 224 (58%) were male. Among these 389 patients, 31 patients had 46 operation-related MEs during their hospital stay and the incidence of operation-related MEs was 12 per 100 patients. Operation-related AEs occurred in 29 patients with 43 events. The most frequent symptoms for operation-related MEs were skin (26%), bleeding (21%), and central nervous system (14%). Among 46 operation-related MEs, 43 (93%) were not intercepted, and they resulted in operation-related AEs that were considered as preventable operation-related AEs. Nine of preventable operation-related AEs (21%) were fatal or life-threatening: five were nerve injury during operation and stroke after neurosurgical operation, and one biliary peritonitis after gastrectomy and cholecystectomy, and tension pneumothorax after lung lobectomy, and two unexpected massive bleeding due to vessels injury.Ninety-three percent of operation-related MEs resulted in operation-related AEs and 21% of them resulted in life-threatening events. Prevention of operation-related MEs should improve the mortality of surgical patients.


GALT-deficient Drosophila disease model. Loss of Drosophila GALT (dGALT) greatly impairs coordinated movement and results in structural overelaboration and architectural abnormalities at the neuromuscular junction (NMJ). Dietary galactose and mutation of galactokinase (dGALK) or UDP-glucose dehydrogenase (sugarless) genes are identified, respectively, as critical environmental and genetic modifiers of behavioral and cellular defects. Assaying the NMJ extracellular synaptomatrix with a broad panel of lectin probes reveals profound alterations in dGALT mutants, including depletion of galactosyl, N-acetylgalactosamine and fucosylated horseradish peroxidase (HRP) moieties, which are differentially corrected by dGALK co-removal and sugarless overexpression. Synaptogenesis relies on trans-synaptic signals modulated by this synaptomatrix carbohydrate environment, and dGALT-null NMJs display striking changes in heparan sulfate proteoglycan (HSPG) co-receptor and Wnt ligand levels, which are also corrected by dGALK co-removal and sugarless overexpression. These results reveal synaptomatrix glycosylation losses, altered trans-synaptic signaling pathway components, defective synaptogenesis and impaired coordinated movement in a CG neurological disease model.Classic galactosemia (CG) is an autosomal recessive disorder resulting from loss of galactose-1-phosphate uridyltransferase (GALT), which catalyzes conversion of galactose-1-phosphate and uridine diphosphate (UDP)-glucose to glucose-1-phosphate and UDP-galactose, immediately upstream of UDP–N-acetylgalactosamine and UDP–N-acetylglucosamine synthesis. These four UDP-sugars are essential donors for driving the synthesis of glycoproteins and glycolipids, which heavily decorate cell surfaces and extracellular spaces. In addition to acute, potentially lethal neonatal symptoms, maturing individuals with CG develop striking neurodevelopmental, motor and cognitive impairments. Previous studies suggest that neurological symptoms are associated with glycosylation defects, with CG recently being described as a congenital disorder of glycosylation (CDG), showing defects in both N- and O-linked glycans. Here, we characterize behavioral traits, synaptic development and glycosylated synaptomatrix formation in a  Drosophila sugarless (sgl) (sgl mutations compromise biosynthesis of the heparan sulfate proteoglycan (HSPG) co-receptor Dally-like protein (Dlp), which is known to regulate trans-synaptic signaling of the Wnt protein Wingless (Wg); such signaling drives neuromuscular junction (NMJ) synaptogenesis  results from loss of galactose-1-phosphate uridyltransferase (GALT), the second enzyme in the Leloir pathway, which acts immediately downstream of galactokinase (GALK), the initial enzyme . GALT mass (sgl) , and sglogenesis . These sAcute CG neonatal symptoms are alleviated by dietary galactose restriction , but matDrosophila screen of glycogenes via RNAi knockdown of N/O-linked glycans, glycosaminoglycans, glycosyltransferases and glycan-binding lectins to test the effects on NMJ structure and function (Drosophila GALT (dGALT) as a potent regulator of NMJ architecture. We therefore set forth to characterize synaptic morphology and glycosylated synaptomatrix composition in the recently established Drosophila CG disease model (dGALT deficiency)  , and to Clinical issueClassic galactosemia (CG) results from complete or almost complete loss of galactose-1-phosphate uridyltransferase (GALT), the second enzyme in the Leloir pathway of galactose metabolism. GALT catalyzes the generation of a glucose precursor and maintains the balance between uridine diphosphate (UDP) sugars, the obligate sugar donors for the synthesis of glycoproteins and glycolipids (glycosylation), which are important components of cell membranes and extracellular matrix. Dietary galactose restriction alleviates neonatal lethal symptoms of CG, but affected individuals still develop substantial neurological complications (including motor and cognitive impairments) of unknown etiology. CG has recently been classified as a congenital disorder of glycosylation (CDG), and glycosylation defects have been suggested as the underlying mechanism for the chronic neurological disease symptoms. Beyond the single CG condition, more than 60 other CDGs have been identified so far. Neurological impairments are common in these CDG disease states, but effective treatments are not yet available.ResultsDrosophila screen of glycosylation-related genes identified GALT as a potent regulator of the structure of the neuromuscular junction . To extend this discovery, GALT-deficient Drosophila  have been used to characterize movement behavior, NMJ synapse structure and function, and the carbohydrate composition of the synapse extracellular environment (i.e. synaptomatrix) in the disease. The results show that loss of GALT activity impairs coordinated movement, causes structural synapse overelaboration, profoundly changes the carbohydrate composition of the NMJ synaptomatrix, and alters the Wnt trans-synaptic co-receptor and ligand abundance (known to control synaptic morphogenesis at the NMJ). In double mutants, the combination of GALT deficiency either with loss of galactokinase  or overexpression of UDP-glucose dehydrogenase  corrects the glycosylation defects, the NMJ architecture alterations and the movement impairments associated with GALT loss in this Drosophila CG model.A recent Implications and future directionsNMJ defects likely underlie the pathogenesis of movement disorders frequently reported in CG-affected individuals, and might also account for similar impairments that characterize other CDG disease states. The findings reported here are the first to reveal NMJ synaptic glycan loss, synaptic architecture defects and trans-synaptic Wnt co-receptor and ligand defects as potential causal factors for the well-known CG-related movement disabilities. Furthermore, the results presented here suggest that targeting galactokinase and UDP-glucose dehydrogenase could modify CG outcome and represent a potential therapeutic approach in this disease. Future investigations are needed to advance the translation of findings on CG-related candidate genes into the development of novel drugs, and to extend this approach to the study of other CDG diseases.dGALT-null mutants display defects in startle-induced, geo-negative climbing behavior (Drosophila Activity Monitoring (DAM) system, which measures movement disruption of an infrared beam (ΔAP2dGALT (null) and C2dGALT (precise-excision genetic control) flies. Both genotypes were entrained to 12:12-hour light:dark cycles for 2 days, and then activity counts were recorded for ≥3 days. dGALT-null mutants were significantly (P<0.01) movement-impaired compared with controls . These results show that removal of dGALT strongly reduces locomotor activity in this CG model.Evidence amassed over decades from individuals with CG documents common movement defects . Similarbehavior . To assared beam . Adult aDrosophila larvae , and compared with a genetic control . Compared with the righting time of controls (17.20±1.03 s), both dGALT  and dGALT/Df  animals were significantly slower and obviously less coordinated  in the ΔAP2dGALT-null background (n=14). Transgenic hGALT expression in the ΔAP2dGALT-null background completely rescued normal coordinated movement compared with the driver-alone control , which was indistinguishable from homozygous ΔAP2dGALT mutants , homozygous-null ΔAP2dGALT and ΔAP2dGALT/Df, and ubiquitous UH1-, neuronal elav- and muscle 24B-Gal4-driven dGALT-RNAi with their respective controls . Confocal images for representative genotypes are shown in Our recent ogenesis . The glua larvae , and thea larvae . We ther mutants . Axonal  mutants . NMJs frdGALT nulls as well as ubiquitous  and tissue-targeted  dGALT-knockdown NMJs all exhibited obvious synaptic structural overelaboration  compared with controls , as well as in the ubiquitous dGALT RNAi-knockdown condition  compared with controls  and muscle -driven dGALT RNAi showed more modest, but significant, increases in bouton number  compared with driver-alone controls  and with ubiquitous dGALT RNAi knockdown  fully rescued both bouton and branch numbers. These results demonstrate that dGALT is necessary for sculpting NMJ synaptic architecture, and that this requirement is fully met with transgenic expression of human GALT.boration , top rowboration . The incn muscle . ImportaC2dGALT; precise excision) to homozygous-null mutant , and UH1-Gal4-driven UAS-dGALT-RNAi to UH1-Gal4/+-alone transgenic control. More than ten NMJs from ≥five different animals were recorded for each of the four genotypes. Mean EJC amplitudes were not significantly different in dGALT nulls compared with controls . Similarly, ubiquitous dGALT knockdown animals also displayed similar synaptic strength to controls . Although there was a trend towards increased neurotransmission in both comparisons, neither change was significant. We therefore conclude that dGALT does not significantly alter neurotransmission strength at the NMJ, but rather plays a specific role in controlling synaptic architecture, which we therefore focused on in subsequent studies. Because exposure to dietary galactose is a well-recognized modifier of acute outcome in CG  and homozygous-null (ΔAP2dGALT) animals were raised on either (1) normal molasses-based food containing negligible galactose levels , and ~120% more time than control animals fed galactose-free food  e levels . We foundGALT-null larvae, and homozygous-dGALT-null larvae raised in the absence of galactose, displayed supernumerary boutons  than did controls , and dietary galactose did not alter these values for either genotype  and eliminated the difference with null mutants , with undetectable enzymatic activity, were raised either on food supplemented with 200 mM galactose or on normal food. Galactose-fed mutants took as much time as unsupplemented mutants to rollover from an inverted to an upright position . Similarly, both NMJ branch number and interbouton distances were not significantly different in the two conditions (data not shown). Animals displayed 2.0±0.19 (n=8) and 2.2±0.17 (n=15) branches under galactose-free and galactose-fed conditions, respectively. Interbouton distance was 0.81±0.08 μm (n=15) and 0.77±0.06 μm (n=8) with and without galactose feeding, respectively. However, galactose-fed dGALK mutants displayed excess NMJ boutons compared with unsupplemented animals  and significantly greater NMJ cumulative length . Because a high-galactose diet can alter glycosylation status, and abnormal glycosylation of the extracellular environment at the NMJ is well known to modulate synaptogenesis  , which wogenesis , we nextDrosophila NMJ is well known to alter synapse formation ], (2) N-acetylgalactosamine residues , and (3) N-acetylglucosamine residues [Wheat germ agglutinin (WGA)]. Representative images and a summary of the major changes are shown in Glycosylation defects in plasma and tissue samples have long been reported in individuals with CG . These gormation , and the mutants , 2. To tdGALT-null larvae were probed with each lectin label (green in n=8), but labeling was dramatically decreased in mutants . We also assayed the NMJ with SBA and WGA lectins, which bind to terminal N-acetylgalactosamine (α and β) and N-acetylglucosamine, respectively, but found no differences between genotypes (data not shown). Finally, we probed dGALT-null NMJs with the commonly employed synaptic marker anti-HRP  but, similar to dGALT mutants, was strikingly reduced in larvae raised on a high-galactose diet . To further test the role of dGALT as a modifier of NMJ glycosylation state, transgenic hGALT rescue animals were compared with two control groups  by assaying WFA expression. The loss of WFA lectin signal in dGALT nulls  was completely rescued with transgenic expression of hGALT  to a level undistinguishable from wild-type controls . Taken together, these results reveal striking alterations in NMJ glycan composition in the absence of dGALT activity, specifically including reductions in galactosyl and N-acetylgalactosamine residues, and fucosylated HRP glycans. Because the NMJ synaptomatrix carbohydrate environment limits synaptic morphogenesis via the modulation of trans-synaptic signaling of the Wnt Wg, the pathway that drives morphological synaptogenesis , dGALT-null NMJs  showed a >50% reduction in Dlp levels , with no significant difference remaining between rescue animals and the genetic control. We next tested Wg ligand levels in the same genotypes , a significant elevation compared with the genetic control , with no significant difference to controls. Taken together, these results reveal changes in Wg co-receptor and ligand levels, with the loss of the HSPG Dlp co-receptor providing a well-established mechanism to explain the elevation of Wg ligand in the NMJ synaptomatrix.In controls, Dlp expression overlapped that of the HRP synaptic marker throughout most of the NMJ, extending slightly beyond the HRP boundaries . Compareenotypes . In contenotypes . Convers P<0.05; , right. dGALK co-removal should ameliorate dGALT-null phenotypes in our CG disease model. To test this hypothesis, we generated dGALT; dGALK double-null mutants, and then retested all of the behavioral, NMJ architecture and glycosylated synaptomatrix phenotypes described above. A summary of these analyses is shown in To explore a systems-level understanding of GALT-dependent functions at the synapse, we used the Search Tool for Retrieval of Interacting Genes (STRING)  databasedGALK fully corrected the behavioral movement defects of dGALT nulls (dGALT larvae again showed a slowed movement time (and movement was uncoordinated) compared with genetic controls , which was fully restored to the wild-type condition in double mutants , a highly significant improvement , bouton number was significantly decreased in double mutants  and was indistinguishable from wild-type animals , NMJ bouton number  or interbouton distance , which were all indistinguishable from controls.First, co-removal of LT nulls . Null dGP<0.001; . Likewisng dGALK . ComparedGALK co-removal would prevent the losses in glycosylation of the NMJ synaptomatrix observed in dGALT nulls, we next probed double mutants with lectins for the three glycans with the largest changes in levels observed in the single mutants , but dGALK co-removal restored ECL labeling to control levels  compared with controls , but WFA labeling was again fully restored to control levels in double mutants . In contrast, the reduced HRP signal observed in dGALT-null NMJs was unaffected by eliminating dGALK in parallel: in the absence of dGALT , there was a highly significant reduction in HRP labeling in mutants compared with controls , but there was no significant improvement with dGALK co-removal . Thus, the double mutant selectively restores synaptomatrix glycosylation state, but does not completely reverse NMJ glycan changes.To test the hypothesis that  mutants : ECL lec mutants . The los-removal . ECL lecls n=19; . Loss ofts n=10; . Loss ofdGALK co-removal also restores Dlp and Wg levels  were significantly elevated towards wild-type values in double mutants  and were no longer significantly different from controls  were reduced towards wild-type levels in dGALT; dGALK double nulls , and were no longer significantly different from controls  to assay the movement behavior, NMJ architecture, glycosylated synaptomatrix and Wnt pathway phenotypes described above. A summary of these data is shown in Our STRING analysis  revealedth dGALT . The sgl UDP-glc , an acti UDP-glc . Importaptor Dlp , which iogenesis , as discogenesis . We thersing sgl  in the dUH1-Gal4 driven UAS-sgl overexpression in dGALT nulls strongly restored coordinated movement , but sgl overexpression strongly decreased movement time, eliminating any significant difference with controls ; mouse anti-Discs-large ; mouse anti-Dally-like-protein  and mouse anti-Wingless . Conjugated lectins used included: Peanut agglutinin , Wisteria floribunda , Erythrina cristagalli lectin , Vicia villosa agglutinin , Soybean agglutinin  and Wheat germ agglutinin , all from Vector Labs. Secondary antibodies used included: Alexa-Fluor-555 donkey anti-mouse (1:300) and streptavidin Alexa-Fluor-488 conjugate (1:250), both from Invitrogen. Primary antibodies and lectins were incubated at 4°C overnight. Secondary antibodies were incubated at RT for 2 hours. Preparations were mounted in Fluoromount G (Electron Microscopy Sciences).Immunocytochemistry confocal imaging was performed as described previously . L3 larvz-stacks were taken with a Zeiss LSM 510 META confocal using 40/63× oil-immersion objectives, with sections starting immediately above and ending immediately below the NMJ. Expression analyses of conjugated lectins and anti-HRP were performed using NIH ImageJ software with the threshold function outlining lectin- or HRP-labeled NMJ within FasII-defined synaptic regions. Anti-Dlp and anti-Wg expression analyses were quantified within HRP-defined synaptic regions. Fluorescence was normalized to control values from the same experiment with all imaging parameters kept constant between compared genotypes. For structural analyses, preparations were double-labeled with anti-HRP and anti-DLG, with counts made at muscle 4 in segments A2/3 on the right and left sides. Data were averaged for each animal or hemisegment to produce one data point for expression and structural analyses, respectively. For structural quantification, a bouton was defined as an axon varicosity >1 μm in minimum diameter, and ≥two boutons on one axon defined an NMJ branch. NMJ cumulative length was measured by combining branch lengths, and inter-bouton distances obtained as an average from single branches with ≥six boutons.All mutant and control larvae were dissected, labeled and imaged in parallel. Two-electrode voltage-clamp (TEVC) electrophysiology was performed as described previously . Brieflyt-tests with Welch correction were applied for normally distributed datasets with unequal standard deviations. For data not normally distributed, pairwise comparisons were done with non-parametric Mann-Whitney tests. One-way analysis of variance (ANOVA) was used for parametric multiple comparisons with Tukey-Kramer post-tests. For nonparametric multiple comparisons, Kruskal-Wallis tests were applied with Dunn’s multiple comparisons. All statistical analyses were performed using GraphPad InStat version 3.0 (GraphPad Software). Significance in figures is presented as P<0.05 (*), P<0.01 (**), P<0.001 (***) and P<0.0001 (****).Behavioral, functional, structural and fluorescence intensity data were averaged per genotype, calculated as a fold-change relative to the mean control value from the same experiment. Unpaired two-tailed


Invasive fungal disease (IFD) causes significant morbidity and mortality in hematologic malignancy patients with high-risk febrile neutropenia (FN). These patients therefore often receive empirical antifungal therapy. Diagnostic test-guided pre-emptive antifungal therapy has been evaluated as an alternative treatment strategy in these patients.2 statistic, and between study τ2. Incorporating these parameters and direct costs of drugs and diagnostic testing, we constructed a comparative costing model for the two strategies. We conducted probabilistic sensitivity analysis on pooled estimates and one-way sensitivity analyses on other key parameters with uncertain estimates.We conducted an electronic search for literature comparing empirical versus pre-emptive antifungal strategies in FN among adult hematologic malignancy patients. We systematically reviewed 9 studies, including randomized-controlled trials, cohort studies, and feasibility studies. Random and fixed-effect models were used to generate pooled relative risk estimates of IFD detection, IFD-related mortality, overall mortality, and rates and duration of antifungal therapy. Heterogeneity was measured via Cochran’s Q test, INine published studies met inclusion criteria. Compared to empirical antifungal therapy, pre-emptive strategies were associated with significantly lower antifungal exposure  and duration without an increase in IFD-related mortality  or overall mortality . The pre-emptive strategy cost $324 less  than the empirical approach per FN episode. However, the cost difference was influenced by relatively small changes in costs of antifungal therapy and diagnostic testing.Compared to empirical antifungal therapy, pre-emptive antifungal therapy in patients with high-risk FN may decrease antifungal use without increasing mortality. We demonstrate a state of economic equipoise between empirical and diagnostic-directed pre-emptive antifungal treatment strategies, influenced by small changes in cost of antifungal therapy and diagnostic testing, in the current literature. This work emphasizes the need for optimization of existing fungal diagnostic strategies, development of more efficient diagnostic strategies, and less toxic and more cost-effective antifungals. Invasive fungal disease (IFD) is an important cause of morbidity and death in hematologic malignancy or hematopoietic stem-cell transplantation (HSCT) patients with high-risk febrile neutropenia (FN) –6. ThesePrompt initiation of appropriate antifungal therapy early in the course of IFD reduces IFD-related mortality. Because of the consequences of not treating IFD early and limitations in existing IFD diagnostic methods, many patients with high-risk neutropenia and persistent or recurrent fevers despite broad-spectrum antibiotic therapy receive empiric antifungal therapy ,9.Aspergillus species and other fungi, including detection of circulating Aspergillus galactomannan , 3126, 32 = 17.0%; τ2 = 0.132). While one study identified a significant difference in overall mortality between pre-emptive and empirical strategies, with decreased mortality with the pre-emptive strategy [2 = 61.9%; τ2 = 0.328).Of the five studies reporting IFD-related mortality, none found a significant difference with empirical versus pre-emptive management  and ther.04–0.71), there w2 = 93.4%; τ2 = 0.503).Five of seven studies assessing antifungal use found a significant decrease in antifungal use in the pre-emptive therapy group , Fig 8 ,26,28–30Overall, the existing literature demonstrates that pre-emptive diagnostic-test driven approaches may increase IFD detection without an increase in IFD-related or overall mortality, compared to empirical therapy. The majority of studies showed a reduction in the proportion of patients receiving antifungals and duration of antifungal exposure using a pre-emptive strategy.Tables Our cost estimates did not change when we derived model parameters only from the most robust randomized controlled trials of empirical versus preemptive antifungal therapy ,31. See Probabilistic sensitivity analysis using 95% confidence intervals derived from our meta-analysis for relative risks of IFD detection and antifungal use resulted in 95% credible intervals for the cost of pre-emptive therapy being $291.88 less to $418.65 more costly than empirical therapy overall, and pre-emptive therapy being $269.00 less to $106.33 more costly than empirical therapy prior to IFD detection. With one-way sensitivity analysis, with the RR of IFD detection comparing pre-emptive and empirical groups increased to a threshold of RR of 2.03, pre-emptive therapy became the more costly strategy. As long as the proportion of patients in the pre-emptive group receiving antifungal treatment was less than or equal to the proportion of people being treated in the empirical therapy group, empirical therapy was always the more costly strategy.Our results were sensitive to changes in the diagnostic testing strategy, antifungal drug costs, antifungal duration, and ratios of incident IFD diagnoses and antifungal use between the pre-emptive and empirical groups. For the diagnostic testing strategy, if the pre-emptive group received an additional chest CT scan per FN episode, the two strategies were nearly equivalent in cost with the empirical group being only $90 more costly overall per FN episode.With regards to antifungal drugs, the pre-emptive strategy became equivalent to empirical therapy (more costly by $12 per patient) using the most conservative estimate of drug costs (voriconazole) at $49 per day. In contrast, using a more expensive drug regimen of liposomal amphotericin B ($785 per day) in the pre-IFD diagnosis period and posaconazole ($172 per day) in the post-IFD diagnosis period made empirical therapy the even more costly strategy by $1,453 per FN episode. At a threshold pre-IFD diagnosis antifungal drug cost of $111 per day and post-IFD diagnosis antifungal drug cost of $221 per day, the two strategies were equivalent in cost. At an antifungal treatment duration threshold of less than 185 days following incident IFD diagnosis, pre-emptive therapy became the more costly strategy.This study provides the first systematic analysis of available evidence comparing empirical and pre-emptive antifungal therapy strategies among hematologic malignancy or HSCT patients with high-risk FN. Overall, pre-emptive therapy was associated with decreased antifungal treatment rates and duration and diagnostic-test driven increased IFD detection without an increase in IFD-related mortality. We incorporated these composite estimates in a cost comparison model and found a state of economic equipoise between empirical and pre-emptive antifungal therapy, slightly favoring pre-emptive therapy as the less costly strategy by $325 per FN episode, but influenced by relatively small changes in the cost of antifungal therapy and diagnostic testing. However, overall costs incorporating treatment for proven and probable IFD were consistently higher with pre-emptive therapy, largely due to the increased diagnosis of incident IFD and the cost of antifungal treatment for diagnosed IFD cases in this group.The influence of diagnostic testing for IFD is evident in our results. Pre-emptive approaches in the studies we examined varied significantly in fungal biomarker and imaging use. In particular, studies we reviewed employed varying combinations of galactomannan, beta-D-glucan, and fungal PCR. Of these, serum galactomannan was the most common biomarker trigger for starting pre-emptive antifungal therapy. Fungal PCR was also used in conjunction with galactomannan in some of these studies, though this assay is subject to variations in internal and external validity . Still, Also important to consider is the clinical and economic significance of IFD diagnoses in the setting of augmented diagnostic surveillance. Higher IFD detection without a corresponding increase in IFD-related mortality with pre-emptive therapy likely reflects the sensitivity of serial, systematically assessed fungal biomarkers and chest imaging for IFD, with initiation of antifungal therapy when there is relatively limited extent of fungal disease. However, higher rate of IFD identification with the pre-emptive strategy led to increased antifungal treatment and overall costs compared to the empirical group.This work also highlights the heterogeneity of antifungal drug treatment for incident IFD. Pre-emptive management led to an overall reduction in antifungal use and duration. However, the cost of specific antifungal agents influenced which approach was more costly. This is particularly relevant given institutional and regional variability in IFD epidemiology, antifungal drug susceptibility, and clinical management of patients with prolonged FN. Specifically, combination antifungal therapy is increasingly used in refractory cases with a recent study suggesting potentially increased efficacy in the treatment of early invasive aspergillosis .Limitations of our systematic review and cost comparison are largely intrinsic to the available literature comparing empirical and pre-emptive antifungal therapy in FN patients. Many of the studies we incorporated into our estimates of IFD detection, IFD-related mortality, antifungal use, and antifungal duration were small, observational studies ,34. Togeet al used parameters derived solely from two small observational studies and assumed significant differences in key patient characteristics and outcomes. While they found that pre-emptive therapy was 2.6% more effective and 33% less costly, their results may have limited generalizability and validity [et al constructed a model that made several assumptions  that are not supported by current literature examining empirical versus pre-emptive antifungal therapy [While simple, our cost comparison model incorporated key differences in antifungal use, diagnostic testing, and clinical outcomes driving cost differences between the two strategies. Compared to the two existing published cost-effectiveness studies comparing empirical and pre-emptive antifungal therapy in FN ,23, our validity . Similar therapy ,29,30,31Overall, we show that a diagnostic test-guided pre-emptive approach to antifungal management in FN is a clinically and economically reasonable alternative to fever-based empirical therapy. Forthcoming results of the EORTC randomized trial of caspofungin for empirical versus diagnostic-guided pre-emptive antifungal therapy in patients with acute myeloid leukemia or myelodysplastic syndrome and the subsequent economic analysis should help further elucidate some of these issues substantially . Our finS1 Dataset(XLSX)Click here for additional data file.S1 File(DOC)Click here for additional data file.S1 Model(XLS)Click here for additional data file.