Phascolarctos cinereus) genome and is currently spreading through the Australian koala population. Understanding how the koala’s immune system responds to KoRV infection is critical for developing an efficacious vaccine to protect koalas. To this end, we analyzed the antibody response of 235 wild koalas, sampled longitudinally over a four-year period, that harbored KoRV-A, and with or without KoRV-B. We found that the majority of the sampled koalas were able to make anti-KoRV antibodies, and that there was a linear increase in anti-KoRV IgG levels in koalas up to approximately seven years of age and then a gradual decrease thereafter. Koalas infected with both KoRV-A and KoRV-B were found to have slightly higher anti-KoRV IgG titers than koalas with KoRV-A alone and there was an inverse relationship between anti-KoRV IgG levels and circulating KoRV viral load. Finally, we identified distinct epitopes on the KoRV envelope protein that were recognized by antibodies. Together, these findings provide insight into the koala’s immune response to KoRV and may be useful in the development of a therapeutic KoRV vaccine.Koala retrovirus (KoRV) is in the process of endogenization into the koala ( A seven year old survey to determine the prevalence of KoRV in Australia previously reported that all koalas tested from northern Australia  contain detectable KoRV provirus, while 15–72% of koalas from southern Australia  also have detectable KoRV provirus5. A total of nine envelope protein variants of KoRV (KoRV-A to KoRV-I) have been identified6, two of which have been widely studied (KoRV-A and KoRV-B). KoRV-A has been shown to use a phosphate transporter (PiT1)7 for cell attachment and entry, while KoRV-B uses the thiamine transporter 1 (THTR1)1. The patterns of KoRV integration in the koala genome suggest that KoRV is currently undergoing endogenization into the koala9, offering a unique opportunity to study the endogenization process of a retrovirus in real time, as well as the host’s immune response to this invasion.Koala retrovirus (KoRV) has been reported to play a key role in neoplasia and chlamydial diseases of both captive and wild koala populations9. KoRV-A integrants in northern koalas display a pattern consistent with that of an endogenous retrovirus  and of an infectious retrovirus (with other loci integrated randomly in infected cells)9. The characterization of replication competent KoRV-A molecular clones further supports the supposition that KoRV-A is infectious in northern koalas11. Collectively, the evidence to date shows KoRV-A as a circulating subtype of KoRV with provirus transmitted both horizontally and vertically in northern koalas. This is quite different to the information available about KoRV-A in southern koalas, where low infection rates and fewer KoRV proviral copies per cell (~0.00013 copies per cell) suggest only exogenous KoRV variants (including KoRV-A) are present in these populations5. New information from a recent study in preprint has raised the possibility of endogenous KoRV in southern koalas, based on detecting apparently defective provirus12. However, until this interesting finding can be investigated more thoroughly, the current body of evidence from southern Australia only supports the presence of exogenous KoRV in this region.Northern koalas appear to harbor endogenous KoRV-A based on conserved genome integration pattern determined by southern blot analysis and relatively high provirus copy number per cell (~165 copies per cell)6. Contrasting with KoRV-A, there has been no evidence to suggest that any of these eight KoRV variants have endogenized in the northern koala population9. Testing of southern koala populations for non-KoRV-A variants has traditionally been limited to conventional PCR for KoRV-B provirus, which has been undetectable to date, leaving the impression that southern koalas harbor only KoRV-A13. However, a recent study has revealed genetic evidence of non-KoRV-A variants in southern koalas, suggesting the presence and diversity of KoRV in the south may be more complicated that currently recognized14.The remaining KoRV variants, KoRV-B to KoRV-I, have been detected and studied in northern koala populations15. While there has been no definitive and direct measurement of koala health outcomes in response to experimental KoRV infection, observational studies in wild and captive koalas do support possible roles for KoRV in koala pathologies. For instance, having detectable levels of KoRV-A provirus in southern koalas has been associated with chlamydial disease13. Similarly, detectable levels of KoRV-B provirus in northern koalas has also been associated with clinical chlamydial disease and neoplasia17. Finally, significant increases in total KoRV viral load (not defined by variant) has also been found to be associated with the development of lymphoma4. These statistically significant associations suggest KoRV presence and expression have some connection to serious health conditions in koalas.The possible involvement of viruses, like KoRV, in the development of disease in koalas, and particularly neoplasia, was first suggested in 196119. While this natural immune response is not always protective, it can confer resistance against the establishment of infection and disease in some individuals. The innate immune system plays a significant role in retrovirus control, with downregulation of nucleic acid-recognizing toll-like receptor (TLRs) 3, 7 and 9 being associated with reactivation of murine leukemia virus (MLV)21. Cell mediated immunity also contributes, with levels of virus-specific cytotoxic T lymphocytes being shown to play a critical role in determining feline leukemia virus (FeLV) infection outcomes in cats23. Adaptive immunity, such as T cell and B cell responses, can also be induced against endogenous retroelements by using experimental immunization, as seen in mouse studies using gp70 to target murine leukemia virus and non-human primate studies using Gag and Env proteins to target simian endogenous retrovirus-K25. Finally, antibodies play a key role in defending against exogenous retroviruses27. Antibody titers against gibbon ape leukemia virus and bovine leukemia virus have been shown to sometimes correlate, inversely, with viral load and disease severity30. Similarly, passive transfer of FeLV neutralizing antibodies via colostrum to kittens has been shown to provide some protection against viremia upon subsequent exposure31. However, antigen-specific antibody titers do not stay static throughout an infection, with age, sex and environmental factors having been shown to influence antigen-specific antibody levels in infected individuals33. Collectively, the antibody response to retrovirus infection is important for virus containment.A common feature of retroviral infections is the ability to trigger a host immune response32 and subsequent reduction in ability to clear new infections34. Similarly, production of new lymphocytes declines with aging36. Overall, there is a general decline in immune system function with age, known as immunosenescence37. The relationship between infection load and age appears to be complicated. Whereas an association between viral load and age has been reported for certain viruses including KoRV4, there appears to be no significant relationship between age and viral load in FeLV, Feline foamy virus, and HTLV39.Aging has been associated with reduction in the number of functional innate immune cells42, very little is known about how the koala’s immune system responds to KoRV infection. A major concern is whether an immune response can be mounted to an endogenous KoRV-A virus. It has been proposed that the expression of endogenous retroviral proteins in embryonic tissues may trigger immune tolerance to these proteins in the developing immune system43. Escape from immune tolerance has been reported for some infectious polytropic viruses, despite the expression of their antigens during ontogenesis44. However, a study of a small group of northern koalas (n = 16) did not find antibodies against KoRV, suggesting that koalas with endogenous KoRV infection may be tolerant to KoRV41. Subsequently, a study of immune cytokine expression in lymphocytes obtained from northern koalas reported an association between altered immune cytokines and KoRV-B infection45, suggesting a role for at least some variants of KoRV in the response to KoRV infection. However, more study into the immune response to KoRV in koalas known to contain both endogenous and exogenous variants of KoRV is needed.While there have been studies aimed at developing vaccines against KoRVTo this end, we tested 235 northern koalas and demonstrated that koalas harboring KoRV-A, with or without KoRV-B, do indeed produce anti-KoRV IgG antibodies. We showed an association between both koala age and the presence of KoRV-B with anti-KoRV IgG levels. We then went on to identify distinct antibody epitopes on the ectodomain of the transmembrane subunit of KoRV envelope protein. A key feature of our study was our ability to identify and monitor cohorts of koalas over time, thus enabling us to test several of our hypothesis on longitudinal samples.Two hundred and thirty-five koalas from Gold Coast City Region (GCCR) and Moreton Bay Region (MBR) populations were screened for KoRV infection using KoRV-A and KoRV-B specific PCR assays, to detect integrated provirus. All 235 koalas tested positive for KoRV-A envelope gene , while only 27% (n = 63/235) of koalas were positive for KoRV-B envelope gene.42  protein (rEnv)42 Fig. , we meas42 Fig. .Figure 1Examining the anti-KoRV IgG titers from the MBR koala population n = 207) in the context of the koala’s age, we found that there was a gradual increase in anti-KoRV IgG levels between ages one and six, peaking at around seven to eight years and then declining in koalas aged nine 12 Fig. . To bett07 in theTo determine if the presence of KoRV-B in koalas carrying KoRV-A has any association with anti-KoRV IgG antibody levels, we compared sera from KoRV-B positive and negative koalas Fig. . Using upol gene and correlated it with serum anti-KoRV IgG levels in paired samples from 34 randomly chosen koalas. These koalas were either positive for KoRV-A and KoRV-B, or positive for KoRV-A only. The results showed a negative correlation between plasma KoRV viral RNA load and anti-KoRV IgG levels in koalas that were positive for KoRV-A only  and for koalas positive for both KoRV-A and KoRV-B . Overall, expressed KoRV viral load declined as anti-KoRV IgG increased   of KoRV env protein were recognised in koalas infected with KoRV-A, we designed 48 overlapping 15mer peptides with three amino acids offsets, spanning the ectodomain of the p15E subunit of KoRV-A env protein , we compared all epitopes recognized by antibodies from six northern koalas in the present study, sampled twice, approximately three years apart (n = 12), with the vaccinated southern koalas (n = 3). For the vaccinated koalas, the response of each pre-vaccination sample was compared against its 8 weeks post-vaccination sample. The results presented for the non-vaccinated koalas represent an average of the frequency of each reported epitope from Fig. We recently reported the development of a tri-adjuvanted recombinant KoRV envelope protein based vaccine in koalas infected with KoRV-A from southern Australia46. Therefore, we screened for antibodies to this epitope in a larger cohort of our koalas (n = 197). A 31-amino acid synthetic peptide spanning the MPER of KoRV p15E was synthesized and used as an antigen to measure antibody levels directed against this region. We found that the pattern of anti-MPER IgG levels was similar to IgG levels against the full length env protein reported in Figs 2 = 0.437, p < 0.0001, n = 197)  have been shown to induce neutralizing antibodies in FeLV, PERV and HIV-1, peptides 42–45 present in the MPER have been shown to induce neutralizing antibodies against the above retroviruses, as well as KoRV, in experimental immunization studies in non-koala models97) Fig. .Figure 816. All samples included in this study were positive for KoRV-A provirus, while only 27% were positive for KoRV-B provirus. This finding is consistent with infection rates in northern koala populations reported in previous studies47.Koala retrovirus (KoRV) poses a significant threat to koala populations. KoRV-A is present in all northern koala populations in Australia, with some koalas in this area co-infected with KoRV-B41. However, a subsequent study using similar methods found that nearly all tested koalas (6 of 7) had detectable antibodies against at least one of the KoRV-A proteins used in the study48. The authors explained that they were able to detect anti-KoRV antibodies because they had access to additional reagents and controls. In the current study, we sought to settle this discrepancy by employing a more sensitive and quantitative serological assay (ELISA) and to assess a much larger number of koalas (n = 235). Using our techniques, we found that most koalas do make a detectable antibody response to KoRV. While a limited number of koalas in our cohort (5%) appeared to make little or no anti-KoRV IgG response, the rest (95%) did make an IgG response specific to KoRV. This is an important finding, as it suggests that koalas possessing an endogenous KoRV variant are not tolerant to KoRV but are capable of making an antibody response against KoRV.To further understand how the koala’s immune system responds to KoRV infection, we investigated whether koalas with both endogenous and exogenous KoRV could make antibodies against KoRV. A previous study, using a western blot assay and small sample numbers (n = 16), suggested that no antibodies were made against KoRV in northern koalas (harboring both endogenous and exogenous KoRV)49 and appears to exist as an endogenous virus that retains infectious properties within northern koalas11. Therefore, it is not surprising that KoRV-A, in contrast to ancient quiescent endogenous retroviruses, does not induce immunological tolerance. The expression of endogenous Jaagsiekte sheep retrovirus, a Betaretrovirus, during early stages of ontogeny is thought to contribute to tolerance and absence of serological response later in life50. It is currently unknown at what stage and in which cells/tissues during ontogenesis endogenous KoRV proteins become expressed. The lack of tolerance suggests that the viral proteins are expressed after the establishment of some immune function or acquisition of passive immunity, possibly via immunoglobulin-rich milk51 from the mother 52.In contrast to most endogenous retroviruses that are millions of years old, KoRV-A is a relatively new germline acquisition 53. Typically, dependent juvenile koalas become physically independent of their mothers around one year of age, reach sexual maturity at approximately two years of age and are considered adults at four years. The average life expectancy for koalas in the wild is around 13 years54. We investigated possible correlations between antibodies against KoRV and koala age. Analysis showed a gradual increase in antibody levels against KoRV as the animals aged and progressed from dependent juveniles to adults, with antibody levels peaking at around six to eight years of age and then declining gradually as the koalas continued to age further. In addition, when we analyzed a set of koalas over time, a similar antibody pattern to the general population was observed. An age-related reduction in antibodies against HERV-K in women has recently been shown, with the authors suggesting anti-HERV-K serum antibodies can be an important parameter reflecting a person’s age53. When we examined specific epitopes over time in our study, we found juvenile animals (aged about one year) and elderly animals (nine years and above) recognized fewer epitopes compared to adult koalas (about four years of age). Since it has been shown that while the total number of B cells is relatively constant for most of life55, it is possible that age-related changes in antibody repertoire quality may be responsible for changes in response to natural infection or vaccination56. Also, aging has been associated with a decreased diversity, and even loss of high affinity antibodies, upon immunization57.A relationship between age and host immunity has been shown in many infection models1. The CETTG motif is present in all infectious gammaretroviruses and has been shown to be more pathogenic than the CETAG/CGTAG motif present in KoRV-A and other endogenous gammaretroviruses7. We hypothesize that the presence of this more pathogenic motif in KoRV-B may lead to the induction of a stronger immune response, and hence the higher levels of anti-KoRV-IgG, seen in KoRV-B positive koalas in this study.In this study, we found that koalas with detectable levels of KoRV-B had slightly higher anti-KoRV IgG levels compared to koalas in which KoRV-B was not detected. A possible explanation for this observation could be that koalas harboring KoRV-B express more virus (due to the presence of more enhancers in its LTRs) and, therefore, more antigens, than those harboring solely KoRV-A. Another possible explanation is the presence of the CETTG motif in KoRV-B env protein58 capable of preventing virus replication59. An inverse relationship has been reported to exist between viral load and immune response in some instances60. Similar to these studies in other retroviruses, we observed an inverse relationship between total circulating KoRV load and anti-KoRV IgG levels. However, when we expanded this population-level survey to individual koalas tested over time, the viral load/anti-KoRV IgG relationship was less clear.Retroviral infections typically result in the induction of virus-specific antibodies62. This offers great promise for the KoRV antibodies to these epitopes; however further experiments should be set up to determine the actual neutralizing potentials of these epitopes in KoRV. A sequence alignment of the epitopes among different KoRV subtypes shows a high degree of sequence conservation. This is important as it suggests that these epitopes could be useful in designing a multivalent/multi-subtype vaccine against KoRV.Three epitopes recognized across all age groups were identified in this study, one within the fusion peptide proximal region and two within the immunosuppressive domain. Interestingly, these regions have been shown to induce neutralizing antibodies following vaccination in other retroviruses, such as FeLV, HIV-1 and PERV66. This sequence is present in KoRV p15E region epitope two (peptides 24–25) and was recognized by koalas in this study. Given the importance of this region to the host-virus interaction for many retroviruses, the strong koala antibody response to the CKS-17 region is highly encouraging and warrants further investigation.CKS-17, with the amino acid sequence LQNRRGLDLLFLKEGGL, is a highly conserved region on the immunosuppressive domain in many retroviruses. It has been shown to possess the ability to suppress cell-mediated immunity, inhibit interleukin 2, interferon gamma and tumor necrosis factor production, as well as inhibit lymphocyte proliferation and B cell activation42. We were interested in determining if the antibodies produced by koalas harboring endogenous and exogenous KoRV from the present study were different from the antibodies induced by vaccination of koalas believed to be harboring only exogenous KoRV. It has been suggested that optimizing vaccines to elicit a broad range of targets on a pathogen might result in a better protective response67. Some epitopes  in the immunosuppressive domain and the MPER region recognized by the vaccinated koalas were also recognized by the non-vaccinated koalas suggesting that natural KoRV infection may induce the same antibodies against KoRV as vaccination. We also found that vaccinated koalas recognized some epitopes (such as epitope 32) that were not recognized by non-vaccinated koalas. It remains to be seen if unique epitopes like this may explain the difference between natural and vaccine-induced response to viral infections.We recently reported an anti-KoRV vaccine based on the recombinant KoRV env protein capable of inducing neutralizing antibodies and reduction of viral load in koalas with KoRV infection68, makes this task even more urgent. This study showed that koalas with both endogenous and exogenous KoRV are not tolerant to KoRV infection, as they are able to make antibodies. We also report an inverse relationship between anti-KoRV IgG and KoRV viral load. These are important findings, as they highlight the possibility of developing a therapeutic vaccine to boost the natural anti-KoRV IgG response that may reduce circulating viral load. The distinct epitopes identified in this study can be used as the basis for the development of a multivalent KoRV vaccine. In conclusion, this study suggests that all koalas with KoRV may benefit from a vaccine designed to boost the natural antibody response in these animals.Critical to developing an efficacious therapeutic vaccine against KoRV is understanding how the koala’s immune system responds to natural KoRV infection. The fact that KoRV is ubiquitous in northern Australia 69. Also included in this study is a previously described group of KoRV-A positive southern koalas (n = 3) sourced from Adelaide, South Australia, and vaccinated with a tri-adjuvanted recombinant KoRV envelope protein vaccine42. These koalas received two doses of the vaccine subcutaneously, four weeks apart. Blood samples for the current study were obtained on day 0 (pre-vaccination) and 8 weeks post-vaccination. All procedures were approved by the relevant authorities . All experiments were performed in accordance with relevant guidelines and regulations.Koalas included in this study were from two wild koala populations, geographically 100 kilometres apart, Gold Coast City Region (GCCR) and Moreton Bay (MBR) in south east Queensland. Samples were collected from a total of 235 koalas between 2012 and 2017; 28 from GCCR and 207 from MBR. These koalas were captured, examined by qualified veterinarians, tagged and released back into the wild. Many of these koalas from the MBR population were recaptured at regular intervals, usually every six months, during the 5-year study period. Blood samples were collected during clinical examination and stored at -20 °C. Serum and plasma were obtained from whole blood and stored at -20 °C. A more detailed description of sampling procedure has been previously reported69. Briefly, DNA was extracted from 200 µl of whole blood using Qiagen’s QIAamp DNA minikit following manufacturer’s instructions. KoRV subtypes A and B were screened for using a universal forward primer (5′-TCCTGGGAACTGGAAAAGAC-3′) and subtype-specific reverse primers . Standard PCR was carried out using the HotStarTaq Plus master mix kit (Qiagen).Using genomic DNA extracted from whole blood, all koalas in this study were screened for KoRV infection following our previously described protocolpol gene as the PCR target was carried out by modifying existing protocols from Waugh et al.16. Briefly, Viral RNA was extracted from plasma stored in RNALater using the Qiagen viral RNA mini kit. Contaminating DNA was removed using TURBO DNA-free . cDNA synthesis of RNA prepared from plasma was conducted using iScriptTM cDNA synthesis kit . Viral RNA load was thereafter quantified using Tarlinton et al.4 pol gene primers (forward: 5′- 5′-TTGGAGGAGGAATACCGATTACAC-3′ and reverse 5′-GCCAGTCCCATACCTGCCTT-3′) using iTaq™ Universal SYBR® Green Supermix . Standards of known concentration (108 to 101 copy numbers) of purified KoRV pol gene PCR product were prepared for each run and the results were normalized against the standard curve. PCR reactions were carried out on a CFX 96 Touch System  with the following cycling conditions: initial denaturation of 95 °C for 5 min, and then 40 cycles of denaturing at 94 °C for 15 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. All procedures were carried out following the manufacturer’s instructions.Quantification of circulating total KoRV viral RNA using the 42 were used in this study. The first ELISA was used for measuring anti-KoRV IgG in koala serum using a recombinant KoRV envelope (rEnv) protein  was interpolated from the line and reported as the end point titre. This was calculated with GraphPad Prism 6 .Two previously described ELISAs specific to anti-KoRV IgGein Fig.  as antig42.A second ELISA measured koala IgG specific to a 31-amino acid (LKERLDKRQL EHQKNLSWYE GWFNRSPWLT T) synthetic peptide of KoRV that spans the membrane proximal external region (MPER) of the KoRV env protein. For this assay we report the optical density for a single dilution (1:1000) of the test serum samples minus the optical density of the blank wells (no sample controls). In order to accurately compare the anti-MPER IgG to the anti-rEnv IgG, we developed a single dilution variant of the anti-rEnv IgG ELISA (1:1000 of test sera), similar to the anti-MPER IgG ELISA. This extra assay was set up to ensure the antibody levels against the different test antigens used for comparison were obtained using similar methodology. Assay background was determined for all assays by calculating the mean plus two times the standard deviation of serum IgG titers of three KoRV negative koalas. The KoRV negative koalas were sourced from Adelaide, South Australia and were screened and confirmed negative for KoRV using a genomic DNA conventional PCR assay targeting the pol gene, and quantitative PCR of viral RNA targeting the KoRV-A and KoRV-B env gene as per42. The plates were incubated for two hours at room temperature and thereafter washed four times with phosphate buffered saline (PBS) containing 0.1% Tween-20 (PBST). Sera was added to the plates at 1:1000 and incubated overnight at 4 °C. The plates were thereafter washed four times with PBST. Sheep anti-Koala IgG70 was added at a dilution of 1:8000 and followed by a one-hour incubation at room temperature and four washes with PBST. Secondary antibody, donkey anti-sheep HRP IgG , was added at a dilution of 1:5000 in PBS, incubated for 1 hour at room temperature and subsequently washed four times with PBST and two times with PBS. Fifty µl of Tetramethylbenzidine Liquid Substrate (Sigma-Aldrich) was added to each well and the reaction was stopped after 10 minutes using 50 μl of 1 M H2SO4. Optical density was read at 450 nm  and titres thereafter calculated (GraphPad Prism 6).Forty-eight overlapping 15-mer peptides with three amino acids offset were made using the amino acid sequence representing the ectodomain of p15E subunit of KoRV env protein Fig. . The pepSupplementary Information


Myocardial fibrosis is a common pathophysiological process that is related to ventricular remodeling in congenital heart disease. However, the presence, characteristics, and clinical significance of myocardial fibrosis in Ebstein’s anomaly have not been fully investigated. This study aimed to evaluate myocardial fibrosis using cardiovascular magnetic resonance (CMR) late gadolinium enhancement (LGE) and T1 mapping techniques, and to explore the significance of myocardial fibrosis in adolescent and adult patients with Ebstein’s anomaly.Forty-four consecutive patients with unrepaired Ebstein’s anomaly , and an equal number of age- and gender-matched controls, were included. A comprehensive CMR protocol consisted of cine, LGE, and T1 mapping by modified Look-Locker inversion recovery (MOLLI) sequences were performed. Ventricular functional parameters, native T1, extracellular volume (ECV), and LGE were analyzed. Associations between myocardial fibrosis and disease severity, ventricular function, and NYHA classification were analyzed.P < 0.001). An increased ECV was found to be independent of the existence of LGE. Positive LGE or higher ECV (≥30%) was associated with larger fRV volume, aRV volume, increased disease severity, and worse NYHA functional class. In addition, ECV was significantly correlated with the LV ejection fraction (P <  0.001).LGE was found in 10 (22.7%) patients. Typical LGE in Ebstein’s anomaly was located in the endocardium of the septum within the right ventricle (RV). The LV ECV of Ebstein’s anomaly were significantly higher than those of the controls (30.0 ± 3.8% vs. 25.3 ± 2.3%, Both focal and diffuse myocardial fibrosis were observed in adolescent and adult patients with Ebstein’s anomaly. Increased diffuse fibrosis is associated with worse LV function, increased Ebstein’s severity, and worse clinical status. Ebstein’s anomaly is a rare congenital heart disease. It is considered a right ventricular (RV) myopathy with failure of delamination of the tricuspid leaflets. Ebstein's anomaly is associated with considerable tricuspid regurgitation and the formation of an atrialized right ventricle (aRV) –4. ComprCompared with the tricuspid valve and RV abnormalities, the left ventricle (LV) of Ebstein’s anomaly is less studied. LV dysfunction was found in 7–50% patients with Ebstein’s anomaly and was associated with worse prognosis after surgery . The mecCardiovascular magnetic resonance (CMR) is a powerful non-invasive imaging modality to evaluate myocardial fibrosis. Late gadolinium enhancement (LGE) is an established CMR technique that provides accurate quantitation of myocardial replacement fibrosis. Recent advances in CMR native T1 mapping have provided us with a groundbreaking tool for quantification of diffuse myocardial fibrosis. The extracellular volume fraction (ECV) calculated from pre and post contrast T1 mapping images showed a strong correlation with collagen volume fraction histologically obtained from endomyocardial biopsies , 14. MyoConsecutive patients with unrepaired Ebstein’s anomaly referred to our institution for clinical evaluation gave their consent and were enrolled into a registry since June 2013. All patients received a comprehensive evaluation including a detailed medical history, physical examination, oxygen saturation test, routine blood examination (including serum creatine), electrocardiography, echocardiography, and CMR imaging. Patients in our registry database from June 2013 to Oct 2016 were reviewed and patients with complete data sets and those older than ten years of age were included in the study. Exclusion criteria were as follows: 1) Contraindications to contrast-enhanced CMR; 2) other coexisting cardiomyopathy, such as hypertrophic, restrictive, or infiltrative cardiomyopathy; 3) a history of known coronary heart disease or myocarditis; 4) other complex congenital cardiac anomalies ; 5) CMR image quality was too poor to analyze due to significant arrhythmia or difficulty with breath-holding. Patients were grouped into Carpentier’s classification of types A to D, based on the mobility of the tricuspid leaflet and the RV shape and contractility on echocardiography .An equal number of age- and gender-matched subjects were included as controls. Healthy adults with no documented cardiovascular diseases or other major illnesses, and with normal echocardiograms, were recruited as volunteers. Subjects younger than 18 years old were selected for CMR examination to screen for myocarditis. Subjects were included as controls if all clinical data, routine blood work, electrocardiography, echocardiography, and CMR examination excluded cardiovascular disease. All patients and controls (or their parents if a subject was younger than 18 years old) gave written informed consent for their data to be included in this study.3 and slice thickness of 8 mm. The inversion time for each patient was chosen at the time of scanning from the TI scout images, with three heartbeats for recovery between each experiment. Native T1 measurements were acquired before injection of gadolinium, with an imaging scheme of 5(3)3), and post contrast T1 measurements were repeated in the same short-axis slices 10–15 min after the administration of gadolinium with a scheme of 4(1)3(1)2. The hematocrit was tested within 24 h of CMR scanning for ECV calculation.CMR was performed on a 3 T scanner  with using a 32-channel phased-array cardiac coil. All images were acquired with electrocardiogram (ECG)-gated breath-hold technique. All patients were scanned according to the standard imaging protocol for Ebstein’s anomaly . The proCMR images were analyzed using a commercially available software package . Biventricular volume and function were obtained by tracing epicardial and endocardial contours manually on both end-diastolic and end-systolic phases. The LV volume, function, and mass were measured based on the consecutive short-axis slices. RV volume and function were derived from the axial views, as described in previous studies , 24. AllLGE images were visually assessed on phase sensitive inversion recovery (PSIR) images at the short- and long-axis views by two experienced CMR imagers . The myocardial T1 calculation was based on the LV short axis MOLLI images with motion correction. The endocardial and epicardial contours were traced manually on the pre-contrast T1-mapping images and post-contrast T1-mapping images. The T1 curve was fitted automatically using the software , and the mean myocardial T1 was obtained. At the same time, the blood T1 was obtained by drawing an ROI in the blood pool within the LV cavity in the pre-contrast and post-contrast T1-mapping images, respectively. The ECV was calculated as :\documenIn our study, the severity index (SI) and total right/left-volume index  were utilized to quantify the severity of EA. SI, described by Fratz et al., was calculated using the area of the RA, aRV, fRV, left atrium (LA), and LV traced in end-diastole on the four-chamber view , 2, 25. Total R/L-Volume Index = volume of 2 test or the Fisher’s exact test as appropriate. Correlations between variables were analyzed by using Pearson’s or Spearman correlation analyses. Univariable factors related to the ECV at p <  0.1 were included in the multivariate stepwise regression analysis. Statistical significance was defined as P <  0.05.Statistical analysis was performed using SPSS 17.0 . Numerical data in normal distribution were expressed as the mean ± standard deviation (SD), or else were expressed as the median with the interquartile range. Continuous data were compared using a paired t-test. Categorical data were expressed as frequencies and compared using a χn = 12), no contrast was used (n = 2), poor image quality caused by due to persistent atrial fibrillation or unable to breath hold (n = 10), and T1 mapping not performed (n = 10). The remaining 44 patients were included in the study. The mean age of the patients was 34.0 ± 16.2 years (range 10–60) years. Seven patients were between 10 and 18 years. Age, gender and BMI were well balanced between patients and the healthy control groups. Among the 44 patients, 29 (65.9%) had an atrial septal defect or patent foramen ovale. Tricuspid regurgitation was graded as severe in 32 (72.7%) and moderate in 12 (27.3%) patients  . LVEF was reduced (< 50%) in 34.1% (n = 15) of the Ebstein’s anomaly patients. The LV mass to volume ratio was not significantly different from the control group. Both total and fRV EDV indices and RV end systolic volume (ESV) index were significantly increased compared with those of the controls. The fRV EF in Ebstein’s anomaly was significantly reduced (P <  0.001).LV end diastolic volume (EDV) index was reduced in Ebstein’s anomaly patients  Table . LV ejecP value < 0.01). The fRV EF was lower in patients with LGE than without. The aRV volume was also significantly larger in the LGE-positive group than the LGE-negative group. Ebstein’s anomaly severity evaluated by the total R/L-volume index was more severe in patients with LGE than without LGE, while the SI was not significantly different between these two groups. Patients with LGE also had a higher NYHA functional class and lower oxygen saturation at rest than patients without LGE . The LV volume and the LV EF were lower in patients with LGE; however, the difference was not significant. All data are shown in Table LGE was seen in 10 out of 44 (22.7%) Ebstein’s anomaly patients. The typical LGE pattern was linear or patchy localized within the RV subendocardium of the basal septum . The septal ECV was higher than that of the free ventricular wall in Ebstein’s anomaly patients and free wall ECV was significantly higher than that of the controls (P < 0.001). Twenty (45.5%) patients had an ECV higher than 30%, which is above the upper limits of normal. Except for one a-60-year old, all subjects in the control group had an ECV less than 30%. In patients with a higher ECV, the Ebstein’s anomaly severity index, NYHA class and tricuspid regurgitation were significantly worse than in patients with a normal ECV. The LVEF was much lower in Ebstein’s anomaly patients with a higher ECV than those with a normal ECV . It is important to recognize that the ECV in patients without LGE was still significantly higher than that of the controls. The same trend was found . Multivariate correlation analysis showed that ECV was independently correlated with LVEF.Among all variables, the ECV of the LV myocardium was significantly correlated with NYHA class, tricuspid regurgitation severity, the LV ESV index, the LV EF, the fRV EDV index, the fRV ESV index, and the fRV EF Table . MoreoveTo the best of our knowledge, this is the first study to investigate diffuse myocardial fibrosis in Ebstein’s anomaly using CMR. The main findings are: (1) both focal myocardial fibrosis and diffuse myocardial fibrosis were detected by CMR in Ebstein’s anomaly patients. (2) Diffuse myocardial fibrosis was more common than focal myocardial fibrosis in Ebstein’s anomaly, and both were associated with the severity of Ebstein’s anomaly. (3) Diffuse myocardial fibrosis demonstrated by T1 mapping was closely associated with LV dysfunction.Ebstein’s anomaly has a wide spectrum of clinical manifestations, from early death in neonates to mild symptoms surviving into adulthood . Heart fThe finding of LV myocardial fibrosis in Ebstein’s anomaly provides another perspective on this question. In early studies, myocardial fibrosis was found in ex-vivo hearts of patients with Ebstein’s anomaly, in which histopathological examination demonstrated myocardial fibrosis existed in the fetus and neonate. Interstitial fibrosis was more common in both the RV LV and sporadic replacement myocardial fibrosis also existed –35. WhetIn the present study, both LGE and elevated ECV were observed in Ebstein’s anomaly patients. The typical LGE pattern found in this study was mostly localized in the endocardium of the aRV septum. This finding was in accordance with a previous case report . In one Both LGE and elevated ECV were related to the severity of Ebstein’s anomaly and ventricular dysfunction in this group of Ebstein’s anomaly patients. This finding emphasizes the importance of focal and diffuse myocardial fibrosis in ventricular remodeling in Ebstein's anomaly. LV remodeling was related to Ebstein’s anomaly severity, which demonstrated a close RV-LV interaction , 38. FurLV dysfunction is a high-risk factor in Ebstein’s anomaly and is associated with higher mortality after surgery . PreservIn our study, ECV was also associated with clinical and imaging-based ventricular function. Thus, ECV is a potential imaging marker for risk stratification in Ebstein’s anomaly patients. Future CMR evaluation for Ebstein’s anomaly should not only include volume and function analysis, but also consider myocardial tissue characterization using LGE and ECV.There are several limitations to our study. First, the present study consisted of both adolescent and adult patients, and does not represent the entire Ebstein’s anomaly spectrum. Second, the ECV of the RV myocardium was not quantified because the RV free wall was too thin in Ebstein’s anomaly for accurate assessment. Third, the cross-sectional study design limited the ability to determine the impact of myocardial fibrosis on the long-term prognosis of Ebstein’s anomaly. Our study indicated that further longitudinal observation is needed to clarify the impact of myocardial fibrosis and remodeling in Ebstein’s anomaly patients.Focal and diffuse myocardial fibrosis assessed by LGE and ECV were observed in Ebstein’s anomaly patients. The elevated fibrosis was associated with worse LV function, greater disease severity, and reduced clinical status in Ebstein’s anomaly patients. LGE or ECV may serve as imaging markers for the severity of Ebstein’s anomaly. Further studies are needed to assess the prognostic significance of these findings.


Transcatheter Mitral Valve Repair (TMVRe) technologies constitute a rapidly expanding field, and have the potential of being adopted as a valuable alternative to surgery in selected patients. TMVRe devices can be distinguished depending on the targeted part of the Mitral Valve (MV) apparatus. Standard classification includes leaflet repair, direct/indirect annuloplasty, chordal repair, and ventricular/chamber remodeling devices. We present the current device situation on chordal repair technologies. Nowadays, transapical off-pump beating heart chordal implantation procedure has become a safe and reproducible option for Degenerative Mitral Regurgitation (DMR). Besides the truly minimally-invasiveness of the procedure, another unique advantage offered by a beating-heart chordal implantation is the real-time assessment of chordal length adjustment during heart cycle with a normally filled left ventricle. Currently, one system is commercially available in Europe, the NeoChord DS 1000  and the Harpoon TDS-5  should become available soon. There is also a diffuse and strong interest to move from a transapical procedure toward a fully transcatheter  procedure as shown by the increased number of preclinical programs under development. Interestingly, to achieve outcomes that equate to those of open surgery in DMR, transcatheter therapies will need to follow rigid indications due to strict patient selection criteria for each device, or adopt multiple techniques in a single repair procedure for complex MV disease. Continuous analysis of current clinical results together with future dedicated trial will be of extreme importance to foster the new and upcoming field of transcatheter MV therapy technology development. Mitral regurgitation (MR) is the most frequent valvular heart disease (VHD) requiring surgery in the United States and the second most common in Europe . CurrentIn general, principles of MVRe include preservation or restoration of leaflet anatomy, creation of a large surface of leaflet coaptation and remodeling of the annulus to provide an optimal and stable orifice area.In recent years, the classical leaflet resection techniques have been challenged by the introduction of more tissue-sparing approaches which include the implantation of artificial ePTFE chords to restore physiological leaflet behavior.The next step in the progress cycle, which is currently underway, is to move from surgical on-pump procedure toward beating-heart transcatheter solutions .TMVRe devices can be distinguished based on the targeted component of the MV apparatus and classified into leaflet repair, annuloplasty, chordal repair, and ventricular/chamber remodeling. This classification can also include some overlap .Transapical approaches have been recently introduced as well as new transfemoral devices currently in the developing phase.The Neochord DS 1000 device  has been the first transapical chordal implantation device available for clinical use in Europe –7. CE maThe procedure is performed under general anesthesia, through a left mini-thoracotomy access in the fifth-intercostal space with 2D/3D real-time TEE guidance. The left ventricle (LV) entry site is identified about 2–4 cm postero-lateral from the real apex in order to obtain a perfect posterior and symmetrical alignment with the papillary muscles . LV naviThe leaflet is pierced and the ePTFE chord is passed through the leaflet and retrieved together with the device from the LV. A girth knot is performed allowing for leaflet fixation of the chord. The procedure is repeated for the total number of Neochordae intended to be implanted. Under 2D and 3D TEE control, all the chords are tensioned until adequate leaflet coaptation is achieved, and the neochordae are then secured to the LV wall using a large Teflon pledget .NeoChord procedural safety has been evaluated by single centers studies showing low mortality  and morbidity, good acute procedural success (98.6%) . PatientMore recently Seeburger and coauthors presented the 5 years results of the 6 patients enrolled at University of Leipzig in the TACT trial. Three patients were converted to surgery because of procedural failure and in another 3 patients residual MR at 5 years was significantly less than moderate and experiencing good clinical condition with no symptoms .Based on the initial experience gained by early adopters the procedure underwent continuous technical refinement. LV access site was modified with a more postero-lateral access, echocardiographic views for navigation and grasping were standardized, tensioning was achieved with the use of tourniquets and epicardial stiff Teflon pledget. Moreover, patient selection criteria were refined combining echocardiographic measurement and morphology description.In particular MV morphology was characterized based on growing complexity as “Type A” isolated central posterior leaflet prolapse/flail, “Type B” posterior multi-segment prolapse/flail, “Type C” anterior or bi-leaflet prolapse/flail, “Type D” para-commissural prolapse/flail or any type of disease with presence of significant leaflet/annular calcifications. Outcomes are strictly connected with the morphological classification , 13.The central echocardiographic selection criteria became the leaflet-to-annulus index (LAI)  that evaAcute echocardiographic data analysis, demonstrated a significant reverse remodeling, both of the antero-posterior annular diameter and of the LV cavity volume; these findings were maintained at 1 year follow-up damage .ex-vivo pulsatile model that reproduces all the steps of the procedure with direct endoscopic and TEE control  is a sheeted 10 Fr device developed for transapical, beating-heart chord implantation. As for Neochord DS 1000, the procedure is performed under 2D and 3D transesophageal echocardiography guidance. Harpoon system allows the implantation of an ePTFE specially designed chord that is fixed on the MV leaflet by using a preformed double-helix coil knot.Harpoon system is composed of a 14 Fr external diameter introducer with an inner hemostatic valve and a delivery system. The introducer sheet is inserted in the LV more anteriorly than in the Neochord procedure and fixed to the epicardial surface by using conventional “U-pledgets” purse strings. The delivery system contains a 21-gauge needle tightly wrapped with a pre-formed ePTFE bulky knot. When the tip of the delivery system is positioned under the target part of the diseased leaflet using 2D TEE guidance the knot is released by the penetration of the needle through the leaflet tissue. The needle is rapidly withdrawn and the ePTFE coil is tightened forming a double-helix on the atrial surface of the leaflet that secures the artificial chord  Figure .The system is then retrieved from the LV leaving outside the two ends of the ePTFE chord. The procedure can be repeated using a different delivery system for each implant. When the desired number of chords is reached, the introducer is removed and the pure-string sutures closed. Under 2D TEE guidance all the chords are tightened together to reach the desired final coaptation and then are secured to the epicardium with a large Teflon pledget as previously described .Harpoon MVRS received CE mark approval in late 2017 but it is not yet commercially available. The TRACER trial  was conducted to test safety and efficacy of the device. Thirty patients were enrolled in 6 different European Centers. All patients presented severe degenerative MR due to isolated P2 disease. Patient population was highly selected based on presence of an adequate ratio between the posterior prolapse length and the corresponding antero-posterior distance between the free edge of the anterior leaflet and the base of the prolapsed posterior leaflet segment. The ratio should be greater than 1.5, meaning that the redundancy of the tissue should be extremely significant.Six-month follow-up data were published , showingMitralStitch  is a transapical device for beating heart chordal implantation. A pledgeted ePTFE chord is implanted directly in the body of the leaflet avoiding direct suture loop or knots . A speciChordArt  is a transcatheter mitral repair system. It consists of 3 components: a proximal nickel-titanium anchor for leaflet securement, a distal ventricular/papillary muscle anchor and the ePTFE chord. This special configuration has been proposed to be easily translated into a percutaneous transfemoral-transeptal delivery catheter. The device is currently under clinical evaluation using traditional surgical on pump approach . The deployment procedure develops in a stepwise fashion. The leaflet is reached by an antegrade approach and grasped. The leaflet is punctured and the needle tip is passed through the leaflet till the papillary muscle where the first anchoring system is implanted. The needle is then retrieved and the leaflet anchor is released restoring normal leaflet coaptation .Clinical data of the first surgically implanted device are not yet available.V-Chordal Adjustable Artificial Chordae System , is a surgical transcatheter technology allowing on-pump chordal implant with off-pump beating heart length adjustment  Figure . ThroughDespite clinical-feasibility study has been completed on 6 patients, the transfemoral approach has not been further developed.Pipeline  is a transcatheter device designed for off-pump, beating heart chordal replacement. As seen for other devices, transfemoral-transseptal way is used to access the left atrium and drive a guidewire across the MV inside the LV.Once driven on the site, Pipeline is advanced up to the papillary muscles. First, a distal ventricular/papillary muscle helical anchor is deployed. The leaflet is than punctured and the leaflet fixation system is folded up by pulling the artificial Neochord forming this way tissue anchoring. The artificial Neochordae results connected at one side to the ventricle wall by means of the distal anchor and at the other side to the leaflet through the auto-deploying system. The third step consists in suture length adjustment and locking. Under 2D/3D TEE guidance a suture lock device is delivered inside the LV. The chord is then tensioned in order to reach the best coaptation and consequent MR reduction. The residual part of the suture is then cut, disconnecting the device from the LV anchor . The devCardioMech  is developing a percutaneous solution for artificial chords implantation. Currently there are no available information about phase of development of the project. The Company website describes a length adjustment device delivered via transvenous-transeptal approach. It comprises a gripper element housing a self-expandable folded anchor made of memory shape material. When the leaflet is grasped, the anchor is unfolded and is secured to the leaflet by piercing it.The catheter device also holds a self-expandable folded papillary anchor made by shape memory metal. The chord extends from the leaflet anchor to the papillary anchor. The chord length is adjustable under real-time echocardiographic guidance. Interestingly the excess of chord length in the atrium is then cut and all catheters are withdrawn .The ChoRe is an underdevelopment device for transfemoral implantation of chords with an epicardial fixation. The procedure steps can be divided in apex fixation, leaflet fixation, and a length adjustment. In the first step of the procedure, a needle puncture the ventricular wall providing externalization of the chords on the epicardium surface together with a folded pledget . In the ex-vivo tests have been performed. A scaled-up size prototype  has been tested ex vivo in bovine hearts  is a concept technology that can hold and capture the entire prolapsing MV leaflet segment restoring its normal geometry  The deviThe wide variability of MV morphologies drives continuous development of technologies to treat the full spectrum of MR pathophysiology. Consequently a variety of transcatheter MV devices for the percutaneous treatment of MR have been developed. The chordal device spectrum is composed of a big player with a solid clinical experience and many new devices that will enter into clinical practice soon as well as new early phase devices that are still under development. Many companies are working on percutaneous solutions in order to minimize invasiveness as well as to create a more physiological fixation at the level of the papillary muscle or in the base of the LV in between the papillary muscles.Careful patient selection remains the basic step foregoing any TMVRe technology, and this concept could be even more relevant considering the Chordal Repair therapy. Because of the multifaceted presentation of MV disease, traditional surgical procedures have always combined different leaflet and annular therapies. The current solid surgical background will stimulate the already presented MV transcatheter repair tool-box concept , 27 for All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.AC received travel grants from NeoChord Inc, GS is the inventor of the NeoChord DS 1000 device and is a shareholder of NeoChord Inc. The remaining 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.


Atrial remodeling by amyloid infiltration may contribute tocardiac output decrease by means of insufficient or nonexistent telediastolic atrialcontraction.3 The onset ofatrial electrical instability, ending in atrial fibrillation, highlights symptomaticworsening and these patients' reserved prognosis.4Immunoglobulin deposition in the myocardium characterizes involvement of cardiacamyloidosis (CA).5 The developmentof three-dimensional echocardiography (3DE) has allowed for a more accurate calculationof right ventricle volume and function in the diverse pathologies involving thatchamber.6Historically, the right cavities of the heart have been neglected in echocardiographicassessments. The complex morphology of the right ventricle (RV) has possibly contributedfor the lack of reproducible data on echocardiographic cutting plans, diversely from theleft ventricle (LV).9Nevertheless, its asymmetric shape, increased by the occurrence of remodeling, asobserved in CA cases, limits a more precise assessment of its volume usingtwo-dimensional echocardiography (2DE).6 On the other hand, using three-dimensional echocardiography (3DE)overcomes these limitations, allowing not only for the accurate assessment of rightatrium volume changes, but also for the detailed description of its size andfunction.9Regarding the importance of assessing the right atrium (RA), the relation between anincrease in its area and adverse clinical outcomes has already been shown.10 fills the gap regarding the use of 3DE to assess the RA for thediagnosis of CA. The authors noted significant increase in the left atrium diameter, inthe interventricular septum thickness and in the LV posterior wall diameter, besides RVsystolic dysfunction in patients with light-chain cardiac amyloidosis (LC-CA), whencompared to healthy control group patients. These findings, compatible with restrictivecardiomyopathy, have been previously described for CA.2 Assessing the RA by three-dimensional Speckle Trackingechocardiography (3DSTE), increased atrial volumes and smaller fractions of total andactive atrial emptying in patients with LC-CA were found, when compared to control grouppatients. Furthermore, according to the authors, findings of reduced values on globalstrain peak and on segmental area, on circumferential strain in many levels, besideschanges in the longitudinal and area strain in atrial contraction, suggest longitudinale circumferential impairment in RA function in its reservoir and active contractionphases, as well as non-uniform atrial dysfunction. Although the authors have not managedto show differences on the stroke-volume values for RA when compared to healthycontrol-group patients, they describe the importance of measuring the RA emptyingfractions and the strain-values for a proper LC-CA diagnosis.In this context, the study by Nemes et al.,11 studied longitudinalstrain in heart cavities with the purpose of checking if change in a given cavity wouldhave a higher prognostic value than traditional echocardiographic parameters regardingthe occurrence of adverse cardiac events. Prognostic relevance was found on strainchanges in the four cavities, also the RA longitudinal strain was capable ofdifferentiating LC-CA from non-obstructive hypertrophic cardiomyopathy.Kado et al.,10 did not permit the conclusion whether the changes described bymeans of 3DSTE would be LC-CA-specific or if they could be found in another type ofinfiltrative/restrictive cardiomyopathy. On the other hand, it drives our attention tothe need of a more detailed assessment on the right side of the heart, regardless of theunderlying disease investigated.However, the drawing of the study by Nemes et al.At last, the appearance of innovations on echodopplercardiography, which always occurtowards diagnosis improvement or accuracy, as well as to make early LC-CA therapy forthe prevention of adverse clinical outcomes, one must not underestimate alreadyestablished, traditional echocardiographic findings for disease assessment.


GAL, LMO4, STAT3, PD-L1, TGFB and TGFBR3) were proposed for PA immunotherapy. The strategy proposed together with the immune-related markers identified in this study provided a useful guidance for the development of novel immunotherapy for PA.Pituitary adenoma (PA) is prevalent in the general population. Due to its severe complications and aggressive infiltration into the surrounding brain structure, the effective management of PA is required. Till now, no drug has been approved for treating non-functional PA, and the removal of cancerous cells from the pituitary is still under experimental investigation. Due to its superior specificity and safety profile, immunotherapy stands as one of the most promising strategies for dealing with PA refractory to the standard treatment, and various studies have been carried out to discover immune-related gene markers as target candidates. However, the lists of gene markers identified among different studies are reported to be highly inconsistent because of the greatly limited number of samples analyzed in each study. It is thus essential to substantially enlarge the sample size and comprehensively assess the robustness of the identified immune-related gene markers. Herein, a novel strategy of direct data integration (DDI) was proposed to combine available PA microarray datasets, which significantly enlarged the sample size. First, the robustness of the gene markers identified by DDI strategy was found to be substantially enhanced compared with that of previous studies. Then, the DDI of all reported PA-related microarray datasets were conducted to achieve a comprehensive identification of PA gene markers, and 66 immune-related genes were discovered as target candidates for PA immunotherapy. Finally, based on the analysis of human protein–protein interaction network, some promising target candidates ( Pituitary adenoma (PA) accounts for approximately 8–15% of intracranial tumors ,3 and itHowever, those approved therapeutics primarily focus on managing the complication of PA rather than PA itself ,18, whicGAL) is substantially suppressed in the tissue of PA patients compared with those in healthy pituitaries [PD-L1, the immunosuppressive protein) are observed in tumor tissue of PA patients [+ lymphocytes [TGFB) was discovered as one of the chief immune suppressive mediators of prolactin (PRL) secretions and PA proliferation [Although no immunotherapy of PA has been approved yet, various studies have been carried out to discover immune-related genes or target candidates. Particularly, as an immunomodulatory peptide, protein galanin  was proposed to combine available PA microarray datasets for significantly enlarging the sample size. To test the impact of the DDI strategy on the classification ability and the robustness of identified DEGs, its performance and disease relevance were first evaluated by comparing with previously published datasets. Then, all currently available PA-related microarray datasets were directly integrated to achieve comprehensive identification of DEGs between PA patients and healthy individuals. Finally, the immune-related genes were annotated from DEGs, which could be further studied as target candidates for PA immunotherapy. The strategy proposed together with the immune-related DEGs identified in this study provided a useful guidance for future immunotherapy.Several studies have been conducted to identify the DEG capable of distinguishing PA patients from healthy people ,52,53. Dp-value < 0.05). Top-ranked DEGs (top five for upregulated and another top five for downregulated) and their relevance to PAs were reviewed by comprehensive literature review. As shown in As the first assessment, the level of PA relevance was reviewed and discussed for three different analytical strategies. Three lists of DEGs were identified using three different strategies by the linear models and empirical Bayes , sensitivity (SEN), specificity (SPE), Matthews correlation coefficient (MCC), receiver operating characteristics (ROC), and area under ROC curve  [ACCs, SENs, SPEs, and MCCs were in the ranges of ~0.67–0.92, ~0.50–0.88, ~0.75–1.00, and ~0.35–0.84 among strategies, respectively. The metrics ACC and MCC were frequently used in current Omics study to evaluate correctness [ACC of DDI strategy (0.92) was substantially higher than that of the single dataset-based strategies (both are 0.67). Similar to ACC, MCC of DDI strategy (0.84) was discovered to be higher than that of the other two .Classification models are frequently constructed in current transcriptomics studies for predicting samples of various disease status ,66 or asC value) ,70,71. Arectness  and stabrectness  of the cACC and MCC, the ROC and AUC were two other popular metrics widely used to assess ability for classification, which were acknowledged to achieve comprehensive performance evaluations. As shown in ROC curves and AUC values of three strategies were compared. Grey diagonals represented an invalid model with the corresponding AUC value equaled 0.5. As shown in AUC value of the DDI strategy (0.97) was substantially higher than that of the others , which were similar to the results assessed by ROC curves. In conclusion, this finding indicated that classification correctness  and prediction stability  of DDI strategy were found consistently better compared with those strategies based on single dataset.Apart from overlap value had been recognized as the quantitative measure of the robustness of identified DEGs [overlap values represented the more robust DEGs identified by a given strategy. Herein, one sub-dataset was first generated by randomly selecting 2/3 of both cases and controls in the studied dataset, and ten iterations of the same procedure generated ten sub-datasets. For each sub-dataset, a list of DEGs were then identified by LMEB method , and the value of overlap between any two sub-datasets was calculated using their corresponding lists of DEGs. In total, there were 45 . The median overlap of DDI strategy (0.69) was found larger than that of the other strategies .Apart from prediction capacity simultaneously assessed by classification correctness and prediction stability, the robustness of identified DEGs was widely accepted as an additional key metric with its underlying theory distinct from that of prediction ability ,74. So fied DEGs . The higoverlap, the statistical difference of 45 overlap values between different analytical strategies was more meaningful to reveal their level of robustness. Therefore, the comprehensive statistical comparison of robustness among strategies was conducted and illustrated in Overlap values of three strategies were colored in blue, green and orange. Apart from the enhanced median value of overlap by DDI strategy, all overlap values of DDI strategy were found significantly higher  than that of the other two. Particularly, as illustrated in p-value) were all lower than 1.00 × 10−3 . Moreover, the majority of the overlap values of DDI strategy were larger than 0.6, while that of the other strategies were lower than 0.6. This finding indicated that the DDI strategy performed better than others in the robustness of the identified DEGs. Additionally, Compared with the median value of D , a total of 370 DEGs were discovered. As shown in TSHB was found to be absent in most PA patients [GAL was reported to be rarely expressed in somatotroph adenoma and prolactinoma [GAL was substantially suppressed in the tissue of PA patients compared with that in healthy pituitary [CEL was identified to regulate the hormone secretion in adenomatous pituitary cells [PRL and under-expression of RPS29 were observed in pituitary adenomas [c-Fos overexpression was found to be much less common in pituitary tumors and did not necessarily correlate with the ability of tumor to become invasive [PMAIP1 as important in the tumorigenesis and progression of PAs [AGR2 level was significantly higher in the serum of PA patients than the patients with other sellar lesions, which suggests AGR2 a potential marker for PAs’ diagnosis [p-values. Due to their close relationship with human immune system and the differential expression between PA patients and healthy individuals, they are potential target candidates for PA immunotherapy.The discussion above testified the effectiveness of the adopted feature selection algorithm and revealed the superior robustness and classification ability of the proposed DDI strategy compared with that based on the single dataset. To maximumly enhance the prediction power of constructed model, the most comprehensive set of PA-related microarray data were collected  and inteD . To the patients . Differeactinoma . The expituitary . The CELry cells . The higadenomas . The c-Finvasive . Pathwayn of PAs . The seriagnosis . By annoMAPK1, FOS and HSP90AA1 were the top three immuno-candidates with the highest connectivity (network degree) to other human proteins. Moreover, galanin (GAL) was identified as greatly downregulated DEGs in the pituitary glands of PA patients. Due to it immunomodulatory property in PA, the GAL could be expected as a promising target candidate for PA immunotherapy, and this finding was in accordance to previous work [LMO4 was identified in this study, which induced STAT3 activation in brain [PD-L1) [PD-L1 was observed in the pituitary gland of PA patients [LMO4/STAT3/PD-L1 signaling could block the immune evasion by recovering the recognition and elimination of cancer cells via CD8+ lymphocytes [TGFB) was reported to be one of the chief immune suppressive mediators of PRL secretion and PA proliferation [TGFBR3, was also discovered in this study as a DEG, which could be another target candidate for developing novel PA immunotherapy.The human protein–protein interaction (PPI) network analyses  further ous work . The dysin brain  and subs [PD-L1) . Since tpatients , the inhphocytes . Furtherferation ,50. Its Pituitary glands were wildly recognized as the major locus of PA’s dysfunction . In thisR environment (available online: http://www.r-project.org). The raw data (the CEL file) was first read, log transformed, and normalized, and all parameters were set as default. Then, outliers in each dataset were checked and removed, and all probe sets were mapped to their corresponding genes by Bioconductor [R package limma [p-value < 0.05 were identified as the differentially expressed genes between case and control samples (The direct data integration (DDI) strategy combining multiple datasets was carried out in the onductor . Averageonductor ,88,89  was conducted by evaluating the relevance level to PAs of the identified DEGs and by assessing the robustness of marker discovery and the classification ability of the constructed models.p-value < 0.05) [p-value < 0.05) [For complex disorders like PA, the identified DEGs are expected to contain a substantial percentage of PA-related genes , and a c < 0.05) . Accordi < 0.05) . The levTP), true negative (TN), false positive (FP), false negative (FN), accuracy (ACC), sensitivity (SEN), specificity (SPE), Matthews correlation coefficient (MCC), receiver operating characteristics (ROC) curve, and the area under ROC curve (AUC). All these metrics were frequently used in current Omics researches [The classification abilities of the DEGs identified by three different strategies , and the overlap value between any two sub-datasets was calculated using their corresponding lists of DEGs identified. In total, there were 45 . Then, GO terms were used to annotate and identify immune-related DEGs. Moreover, the human protein–protein interaction (PPI) network analysis was introduced to explore the connection among identified DEGs [Due to the substantially enhanced robustness and classification capacity of the DDI strategy discovered in this study, the DDI of all seven datasets  was condied DEGs . In partied DEGs  was adopied DEGs  for visuGAL, LMO4, STAT3, PD-L1, TGFB and TGFBR3) were further proposed for PA immunotherapy.In this study, the direct data integration strategy was proposed, and its classification ability and robustness were assessed in this study. Compared with the traditional strategy based on a single dataset, the DDI strategy was found to greatly enhance the robustness of identified DEGs, which may be attributed to the significant enlargement of the sample size. Based on the comprehensive analysis of the available PA-related transcriptomics data, 370 DEGs were identified, and 66 immune-related DEGs were annotated and proposed as target candidates for PA immunotherapy. Using the PPI network analysis and literature review, several promising candidate targets (


Intraoperative or post procedure right ventricular (RV) dysfunction confers a poor prognosis in the post-operative period. Conventional predictors for RV function are limited due the effect of cardiac surgery on traditional RV indices; novel echocardiographic techniques hold the promise to improve RV functional stratification.Comprehensive echocardiographic data were collected prospectively during elective cardiac surgery. Tricuspid annular plane systolic excursion (TAPSE), peak RV systolic velocity (S′), and RV fractional area change (FAC) were quantified on transesophageal echo (TEE). RV global and regional  longitudinal strain was quantified using speckle-tracking echo in RV-focused views. Two intraoperative time points were used for comparison: pre-sternotomy (baseline) and after chest closure.p < 0.001 for all). However, left ventricular (LV) and RV hemodynamic parameters remained unchanged (LV ejection fraction (EF): 56.8 ± 13.0 vs. 55.8 ± 12.9%; p = 0.40, pulmonary artery systolic pressure (PASP): 26.5 ± 7.4 vs 27.3 ± 6.7 mmHg; p = 0.13). Speckle tracking echocardiographic data demonstrated a significant decline in RV global longitudinal strain (GLS) . Pre-procedure FAC, GLS and free wall strain predicted RV dysfunction at chest closure , whereas traditional linear RV indices such as TAPSE and RV S′ at baseline had no impact on intraoperative RV dysfunction (p = NS for both).The population was comprised of 53 patients undergoing cardiac surgery , among whom 38% had impaired RV function at baseline defined as RV FAC < 35%. All conventional RV functional indices including TAPSE, S′ and FAC declined immediately following CPB (1.5 ± 0.3 vs.1.1 ± 0.3 cm, 8.0 ± 2.1 vs. 6.2 ± 2.5 cm/s, 36.8 ± 9.3 vs. 29.3 ± 10.6%; Global and regional RV function, as measured by 2D indices and strain, acutely decline intraoperatively. Impaired RV strain is associated with intraoperative RV functional decline and provides incremental value to traditional RV indices in predicting those who will develop RV dysfunction. RV dysfunction is common; it occurs in nearly one fourth of patients who undergo cardiac surgery. Intraoperative or post procedure RV dysfunction is linked to high post-operative mortality , 2. HoweMyocardial deformation via two-dimensional speckle tracking echocardiography (2D STE) provides incremental value in prognostic stratification as compared to traditional indices in a wide range of cardiac conditions including heart failure, coronary artery disease and pulmonary hypertension –9. HowevPatients were enrolled prospectively from 11/2017 to the present as part of an established protocol  at Weill Cornell Medical College .Comprehensive TEEs were performed prior to sternotomy (baseline) and after chest closure (post procedure) using commercial equipment  and Phillips iE33  echo systems). TEEs were interpreted by experienced investigators within a high-volume laboratory, for which expertise and reproducibility for quantitative RV indices have been validated , 14.RV systolic function was quantified using tricuspid annular plane systolic excursion (TAPSE), right ventricular systolic myocardial velocity (RV-S′) and fractional area of change (FAC), which were acquired in accordance with consensus guidelines . TAPSE w2D echocardiographic images were analyzed offline for deformation analysis . Strain measurements were performed by an investigator experienced in the interpretation of echocardiographic images, blinded to the results of the 2D measurements. Each image was acquired in the mid-esophageal four-chamber view of the whole RV cavity and with a frame rate > 50 Hz for analysis of RV strain. The RV end-diastolic endocardial border was manually traced and tracked automatically frame-by-frame throughout the cardiac cycle. Endocardial contour was manually adjusted when necessary to optimize tracking.p < 0.05 was considered indicative of statistical significance.Comparisons between groups were made using Student’s t-test (expressed as mean ± standard deviation) for continuous variables. Categorical variables were compared using Chi-square or, when fewer than 5 expected outcomes per cell, Fisher’s exact test. Bivariate correlation coefficients, as well as regression analyses were used to evaluate univariable associations between continuous variables. Multivariate modeling was performed via logistic regression and echo indices were tested as continuous variables. Inter-rater and intra reliability was calculated using the intraclass-correlation coefficient. Statistical calculations were performed using SPSS 22.0 . Two-sided p = 0.15).The study population included 53 patients who underwent cardiac surgery with dedicated TEE for RV assessment at baseline and post procedure. Intraoperative RV dysfunction was defined as RV FAC < 35% post procedure. In this population, 38% had RV dysfunction at baseline and over two thirds (70%) had some degree of RV dysfunction post procedure. Table p < 0.05 for all). In support of the concept that RV functional decline is driven predominantly by effects on the RV free wall, there was no significant change in post procedure interventricular septal deformation (p = 0.23) . On the other hand, traditional linear RV indices such as TAPSE and S′ had no impact on post chest closure RV dysfunction . Figure Table 23) Fig. . When exp = 0.04) even after controlling for septal wall strain. These results indicate that post procedure RV dysfunction is closely linked to altered RV free wall contraction rather than interventricular septal function. Table Regarding LV parameters, there were no differences in baseline or post chest closure LV volumes and LV ejection fraction when stratified by intraoperative RV dysfunction. This finding adds to the concept that intraoperative RV dysfunction occurs independently of LV performance. Hemodynamic indices, including pre and post pulmonary artery (PA) and right atrial (RA) pressures, also had no association with development of RV dysfunction after chest closure. Table The primary aim of this study was to characterize and predict the acute intraoperative change in RV function using established deformational RV techniques. The findings of this study are as follows: 1. RV dysfunction occurs in over two thirds of patients undergoing cardiac surgery and is associated with impaired RV global longitudinal strain but not conventional 2D RV parameters at baseline. 2. RV functional decline is predominantly associated with impairments in RV free wall deformation rather than that of the interventricular septum. 3.Whereas deformational indices predict intraoperative RV dysfunction, traditional linear RV indices had no association with post-operative RV dysfunction.While RV function is an important determinant of cardiac surgical outcomes, there are few known predictors of acute intraoperative dysfunction. Studies demonstrate that RV FAC < 35% is associated with the greatest risk of post-operative mortality , 17 and Previous studies demonstrate a decline in RV function measured by 2D indices including TAPSE and S′ with transthoracic echocardiography (TTE) 3–6 months after surgery. This may be the result of intraoperative factors such as ischemia, pericardial opening, and myocardial stunning secondary to poor protection during CPB . HoweverAt present, 2D STE is a relatively new imaging technique that is not routinely used intraoperatively. Unlike Doppler interrogation, strain measured by 2D is angle independent. Therefore, it may be more accurate and easily applied than tissue Doppler interrogation (TDI). However, 2D STE is dependent on image quality and has lower temporal resolution than TDI. While 2D STE is used to demonstrate subclinical LV dysfunction independent of changes in ejection fraction, it is not yet routinely used to characterize RV dysfunction. Our study demonstrates that it is feasible to use strain as a comparable index to predict RV function in the intraoperative period. We found that free-wall strain, septal strain, global strain, and baseline deformation predicted intraoperative dysfunction .Our study had several limitations. First, 3D RV imaging was not performed routinely as part of this protocol and therefore the effect of cardiac surgery on RV volumes and global EF is not assessed here. Second, the impact of RV dysfunction on post-operative morbidity and mortality is not assessed though it should be noted that intraoperative RV dysfunction has been linked to adverse outcomes in prior studies , 2. LastGlobal and regional RV function, as measured by 2D indices and strain, acutely decline intraoperatively. Moreover, 2D STE of the RV at baseline predicts intraoperative RV dysfunction whereas conventional techniques do not. These findings add to the growing body of literature demonstrating that RV function is significantly impaired during cardiac surgery and suggests that deformational imaging provides incremental value in predicting those who will develop RV dysfunction. The ability to predict this dysfunction could help identify those patients in whom support is needed intraoperatively.