Type II alveolar epithelial cell (AEC) apoptosis is a prominent feature of fibrotic lung diseases and animal models of pulmonary fibrosis. While there is growing recognition of the importance of AEC injury and apoptosis as a causal factor in fibrosis, the underlying mechanisms that link these processes remain unknown. We have previously shown that targeting the type II alveolar epithelium for injury by repetitively administering diphtheria toxin to transgenic mice expressing the diphtheria toxin receptor off of the surfactant protein C promoter (SPC-DTR) develop lung fibrosis, confirming that AEC injury is sufficient to cause fibrosis. In the present study, we find that SPC-DTR mice develop increased activation of caspase 3/7 after initiation of diphtheria toxin treatment consistent with apoptosis within AECs. We also find evidence of efferocytosis, the uptake of apoptotic cells, by alveolar macrophages in this model. To determine the importance of efferocytosis in lung fibrosis, we treated cultured alveolar macrophages with apoptotic type II AECs and found that the uptake induced pro-fibrotic gene expression. We also found that the repetitive intrapulmonary administration of apoptotic type II AEC or MLE-12 cells induces lung fibrosis. Finally, mice lacking a key efferocytosis receptor, CD36, developed attenuated fibrosis in response to apoptotic MLE-12 cells. Collectively, these studies support a novel mechanism linking AEC apoptosis with macrophage pro-fibrotic activation via efferocytosis and reveal previously unrecognized therapeutic targets. Mounting evidence implicates defects in the type II alveolar epithelial cell (AEC) in disease pathogenesis6. For example, histopathologic abnormalities of the epithelium including apoptosis are observed in tissue sections from IPF patients and in animal models of pulmonary fibrosis9. Furthermore, mutations in type II AEC genes including surfactant proteins A and C are linked to familial disease10. Finally, transgenic animal experiments from our laboratory confirm that targeted injury to the type II alveolar epithelium is sufficient to initiate lung scarring11.Progressive alveolar fibrosis is a serious complication of certain systemic inflammatory disorders, inorganic and organic dust exposures, drug toxicity and primary diseases of the lung including idiopathic pulmonary fibrosis (IPF)Despite the substantial evidence linking type II AEC injury/death to the development of fibrosis, the pathways that translate an epithelial insult into lung collagen accumulation have not been well-characterized. Possible mechanisms by which damage to the alveolar epithelium lead to fibrosis have focused on either loss of anti-fibrotic functions supplied by healthy cells or an up-regulation of pro-fibrotic factors from the injured AECs. An alternative mechanism supported by emerging evidence suggests that the apoptotic AECs can directly trigger progressive fibrosis by inducing a response in neighboring cells.12. For example, in models of acute lung injury, efferocytosis of apoptotic neutrophils has emerged as a key pathway in regulating macrophage function and restoring homeostasis by promoting release of anti-inflammatory cytokines13. The ingestion of apoptotic neutrophils is well studied and involves protein receptors expressed on the surface of the ingesting cells and the apoptotic bodies12. Of note, there is considerable overlap between anti-inflammatory and pro-fibrotic pathways as exemplified by one report that found the anti-inflammatory effects of macrophages which had ingested apoptotic cells resulted from the increased expression of TGFβ1 (a well-established pro-fibrotic cytokine)14. Further evidence linking apoptotic cells with lung fibrosis comes from a report in which the administration of a single dose of lavaged alveolar cells (presumably macrophages) induced to undergo apoptosis caused a fibrotic response in mice15. Although much less is known about the fate of apoptotic AECs and whether their uptake by macrophages might be an important inciting event in fibrosis, we hypothesized that the efferocytosis of apoptotic type II AECs would significantly contribute to the initiation of fibrosis following lung injury. To test this hypothesis, we employed a transgenic model of fibrosis in which mice engineered to express the diphtheria toxin receptor (DTR) on their type II AECs are treated with repeated doses of diphtheria toxin (DT)11. We also directly administered repeated doses of apoptotic AECs into the lungs of healthy mice. We found that targeted epithelial injury led to apoptosis and evidence of macrophage ingestion of apoptotic cells. We also determined that the intrapulmonary administration of apoptotic AECs was sufficient to cause lung fibrosis through CD36-dependent efferocytosis. Together these findings indicate that the uptake of apoptotic type II AECs by lung macrophages in the setting of lung injury is a critical initiating step in fibrosis pathogenesis.Cellular apoptosis terminates with fragmentation resulting in formation of vesicles termed apoptotic bodies. Apoptotic bodies are characterized in part by the appearance of phosphatidylserine on the outer leaflet of the lipid bilayer which serves as a recognition signal for phagocytic cells to ingest the cellular debris. Apoptotic cells and bodies modulate cell behavior as they undergo phagocytosis in a process known as efferocytosis11. Control or SPC-DTR mice were treated with daily IP injections of DT at the timepoints indicated. CD36-null mice were purchased from Jackson Laboratories and bred in our animal facilities. Oropharyngeal delivery of apoptotic cells to mice was performed following the method of Lakotos et al. 16. For BAL collection, mice were sacrificed at the timepoints indicated and lungs lavaged with 1 mL of PBS.All animals were housed in a specific pathogen-free environment until the day of sacrifice, and each in vivoll murine experiments was approved by the University of Michigan Animal Care and Use Committee. Wild-type and transgenic mice were on a C57Bl/6 background. SPC-DTR mice in which expression of the diphtheria toxin receptor is regulated by a modified surfactant protein-C promoter are previously describedAdGFP was purchased from University of Iowa Viral Vector Core. Matrigel, biotin-conjugated rat anti-mouse CD45 antibody, biotin-conjugated rate anti-mouse CD16/32 antibody, and PE-conjugated rat anti-mouse CD45 antibody were purchased from BD Biosciences. Streptavidin conjugated and magnetic separator are from ThermoFisher. Mouse/Rat/Porcine/Canine TGFβ ELISA kit was purchased from R&D Systems. Low melting point agarose is from Life Technologies. Small airway growth media (SAGM) is from Lonza. Keritinocyte growth factor (KGF) is from Peprotech. Pro-surfactant protein-C (pro-SPC) antibody is from EMD Millipore. All other reagents are from Sigma Pharmaceuticals.18. Briefly, mice were sacrificed at the timpoints indicated. Lungs were removed, homogenized and incubated in 12 N HCl at 120 °C for 16 h. Aliquots of each sample were mixed with citrate buffer and chloramine T solution. Erlich’s solution was added and each sample was incubated for 15 min at 65 °C. Absorbance at 540 nm was measured and the hydroxyproline concentration was determined against a standard curve.Lung collagen content was determined by an assay for hydroxyproline as previously described18. Briefly, mice were sacrificed and lungs were injected with dispase followed by low melting point agarose. Lung were removed and digested in dispase. The crude dissected lungs were filtered to remove larger chunks and the resulting cells were negatively selected with anti-CD45 and anti-CD16/32 biotin-conjugated antibiodies using streptavidin-magnetic beads. The resulting AECs were further negatively selected by incubating on a sterile petri dish for 1–2 h at 37 °C. Primary AECs were cultured in SAGM supplemented with KGF on Matrigel-coated plates. In some experiments, cells were treated with AdGFP (50 pfu/cell) for one day. The cells were rinsed to remove any free adenovirus and were covered with fresh media for 24 h prior to induction of apoptosis. MLE-12 cells were purchased from ATCC and cultured in HITES media as previously described11. Jurkat cells were purchased from the ATCC and cultured in RPMI per ATCC protocol. Primary murine alveolar macrophages were isolated by serial bronchoalveolar lavage as previously described19. Breifly, mice were anesthetized and lungs were exposed. Lungs were sequentially lavaged 10 times with 1 ml of EDTA in PBS. The pooled lavage fluid was centrifuged at 100 x g and the cell pellet was resuspended and cultured in DMEM supplemented with 10% FBS. All cells were cultured in a 37 °C, 5% CO2, incubator.Murine primary AECs were isolated and cultured as previously descibed5 microjoules) using a UV Stratralinker 1800 (Stratagene). UV treated cells were placed back in the tissue culture incubator for 24 h to ensure sufficient time for apoptotic body generation. Conditioned media containing apoptotic cells was then collected and centrifuged at 1200 x g to isolate the apoptotic cells. The pellet was resuspended in 50 µl of PBS for delivery to mice. Live AECs (5 × 105) or apoptotic AECs (5 × 105) or PBS control were delivered to mice by oropharyngeal aspiration as previously described20 to allow for repetitive dosing. Mice were treated with daily doses for seven days. Mice were sacrificed 21 days after the initial dose for assessment of lung fibrosis.For intrapulmonary delivery of apoptotic bodies derived from primary AEC, cultured primary AECs were subjected to 30 min of UV light  radiation for a total of 10.0 × 10Level of active TGFβ was determined from conditioned media or BAL samples using the murine/mouse/rat/porcine/canine TGFβ ELISA kit per manufacturers protocol and values quantified against a standard curve.RNA was isolated from cells with TRIzol (Life Technologies) following manufacturer’s instructions. Reverse transcription was performed with the SuperScript III first-strand synthesis kit (Life Technologies) and RT-qPCR was performed using the POWER SYBR green PCR mastermix kit (Applied Biosystems) and Applied Biosystems 7000 sequence detection system. The relative expression levels of gene in fold changes were calculated against GAPDH. Primers sequences are GAPDH forward: 5’-AACTTTGGCATTGTGGAAGG-3’, GAPDH reverse: 5’-ACACATTGGGGGTAGGAACA-3’, TGFβ forward: 5’-ATCCTGTCCAAACTAAGGCTCG-3’, TGFβ reverse: 5’-ACCTCTTTAGCATAGTAGTCCGC-3’, arginase forward: 5’-CAGAAGAATGGAAGAGTCAG-3’, arginase reverse: 5’-CAGATATGCAGGGAGTCAC-3’, iNOS forward: 5’-CCCTTCAATGGTTGGTACATGG-3’ and iNOS reverse: 5’-ACATTGATCTCCGTGACAGCC-3’.21. Briefly, cells were resuspended in blocking buffer containing 1% BSA in PBS for 30 min. Cells were then incubated in PE-conjugated rat anti-mouse CD45 antibody in blocking buffer for 30 min. Cells were washed and analyzed by flow cytometry using an attune flow cytometer (Life Technologies). Data was analyzed using FloJo (Treestar) software.Detection of efferocytosis by uptake of GFP-positive apoptotic lung epithelial cells by non-epithelial, CD45-positive cells was performed on BAL cells by flow cytometry using a modified previously reported protocol20.Surface expression of phosphatydylserine was determined using TACS Annexin V-FITC Apoptosis detection kit (R&D Systems) per manufacturer’s protocol. Terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) was performed on lung sections using the in situ death detection kit and TMR red as previously describedt-test (2-tailed) was used to determine differences for experiments between two groups and 1-way ANOVA was used to determine differences for experiments with more than two groups. P value of < 0.05 was accepted as statistically significant.Data are expressed as mean values ± SEM. Student’s 11. Treatment of the SPC-DTR mice with repeated daily doses of DT for 14 days results in the devlopment of pulmonary fibrosis. In this model, the fate of the targeted type II AECs is unknown. To determine whether the DT-mediated injury is associated with increased caspase-mediated apoptosis, we harvested lungs from SPC-DTR mice two days after initiation of DT treatment. A control group of WT animals was treated with PBS. Caspase-mediated apoptosis was measured in whole lung lysates with an assay for active caspase 3/7. We found that DT treatment resulted in a marked increase in caspase 3/7 activity in SPC-DTR mice  in which the murine surfactant promoter C promoter drives diphtheria toxin receptor expression in a type II AEC-specific mannerice Fig.  and TUNEice Fig. . CollectPrior studies demonstrate that the ingestion of apoptotic cells by macrophage leads to a phenotypic switch. To determine if the efferocytosis of apoptotic AECs is sufficent to induce a profibrotic phenotype in alveolar macrophages, we induced apoptosis in cultured GFP-labeled primary type II AECs via exposure to UV radiation. Surface expression of phosphatidylserine was confirmed by flow cytometry  relies on surface expression of phophatidylserine  on the apoptotic cell as a recognition signal for engulfment. Outer leaflet phosphatidylserine expression occurs early in the apoptotic response and persists through the late stages as the cell becomes more and more fragmented, suggesting that macrophages can ingest a broad range of cells and cell debris. In our studies, cells treated with UV light to induce apoptosis were collected after a low speed centrifugation. This protocol likely excluded small bodies from our apoptotic cell preps, but we cannot exclude the possibility that fragments from later stages of apoptosis were included in our in vitro and in vivo experiments. Efferocytosis provides a homeostatic function in tissues by preventing the dead cells from releasing pro-inflammatory molecules and antigens that have the potential to cause autoimmunity12. In relation to this homeostatic function, the engulfment of apoptotic cells classically induces an anti-inflammatory or wound healing phenotype in the ingesting cell. For example, previous reports demonstrate that the uptake of apoptotic cells by macrophages results in the increased expression of TGFβ, a growth factor with both anti-inflammatory and pro-fibrotic activities13. Based on this mechanistic link between efferocytosis and TGβ production, we hypothesized that persistent alveolar epithelial cell apoptosis, as occurs in lung fibrosis, might lead to protracted TGFβ expression as a result of ongoing ingestion of apoptotic cells by alveolar macrophages and that this macrophage response might translate into fibrosis rather than tissue homeostasis. In deed, we found that repetitive doses of either primary type II AECs or MLE12 cells resulted in lung fibrosis. Our observation that apoptic cell instillation  drives fibrosis is consistent with a report in rats in which a single adminsitration of apoptotic lavaged cells (presumably primarily macrophages) caused lung fibrosis as assessed by sirius red staining of tissue sections28. Although we have not assessed the minimum frequency and/or number of apoptotic type II AEC administrations required for the induction of fibrosis in our mouse model, it is remarkable that a single instillation of the apoptotic lavaged cells in rats was sufficient to cause pathology. The authors did find evidence of persistent apoptosis in the rat lungs following the intratracheal delivery of apoptotic cells, and we speculate that this secondary apoptosis may be critical to the promotion of fibrogenesis. Importantly, the authors also detected persistently increased levels of TGFβ in BAL fluid from the rats at the same late time points where they observed evidence of ongoing lung cell apoptosis27Apoptosis is a regulated process that can be initiated by various insults or other stimuli22. In this report, a single administration of apoptotic cells was delivered on day 2 after bleomycin, and the protection against fibrosis was mechanistically linked to an upregulation of hepatocyte growth factor expression that was detectable by day 3 and persisted through day 21. Interestingly, lavage fluid TGFβ levels were suppressed at day 14 and day 21 in the mice that received the apoptoic Jurkat cells, and on day 7, several markers of apoptosis were also decreased. In a follow up study, the apoptotic Jurkat cells were shown to protect against fibrosis by signaling through PPARgamma29. Methodological differences between our study and the findings of Lee and Yoon and colleagues likely explain the discrepant results. These methodologic differences include the apoptotic cell type (type II AECs versus Jurkat cells), the number of apoptotic cell administrations , and the presence/absence of an initiating injury to the lung. The funciton of impaired or dysregulated efferocytosis in human disease including IPF is an active area of investigation30. Notably, alveolar macrophages are not the only cell type which can engulf apoptotic cells27. Furthermore recruited non-resident macrophages or other inflammatory cell types may also engulf apoptotic cells and contribute significantly to the fibrotic process31. Studies of the mechansims involved in regulating expression of efferocytosis receptors and recruitment of different cell types with potentially different responses to apoptotic cell ingestion are currently underway.In contrast to our study results in which the intrapulmonary administration of apoptotic type II AECs elicited a fibrotic response, the intrapulmonary instillation of apoptotic Jurkat cells was found to protect against lung scarring induced by bleomycin injury32. In this same study, it was noted that, in more traditional models of lung fibrosis such as bleomycin injury, there is an accumulation of lipid rich macrophages. This raises the intriguing possibility that AEC apoptosis, which is known to involve activation of ROS, leads to a unique apoptotic body which contains high levels of pro-fibrotic oxidized phospholipids. Notably, CD36 has been identified both as a receptor for efferocytosis as well as a receptor for free phospholipids, and we demonstrate that this receptor is critical to the development of apoptotic type II AEC-induced fibrosis. The importance of CD36 in our model is consistent with findings following bleomycin-induced injury15 and other models of fibrosis33. Although bleomycin is known to induce AEC apoptosis, in this report we show that the apoptotic cells provoke fibrosis in a CD36-dependent manner in a model in which healthy AECs are preserved at the onset of injury. Although our studies focus on alveolar macrophages, many different cell types are known to engulf apoptotic cells/bodies and likley contribute to the fibrotic response. After efferocytosis, the engulfed particle is rapidly degraded. Therefore, our observation that macrophages from CD36-deficient mice contained fewer apoptotic cell fragments could be explained by mechanisms other than impaired uptake. For example, it is possible that CD36 deficiency simply slows the rate of efferocytosis or accelerates degradation of the engulfed particle. The mechanistic role for CD36 and other efferocytosis receptors in mediating fibrosis through the rate of uptake of apoptotic cells/bodies, the rate of degradation of the engulfed particle, and the regulation of phenotypic responses in different cell types are active areas of investigation in our laboratories.The disparate results observed with apoptotic Jurkat cells and type II AECs suggests that the source of apoptotic cells is critical to the down-stream consequences. A number of cell serface receptors have been identified to be important for uptake of leukocyte-derived apoptotic bodies in the context of acute injuries. In contrast, the relevance and importance of specific efferocytosis receptors in the context of AEC-derived apoptotic cells during pulmonary fibrosis has not been studied. In addition to their cell surface receptors for efferocytosis, epithelial cells and leukocytes exhibit distinct functions which could result in differences in the bioactivity of the derived apoptotic cells. Even among epithelial cells, AECs may be fairly unique given their role in generating phospholipid rich surfactant. Recently, Summers and colleagues reported that intrapulmonary delivery of oxidized phospholipids was sufficient to induce pulmonary fibrosis in an animal modelIn summary, we demonstrate that AEC apoptosis promotes fibrosis, in part, through efferocytosis which causes the pro-fibrotic activation of macrophages. Deletion of a key efferocytosis receptor, CD36, significantly blocks efferocytosis-induced fibrosis. Since AEC apoptosis is a prominent feature of fibrotic lung diseases, this AEC apoptosis-efferocytosis-macrophage activation pathway offers novel therapeutic targets.Supplemental Figure LegendsSupplemental Figure 1Supplemental Figure 2Supplemental Figure 3Supplemental Figure 4


There are no internationally agreed upon clinical guidelines as to which women with gynecological cancer would benefit from Lynch syndrome screening or how best to manage the risk of gynecological cancer in women with Lynch syndrome. The Manchester International Consensus Group was convened in April 2017 to address this unmet need. The aim of the Group was to develop clear and comprehensive clinical guidance regarding the management of the gynecological sequelae of Lynch syndrome based on existing evidence and expert opinion from medical professionals and patients.Stakeholders from Europe and North America worked together over a two-day workshop to achieve consensus on best practice.Guidance was developed in four key areas: (1) whether women with gynecological cancer should be screened for Lynch syndrome and (2) how this should be done, (3) whether there was a role for gynecological surveillance in women at risk of Lynch syndrome, and (4) what preventive measures should be recommended for women with Lynch syndrome to reduce their risk of gynecological cancer.This document provides comprehensive clinical guidance that can be referenced by both patients and clinicians so that women with Lynch syndrome can expect and receive appropriate standards of care. Gynecological cancers are often the sentinel Lynch syndrome event in women and have an excellent prognosis.2 This provides an opportunity to diagnose women before further oncological sequelae affect them or their family. Early diagnosis allows women to be enrolled in cancer surveillance programs and enables cascade testing for at-risk relatives. There is well-documented survival advantage for those with Lynch syndrome who are compliant with CRC surveillance.3 Further, early identification of Lynch syndrome can enable the uptake of cancer prevention strategies, including aspirin and risk-reducing surgery.5 A timely diagnosis may also have cancer prognosis and treatment implications. For example, MMR-deficient tumors are susceptible to immune checkpoint inhibition through PD-1 blockade.6 These considerations necessitate guidelines to direct the identification and care of individuals affected by Lynch syndrome. Although CRC clinical guidance is widely available, the same is not true for gynecological cancers , endometrial (EC), and ovarian cancer (OC).n = 2), patient support group representatives (n = 2), gynecological oncology surgeons (n = 12), gynecology nurse specialists (n = 5), clinical geneticists (n = 10), genetic counselors (n = 2), medical oncologists (n = 1), colorectal surgeons (n = 2), gastroenterologists (n = 1), histopathologists (n = 10), genetic pathologists (n = 1), health economists (n = 1), and epidemiologists (n = 1).The meeting was held on 24–25 April 2017. Fifty stakeholders attended from across Europe and North America, including patients , microsatellite instability (MSI), MLH1 methylation testing, and direct germline sequencing for pathological variants of the MMR genes by next-generation sequencing (NGS) for Lynch syndrome testing  are shown in eTable One in 279 of the general population carry pathogenic variants in one of the 10 Given similar rates of Lynch syndrome in EC and CRC, and the potential to reduce mortality through colorectal surveillance and cascade testing of relatives, the Consensus Group strongly recommends that women with EC should also be screened for Lynch syndrome.Current UK guidance from the National Institute for Health and Care Excellence (NICE) supports the universal screening of CRC for Lynch syndrome.11 Hampel et al. found 20% of proven Lynch cases presented >60 years.12 While older patients may have a lower risk of Lynch syndrome, and less potential to benefit from risk-reducing measures, they may also have younger relatives who could benefit from the identification of family pathogenic variants. Further, targeted screening has its own challenges, particularly that screening is not conducted despite being indicated.Restricting screening to those with a higher pretest probability of Lynch syndrome  is likely to reduce the resource burden, although at the cost of missing more Lynch syndrome cases. One study found only 25% of IHC MMR-deficient tumors were in women <50 years.13 Newer prediction tools, MMRpredict1,26, MMRpro, and PREMM5, have increased sensitivity and specificity.16 However, they rely on accurate family self-reported history being recorded by the clinician. A quality-controlled family history is time consuming, outside the scope of many busy clinical settings, and does not meet the specificity or sensitivity needed for a first-line test to identify MMR pathogenic variant carriers,17 however, in the absence of tumor material, such prediction models can be useful to guide germline testing.Amsterdam-II and Bethesda criteria are family history–based prediction tools designed to target Lynch syndrome screening in CRC. Extrapolation of these tools to EC has shown specificity of 61% and 49% for Amsterdam-II and Bethesda criteria, respectively.18 A low body mass index (BMI) increases the likelihood of Lynch syndrome being the underlying cause in EC.19 In CRC, restricting screening for Lynch syndrome to certain high-risk pathological features is not sufficiently sensitive.20Restricting Lynch syndrome screening to tumors with certain pathological phenotypes, for example endometrioid or clear cell morphology, tumors of the lower uterine segment, and those with heavy infiltrates of tumor-associated T-lymphocytes, has not been tested prospectively as a means to direct Lynch syndrome screening.21 Lynch syndrome–associated OC is predominantly endometrioid, presenting at an earlier age and stage than sporadic OC, with improved survival rates.22 Lynch syndrome is found in 7% of women with synchronous EC and OC.23 Many professional organizations now recommend testing all epithelial OC patients for BRCA1/2 pathological variants.25 Given the similar cumulative risk of OC in Lynch syndrome, testing premenopausal women with epithelial OC for both BRCA1/2 and Lynch syndrome is appropriate. This is even more persuasive in an era of panel gene testing where there is no additional cost to add more genes.With regard to Lynch syndrome–associated OC, there is minimal evidence to guide clinical care. A single-center study found 21% of nonserous epithelial OC to be MMR deficient by IHC.26There is no evidence to support a link between Lynch syndrome and other gynecological cancers, neither myometrial, nor squamous cancers of the vulva, vagina, or cervix, in which the most important etiological driver is persistent infection with high-risk human papillomavirus (HPV). Thus screening for Lynch syndrome in women affected by cancers of the lower genital tract is not recommended, with the exception of (HPV-independent) endocervical adenocarcinomas, given the difficulty of distinguishing them from lower uterine segment endometrial cancers.Box 1. Consensus recommendations for screening women with gynecological cancer for Lynch syndromeWhere resources are available, the Consensus Group strongly recommends universal screening of endometrial cancer for Lynch syndrome (grade B).Where resources are restricted, the Consensus Group strongly recommends screening for Lynch syndrome in endometrial cancer where•Women are diagnosed at 60 years of age or younger (grade B).•Women diagnosed at any age have one or more of the following risk factors: a personal history of metachronous or synchronous Lynch syndrome–associated cancer, a first-degree relative with Lynch syndrome–associated cancer at 60 years of age or younger, pathological features strongly suggestive of a Lynch syndrome–associated cancer (grade B).The Consensus Group recommends screening for Lynch syndrome in ovarian cancer where•Women are diagnosed at 50 years of age or younger (grade C).•Women diagnosed at any age have nonserous and nonmucinous histology (grade C).27 Thus, MMR IHC/MSI testing should form part of standard patient care and prior consent is not required. When germline testing is recommended, informed consent should be sought and patients are entitled to receive specialist counseling.Screening women with gynecological cancer for Lynch syndrome is a multidisciplinary responsibility. Health-care systems require robust procedures for quality-assured tumor testing and communication of results. Tissue analysis to triage women for Lynch syndrome testing highlights the potential of having Lynch syndrome rather than diagnosing it. Furthermore, identifying MMR-defective status provides important  prognostic information and can direct, for example, immunotherapy treatment strategies.28 It should be noted that exon panel NGS will not identify MLH1 silencing due to methylation.29 Sequencing of PMS2 is problematic due to the presence of numerous pseudogenes.30 NGS is expensive and many hospitals have limited access to it. Thus most studies and current clinical practice employ a screening triage with the use of IHC and/or MSI before germline NGS and large rearrangement testing  MSH6 or PMS2 loss.33 The first is important because POLE pathogenic variants have prognostic implications; the last because MSI triage may miss Lynch syndrome cases due to MSH6 pathogenic variants.34 For IHC, sensitivity and specificity range between 86–100% and 48–67%, respectively.36 For MSI, sensitivity and specificity are similar at 77–100% and 38–81% (refs. 36). Where there is a strong family history and where EC/OC presents under the age of 50 years with normal IHC and/or MSI, there is still an argument for definitive NGS.37 Investigations should be performed in an agreed stepwise and protocol-driven manner.10There is good concordance between MSI and IHC analysis.MSH6 have been shown to associate with tumor MSS.38 The identification of MLH1 protein loss enables methylation analysis, which can exclude women with somatic MLH1 loss from unnecessary NGS.38 Methylation-specific polymerase chain reaction (PCR) is simple and cost-effective, however validation work has been almost exclusively in the CRC population.40 Methylation-specific PCR is not widely available, with only specialist laboratories offering this test. The molecular mechanism for the strong association of BRAF variant with CRC harboring somatic MLH1 hypermethylation is incompletely understood but appears to be tissue/tumor-specific; unlike algorithms in use for CRC, BRAF immunohistochemistry or sequencing cannot be used as a proxy for somatic MLH1 hypermethylation in gynecological cancers, as oncogenic BRAF variants occur so rarely in these.41 Therefore, moving straight to germline NGS on the basis of IHC MLH1 loss is also an option, although an expensive one.IHC analysis has the advantage of identifying the specific protein that has been lost, thus indicating the potentially mutated gene. Furthermore, some pathogenic variants in 42 However, the accuracy of this system is unproven in gynecological cancers.43 The interpretation of stained slides requires an experienced senior clinician, and IHC more frequently needs to be repeated due to uninterpretable staining patterns in EC compared with CRC.44 Furthermore, the effect of neoadjuvant treatment on tissue analysis for MMR dysfunction has not been defined in EC.45 A reporting proforma is shown in eFigure The MMR proteins form heterodimers, with MLH1 pairing with PMS2 and MSH2 pairing with MSH6. These proteins are unstable in their unpaired state, and while MLH1 and MSH2 can form stable heterodimers with other proteins, PMS2 and MSH6 can only dimerize with MLH1 and MSH2 respectively. It has therefore been proposed that IHC analysis can be performed by testing only two of the four MMR proteins, PMS2 and MSH6, because loss of MLH1 and MSH2 leads to loss of their heterodimer partner (PMS2 and MSH6 respectively).A testing strategy using IHC and MLH1 methylation is likely to be more cost-effective than strategies using MSI testing  and strategies not using MLH1 methylation (because methylation testing is cheaper than NGS and excludes a significant proportion of sporadic cases). Documented IHC results can also help interpret NGS results.Box 2. Consensus recommendations for the methodology used to screen women with gynecological cancer for Lynch syndromestrongly recommends that quality-assured processes for the identification, screening, and reporting of tests for Lynch syndrome are piloted and audited before they are implemented at a local level (grade B).The Consensus Group strongly recommends that tumor MMR or MSI status is used to identify women for germline MMR testing. There is no evidence to advocate MSI over MMR immunohistochemistry or vice versa (grade B).The Consensus Group recommends testing for all four MMR proteins using formalin-fixed paraffin-embedded biopsy or resected tumor specimens (grade B).Where MMR immunohistochemistry is performed, the Consensus Group strongly recommends that immunohistochemistry testing is tightly regulated and protocol-driven to ensure interpretable, quality-assured results that can be understood by nonspecialist clinicians (grade C).The Consensus Group recommends the use of promoter methylation-specific PCR to identify probable cases of MLH1 silencing and therefore further filter the number of samples requiring NGS, while noting that a small proportion of such cases are due to constitutional methylation or pathogenic variants involving MLH1 (grade B). BRAF immunohistochemistry or sequencing cannot be used as a proxy for somatic MLH1 hypermethylation in gynecological cancers (grade B).Where MLH1 protein loss is identified or initial screen suggests microsatellite instability high (MSI-H), the Consensus Group strongly recommends somatic NGS on formalin-fixed paraffin-embedded or fresh frozen tumor tissue of nonmucinous invasive epithelial tumors, which should include BRCA1/2 and Lynch syndrome genes (grade B/C).For ovarian cancer, the Consensus Group strongly recommends that explicit patient consent is sought before germline testing (grade C).The Consensus Group strongly recommends appropriate referral pathways are established with Clinical Genetics before screening is initiated. This includes those with a positive triage test who are negative for a germline Lynch syndrome pathogenic variant (grade B/C).The Consensus Group recommends that clinicians seek to establish the existence of other somatic or germline pathogenic variants, such as biallelic MuTYH, POLE, and/or double somatic MMR pathogenic variants, which may have prognostic implications (grade B).Where tumor tests suggest Lynch syndrome but there is no Lynch syndrome–associated pathogenic variant on germline NGS, the Consensus Group All recommendations are based on current technology. As this evolves so should clinical practice.MMR pathogenic variant carriers opt to undergo gynecological surveillance in lieu of, or whilst awaiting, risk-reducing surgery. The aim of surveillance is to detect premalignant disease or early stage cancer, with the ultimate aim of improving morbidity and mortality from the malignant gynecological  sequelae of Lynch syndrome.Many female 46 Survival data are limited and mortality data are lacking. However, both preinvasive  and stage 1 disease have been diagnosed in asymptomatic women undergoing EC surveillance  (ref. 48). It is important to note that EC survival rates in women with Lynch syndrome are extremely good, with 10-year survival of 98% in those undergoing surveillance;3 comparative data for LS-EC in women not undergoing surveillance are not available.The data relating to gynecological surveillance in Lynch syndrome are generally of low quality, with single-center, retrospective studies predominating eTable . Some ste eTable . One stu50 The Consensus Group was strongly supportive of the need for rapid access facilities being available for women with Lynch syndrome and that suspicious symptoms or signs of malignant gynecological disease should not await routine review in clinic.The Consensus Group acknowledged that ultrasonography, biopsy, and hysteroscopy could detect EC and premalignant pathological abnormalities. However, there is no evidence that this leads to a stage shift or improved survival in women with Lynch syndrome–associated EC. Furthermore, many patients identified during gynecological surveillance are symptomatic of endometrial pathology eTable . Patient46 Large randomized controlled trials (RCTs) of general population screening for OC through ultrasound scanning (USS) or multimodal screening  have so far failed to demonstrate a statistically significant mortality benefit.52 However, screening in high-risk BRCA1/2 pathogenic variant carriers with 4-monthly CA125 analyzed by an algorithm and reflex USS does lead to a stage shift in the disease detected; whether this translates into mortality benefit has yet to be established.53 Extrapolation of OC screening research to the LS population is limited by the known biological differences between LS-associated and sporadic/BRCA1/2-associated OC.54Regarding OC, there is currently insufficient evidence that surveillance is of benefit.Whilst there is true equipoise in the literature, high quality research regarding the value of gynecological surveillance in Lynch syndrome could be performed. The options for study design could include a cluster RCT or a centralized repository for routine data collection from local surveillance programs. Studies should be adequately powered to determine whether surveillance picks up earlier disease with benefits for patient outcomes, as well as assessing its psychological impact and cost-effectiveness.Box 3. Consensus recommendations for gynecological surveillance in women at risk of Lynch syndromestrongly recommends that all women be informed of their MMR pathogenic variant-specific risk of gynecological cancer, specifically endometrial and ovarian cancer, interpreted in the context of their family history (grade C).The Consensus Group recommends that women at risk of Lynch syndrome who have not experienced gynecological cancer undergo optional annual review from the age of 25 with an appropriate clinician to discuss red flag symptoms for endometrial and ovarian cancer,a and where contraceptive and fertility needs are raised. A gynecological referral should be made if there is a specific need (grade C).The Consensus Group MMR pathogenic variant carriers (grade C), due to insufficient evidence that this improves outcomes over symptom awareness and urgent investigation of red flag symptoms. The Consensus Group strongly recommends further research in this area be prioritized by funding bodies. Furthermore, where good quality endometrial cancer surveillance programs already exist, systematic collection of cancer outcomes should be undertaken.The Consensus Group does not recommend invasive gynecological surveillance in strongly recommends that women participate in routine cervical screening in line with local cervical screening programs (grade A).The Consensus Group aRed flag symptoms of gynecological cancer include abnormal bleeding, weight loss, bloating, change in bowel habit, recurrent urinary symptoms, and abdominal pain.505 Surgery is not without risk and potential long-term side effects, however, and preoperative counseling is important. The laparoscopic approach is associated with less postoperative pain, quicker recovery, and improved short-term quality of life, making it the preferred approach in uncomplicated cases, where resources permit.55 Surgical menopause follows risk-reducing  oophorectomy in premenopausal women. This is associated with vasomotor symptoms, urogenital dryness and atrophy, reduced sexual function, emotional lability, and cognitive decline, as well as increased risks of osteoporosis, cardiovascular disease and CRC.56 Thus prescription of estrogen-only hormone replacement therapy (HRT) until at least natural menopause age (~ 51 years) is strongly recommended to prevent these sequelae. There is some evidence that prior hysterectomy may be associated with greater discomfort during intubation at colonoscopy, and lower cecal intubation rates.57 Thus it has been suggested that women undergoing colonoscopic surveillance following hysterectomy undergo specific preprocedure counseling and measures to reduce procedural discomfort.58Risk-reducing total hysterectomy and bilateral salpingo-oophorectomy prevents gynecological cancer in women at risk of Lynch syndrome.59 A woman’s personal risk should be used to provide individualized counseling regarding the need for risk-reducing surgery and the optimal timing of this. According to the Prospective Lynch Syndrome Database , the lifetime risk of EC in women with MSH2, MLH1, and MSH6 pathogenic variants is 57%, 43%, and 46%, respectively. The cumulative risk of EC at 40 years of age is 2%, 3%, and 0%, respectively. Lifetime risk for OC in women with MSH2, MLH1, and MSH6 pathogenic variants is 17%, 10%, and 13%, respectively. The cumulative risk of OC at 40 years of age is 4%, 3%, and 4%, respectively.59 Thus MSH6 pathogenic variant carriers may consider undergoing risk-reducing surgery after the age of 40 years, while women with pathogenic variants in either MSH2 or MLH1 may consider risk-reducing surgery at around 35 years of age assuming their childbearing is complete.60 Risk-reducing surgery at 40 years of age is a cost-effective strategy.61 The risk of gynecological cancer in PMS2 carriers is low; however, patient representatives with PMS2 pathogenic variants felt strongly that they should be offered risk-reducing surgery alongside other women with Lynch syndrome.59There are good quality prospective data outlining the cancer risk associated with specific pathogenic variants and the age at which these occur.62 The POET trial looked to explore the use of the IUS for the prevention of EC in women with Lynch syndrome. This trial closed due to poor recruitment without results. Another study showed an antiproliferative effect of exogenous progesterone on the endometrium of women with Lynch syndrome, suggesting that these agents could be useful for the chemoprevention of EC.63 Exogenous hormones may also protect against CRC.64There is limited evidence as to how reproductive and lifestyle factors impact on gynecological cancer risk in Lynch syndrome. One study suggested that hormonal influences do modulate cancer risk, however. In addition to combined oral contraceptives, progestin-only methods  may protect, but there is little supporting evidence.65 The main toxic effects of aspirin are gastrointestinal. Major gastrointestinal bleeds (those requiring transfusion) are increased in aspirin-takers with an odds ratio (OR) of 1.5–2 (ref. 66). However, the absolute rates are small and mainly affect older individuals. Because gastrointestinal toxicity is dose dependent, the optimal dose for cancer risk reduction is being explored through the CaPP3 study of 100 mg, 300 mg, or 600 mg/day (http://www.capp3.org/).Aspirin reduces incidence of Lynch syndrome–associated EC and other cancers.67 Despite lack of robust evidence, women are advised to eat a healthy diet, avoid obesity, take regular exercise, avoid smoking, only drink alcohol in moderation, and avoid known carcinogens as part of maintaining healthy lifestyles.Smoking, alcohol use, and increased BMI may increase the risk of CRC in individuals with Lynch syndrome; however, the impact of lifestyle factors on gynecological cancer risk is unknown. Aspirin may “normalize” EC risk in obese women with Lynch syndrome.Box 4. Consensus recommendations for gynecological cancer risk reduction in Lynch syndromestrongly recommends that risk-reducing total hysterectomy and bilateral salpingo-oophorectomy is offered no earlier than 35–40 years of age following completion of childbearing, in proven MLH1, MSH2, and MSH6 pathogenic variant carriers (grade B). A woman’s personal risk should be used to provide individualized counseling regarding optimal timing of the procedure. The strength of evidence is insufficient to strongly recommend risk-reducing surgery in PMS2 pathogenic variant carriers.The Consensus Group strongly recommends high quality preoperative multidisciplinary counseling supplemented by patient-friendly written information regarding the risks and benefits of risk-reducing surgery (grade C).The Consensus Group recommends preoperative endometrial biopsy and pelvic ultrasound to identify occult gynecological cancer, particularly if a woman is symptomatic (grade C).The Consensus Group strongly recommends that women who are not up-to-date with colorectal surveillance are offered colonoscopy at the time of their risk-reducing surgery (grade B).The Consensus Group recommends that surgery is offered at specialist surgical center, although women at low surgical risk should be able to choose local care (grade C).The Consensus Group strongly recommends all pathological assessment and reporting is carried out at a specialist gynecological pathology center and that the entire endometrium is sampled in the case of prophylactic hysterectomy (grade C).The Consensus Group strongly recommends that women who undergo risk-reducing hysterectomy and removal of their ovaries are offered estrogen-only hormone replacement therapy (HRT)  until the natural age of the menopause (age 51 years) or, in consultation with their specialist, until they wish to stop (grade B).The Consensus Group recommends that risk-reducing colorectal and gynecological surgery is carried out at the same time, when indicated and where possible (grade C).aThe Consensus Group recommends that surgery for colorectal cancer and risk-reducing hysterectomy is carried out at the same time, when indicated and where possible (grade C).The Consensus Group recommends female carriers of MMR pathogenic variants are given the opportunity to discuss their fertility and contraceptive needs with a specialist (grade C).Where risk-reducing surgery has been declined or is not yet appropriate, the Consensus Group recommends that the combined oral contraceptive pill is considered for women at risk of Lynch syndrome and wishing contraception because it reduces endometrial and ovarian cancer risk (grade B).The Consensus Group strongly recommends that MMR pathogenic variant carriers take aspirin chemoprevention to reduce their risk of colorectal and other cancers (grade A), within the context of a clinical trial , or through discussion with their doctor.The Consensus Group recommends that women at risk of Lynch syndrome maintain a healthy body mass index (BMI) (grade B).The Consensus Group recommends that women eat a healthy diet, take regular exercise, do not smoke cigarettes, drink alcohol in moderation or not at all, and avoid known carcinogens  as part of maintaining a healthy lifestyle (grade B).The Consensus Group aRisk-reducing colorectal surgery here refers to an extended colectomy at the time of a colorectal cancer or in the event of any other benign indication for a colonic resection such a multiple (right-sided) recurrent polyps.This is the first gynecology-focused internationally agreed-upon clinical guideline for the care of women with or at risk of Lynch syndrome.Our key recommendations are as follows: (1) all stakeholders should be informed of the impact of Lynch syndrome on gynecological cancer risk; (2) systems should be established to screen for Lynch syndrome in women with endometrial cancer; (3) women at risk of Lynch syndrome should be offered risk-reducing hysterectomy with bilateral salpingo-oophorectomy, at a time appropriate to them; and (4) further research is required to establish the value of gynecological cancer surveillance in Lynch syndrome and to explore other key areas where there is currently deficient evidence to define appropriate standards of care. Screening for Lynch syndrome is only recommended if effective management exists to benefit those who screen positive.The strength of our guidance comes from the broad and expert medical specialty representation that forms our Consensus Group. All relevant stakeholders, including patients and patient advocates, were given equal voice during discussion. Despite different perspectives and expertise, we were able to achieve a consensus view on topics of international importance. By focusing solely on the gynecological aspects of Lynch syndrome, we were able to provide the most comprehensive guidelines yet for the empowerment of both clinicians and patients. This was all achieved without corporate sponsorship.The major limitation of our work is the lack of robust evidence on which to base our discussions and recommendations. It is hoped that these guidelines will provide the much-needed impetus to inspire researchers and funders to undertake and commission high quality research to fill these gaps in our understanding.Supplemental material


As-prepared composite films were reduced in hydrogen to induce texture growth. Structural, magnetic property and surface morphology of the films were characterized by X-ray diffraction (XRD), vibrating sample magnetometer (VSM) and scanning electron microscope (SEM). These experimental data indicate that integrated intensity ratio I(200)/I(110) of diffraction peaks (200) and (110) of FeCo firstly increases and then decreases, while the coercivity first decreases and then increases with increasing Pt doping content. The specimen with less Pt doping content has a large I(200)/I(110) value and small coervicity value, which is closely related with strong (200) texture in FeCo thin film. These results indicate that fcc-Pt is also in favor of promoting (200) FeCo texture like Al or Cu elements, and this similar trends of Pt and Al originate from their similar atomic radius and crystal cell volume.We prepared FeCoPt/SiO Magnetic nanomaterials have wide applications in the fabrication of optical and electronic devices, since their remarkable magnetic properties can improve the performance of those devices, for examples in power generation, conditioning, conversion, transportation, and other energy-use sectors of the economy13. Nanometer-sized FeCo alloys have been paid enough attention by many researchers recently since they possess much better soft magnetism such as low Hc (coercivity), high μ (permeability), hig h Tc (Curie temperature), high Ms (saturation magnetization) and high tensile strength17. Due to the formation of the ordered B2 state18, near equiatomic FeCo alloys exhibit very good soft magnetic properties, while they are extremely brittle at room temperature19, on the other hand, the coercivity of FeCo alloy films prepared by a conventional sputtering method is quite high, which is naturally not applicable for high frequency application20. So a suitable non-magnetic matrix and a strong (200) FeCo texture are vital on improving FeCo soft magnetism and further applications. Our teams have devoted some years to investigating how to improve (200) FeCo texture growth21 by choosing different metals as seedlayer or underlayer  and doping elements  into FeCo crystal lattice and also by choosing various preparing methods. Sol-gel technique is not only commonly easy-control to achieve uniform-doping among raw materials but also combines a superior advantage of low-cost.Compared with traditional materials, nanomaterials have received considerable attention due to their unique physical and chemical propertiesIn this work we chose Pt metal as doping element in view of the tiny structural difference between fcc-Pt, fcc-Al, fcc-Cu and bcc-FeCo, for the purpose to investigate the influence of deferent elements on FeCo grains’ preferred orientation.3)2·6H2O), platinum nitrate (Pt(NO3)2) and iron nitrate (Fe(NO3)3·9H2O) were dissolved into absolute ethyl alcohol under continuously magnetic stirring, then some mixed solution of glycol and ethyl orthosilicate were dropwise-added into as-prepared initial solution, then some nitrate was added to get transparent sol. The sol was ultrasonic vibrated for 1 hour and aged at room temperature for 24 hours, then spin-coated onto silicon substrates, as-prepared films were annealed in hydrogen for 1 hour to obtain FeCoPt/SiO2 thin films. The Pt doping content was set as 0, 0.5, 1, 3 and 5 wt% in FeCo/SiO2 composite films, the corresponding films were marked as a, b, c, d and e, respectively.Cobalt nitrate /I(110). In Fig. I(200)/I(110) and Pt doping content. It displays that the value of I(200)/I(110) reduces when Pt doping content increases. The maximum value of I(200)/I(110) appears when Pt doping is 0.5 wt%. This indicates that a strong (200) texture of FeCo arises on the smallest Pt doping. Samples with Pt doping follow the same trend as Al element both as doping and underlayer25. It is obvious that both metal Al and Pt can promote FeCo (200) preferred orientation, but this promotion was reduced when more Al or Pt were doped into or deposited. While for samples underlayered with Cu and Co23, they present a different trend. For example, the more is the thickness of Co, the higher is the value of I(200)/I(110), which means more Co content is benefit for improving FeCo (200) preferred orientation. This various doping effect is dominantly derived from their structural and crystal difference between those metals. It is due to the similar atomic radius and crystal cell volume values between Al and Pt, so they can result in similar effects among FeCo grains. The corresponding parameters are listed in Table We calculated the integrated intensity ratio of diffraction peaks 200) to 110), which is noted as 00 to /d0 values, which is dominant in promoting preferred orientation in (200) planes or (110) planes26. It is also noted that the more element doping whether Al or Pt are introduced, the more relative lattice strain are formed, so appropriate choosing of doping element and doping content is important in promoting (200) texture in FeCo thin films. For the samples underlayered with Cu or Co, they display different changing trend due to their structural and crystal difference from Al and Pt.For Pt element, it has similar atomic radius and crystal cell volume with Al element, so they play similar role in doping into FeCo alloys’ crystal lattices, thus cause similar relative lattice strain between FeCo grains, resulting in similar relative lattice deformation  is higher than H‖c (in-plane), this shows that an out-of-plane magnetic anisotropy is dominating for all sample films. It can be shown from previous XRD results, there exists the smallest strain between FeCoPt/SiO2 film so presenting a strong (200) texture when Pt doping content is less than 1 wt%, this leads to expressing the best soft magnetic property with less coercivity Hc values. So Pt is appropriate to be chosen as doping element for improving FeCo (200) texture growth as Al element.Figure This kind of similar atomic and crystal structure between Pt and Al elements produce similar relative lattice strain between FeCo grains when they act with FeCo particles whether as doping elements into FeCo crystal lattice or as underlayers being deposited under FeCo layers, so they play similar role in promoting (200) texture in FeCo thin films, which is also the key factor in governing their magnetic properties. For samples underlayered with Co or Co element, whether the values of coercivity in-plane and out-of-plane, or the changing trends follow the different ways, originating from their structural and crystal difference with Al and Pt elements.Figure 2 thin films follows the similar trends with the increasing of Pt doping content.Pt metal is proved to be a good doping element as Al is introduced into FeCo alloys to promote (200) preferred orientation. It is due to similar atomic radius and crystal cell volume of Pt and Al that the change of structural and magnetic properties of FeCoPt/SiO


Mycobacterium tuberculosis ; the WHO has established this disease as high priority due to its ensuing mortality. Mtb uses a range of mechanisms for preventing its elimination by an infected host; new, viable alternatives for blocking the host-pathogen interaction are thus sought constantly. This article updates our laboratory's systematic search for antigens using bioinformatics tools to clarify the Mtb H37Rv Rv3632 protein's topology and location. This article reports a C-terminal region consisting of peptides 39255 and 39256 (81Thr-Arg114) having high specific binding regarding two infection-related cell lines (A549 and U937); they inhibited mycobacterial entry to U937 cells in a concentration-dependent manner. Rv3632 forms part of the mycobacterial cell envelope, formed by six linear synthetic peptides. Circular dichroism enabled determining the protein's secondary structure. It was also found that peptide 39254 (61Gly-Thr83) was a HABP for alveolar epithelial cells and inhibited mycobacteria entry to these cells regardless of concentration. Sera from active or latent tuberculosis patients did not recognise HABPs 39254 and 39256. These sequences represent a promising approach aiming at their ongoing modification and for including them when designing a multi-epitope, anti-tuberculosis vaccine.Tuberculosis is an infectious disease caused by Tuberculosis has had a high incidence regarding the world's population; it affected more than 10 million people in 2017 and was the second cause of death by an infectious agent . hsp65 gene expression of around 439 bp , identified as B-cell epitope  was only a HABP for the A549 alveolar epithelial cell line.The Rv3632 protein sequence consisted of 114 aa; six 20-aa-long peptide sequences were obtained from them for identifying these fragments' interaction with A549 epithelial cell (ATCC CLL-185) and U937 monocyte-derived macrophage (ATCC CRL-2367) receptors. D) were in the 1,800-2,250 nM range and Hill coefficients (nH) for these interactions were >1, indicating positive cooperativity for HABP binding. Between 1.9 x 106 and 2.7 x 106 receptor sites were calculated per cell for peptide 39255 and around 10 times more sites for HABP 39256 binding. Future studies should be aiming at determining the nature of HABP receptor sites  secondary structure had 2% folded β-sheets and peptide 39255 (81TLSTYMRFKDLELRYARIAR100) 1.5%  1.5% . Such da regions .Phyre2 modelling of protein 3D structure and Swiss model validation giving a 0.121 QMEAN score led to a reliable structure, 88.5% of the residues being located in the Ramachandran plot's most favoured regions . RegardiμM concentrations) were brought into contact with epithelial and (A549) macrophage cell lines (U937) to evaluate their capability for specifically inhibiting Mtb H37Rv entry. Cytochalasin D was used as inhibition control and peptide 39253 (41LAGIYAVLRPDDTTVVANWF60) which had no binding as negative control. Cytochalasin inhibited mycobacterial entry to A549 epithelial cells by 60% and U937 monocyte-derived macrophages by 80%. HABPs 39255 and 39256 inhibited mycobacteria entry to U937 macrophages by up to 80% in a concentration-dependent manner and inhibited entry to A549 cells inversely to peptide concentration. Alveolar epithelial HABP 39254 inhibited mycobacterial entry inversely to peptide concentration  (not considered as a HABP) was recognised by LTB donor sera and had a significant difference regarding recognition of HD sera and ATB patients' sera. HABP 39255 (81TLSTYMRFKDLELRYARIAR100) was recognised by antibodies from LTB and ATB patients and HD, no significant differences being observed between the populations studied. HABPs 39254 and 39256 were not recognised by the sera being used, which might have been associated with Mtb H37Rv binding and entry to cell lines. Mtb H37Rv lysate was recognised by ATB patients' sera, whilst the culture supernatant was not recognised by the sera being used.In our experience , HABPs neither are recognised by infected individuals' sera nor do they induce an immune response. Their sequences must thus be modified to make them immunogenic and antigenic, thereby triggering a protective immune response , 43. It Tuberculosis continues to be one of the diseases having the highest impact on public health worldwide and the BCG vaccine has proved ineffective in protecting adults against pulmonary tuberculosis. As the amount of patients diagnosed with the tubercle bacillus continues to grow, the search for fresh vaccine alternatives continues to be an urgent item on health agendas globally. FIDIC's research represents a promising advance in designing a synthetic vaccine intended to control the pathogen-host interaction by blocking specific receptors by using synthetic peptides for a large range of aetiological agents. Mtb bacterial envelope which are involved in the host-pathogen interaction and which can be included when designing a multi-epitope synthetic vaccine against TB. This study has thus proposed Rv3632-derived HABPs as candidate antigens for blocking specific interactions between target cell receptors and mycobacteria. Despite not having an established function to date, it was found that the rv3632 gene is present and is transcribed in TCM complex strains and in Mtb lineages (being conserved amongst them), thereby suggesting that Rv3632 could be involved in the aetiological agent's virulence.This study contributes towards the search for protein sequences on113Cys into account. Although STRING v10.5 predicted associations involving Rv3629c (39kDa), Rv3630 (43kDa), Rv3631 (26kDa), and Rv3633 (33kDa) being co-expressed and having a synergistic effect which has been described in arabinogalactan production by interaction with PPGs or Rv3631, such associations would not be responsible for the experimental results reported here.TBpred is a tool which can be widely used for the functional prediction of mycobacterial proteins since it predicts four subcellular locations  . Rv3632 61Gly and Tyr114 was identified when screening Rv3632 peptides' 114 aa in terms of interaction with infection target cells . When evaluating these HABPs' ability to block Mtb H37Rv entry it was clear that 39255 and 39256 inhibited Mtb entry to the U937 cell line in a concentration-dependent manner, having > 50% inhibition at 200 μM concentration, without having a cytotoxic effect. Regarding A549 epithelial cells, HABPs 39254, 39255, and 39256 inhibited entry by ≥ 30% at 200 μM regardless of concentration. Such pattern may be related to the peptide cytotoxic effect observed for this cell line; although cytotoxicity may influence entry inhibition data, the inhibitory activity of these HABPs is relevant to our approach. Peptide 39253 (considered as a control in this assay because it did not bind to target cells) did not influence Mtb entry to target cells, which was to be expected for non-binding sequences. Only HABPs characterised by inhibiting Mtb entry to infection target cells would be included when designing a synthetic peptide-based, multi-epitope anti-TB vaccine. Although receptor-ligand assays have highlighted positive cooperation and identified a large amount of binding sites, possible cell receptor nature and identity have not yet been clarified.A highly specific binding region consisting of the 55 aa between  Mtb infection during the disease's active phase [ Mtb strains have not been immunogenic. The ELISA assays used here determined that HABPs 39254 and 39256 (which inhibited Mtb entry in vitro) were not recognised by TB patients' antibodies and might not have been producing any type of protective response against Mtb. These two HABPs highlighted a previously described phenomenon known as conserved antigens' immunological silence [Studies have highlighted a humoral immune response in controllingve phase , 45; how silence .Both of the bioinformatics approach and the experimental evidence were similar in terms of these binding sequences' secondary structure elements; alpha helices predominated throughout the entire protein and the high specific binding sequences were exposed. As our approach to the search for antigens which might be included when designing a synthetic anti-tuberculosis vaccine has been based on results obtained so far regarding the malaria model, it is proposed here that HABP structures must be modified by specifically replacing aa in the peptides' original sequences. This ensures a poly-proline-type structure having a better fit into the major histocompatibility (MHC) class II complex, thereby improving immunogenic and protective characteristics . Mtb and which is mandatory within Colombia's healthcare plan). Interestingly, there was differential recognition of peptide 39252 by HD individuals' sera, thereby enabling it to be differentiated from the sera of individuals classified as ATB and LTB. Its sequence can thus be considered when developing serodiagnostic tests for TB.Healthy donors (HD) had antibodies against Rv3632 peptides; this may have resulted from cross-reactivity because they might have been in contact with environmental mycobacteria or the simple fact of HD having been vaccinated with BCG  was identified as being relevant in interaction with target cells capable of inhibiting Mtb entry to target cells and specifically binding to them. This sequence was conserved and not recognised by antibodies, which is why it has been proposed that this peptide should be modified to increase its immunogenicity, thereby enabling it be recognised by the host's immune system to include it when designing a synthetic anti-tuberculosis vaccine.It can thus be concluded that Rv3632 was exposed on bacterial surface, taking into account that our interest lay in blocking the host-pathogen interaction; it is thus feasible that it may be involved in contact with


Elective cardiac surgery has low procedural complications. However, about 40% of patients develop extracardiac complications including delirium and acute kidney injury. We hypothesized that inflammatory processes and immune cell activation might be associated with these complications. We therefore prospectively included 104 patients undergoing cardiac surgery in our study. We assessed peripheral blood leukocyte populations by flow cytometry and circulating cytokines before operation, after surgery and at days one and four post-operatively. Patients undergoing cardiac surgery showed significantly elevated leukocytes and neutrophils after surgery. On the contrary, monocytes decreased after surgery and significantly increased at days 1 and 4, particularly classical  and intermediate  monocytes. While peripheral leukocyte subsets were unaltered in patients with infectious (n = 15) or cardiac complications (n = 31), post-operative leukocytes (p = 0.0016), neutrophils (p = 0.0061) and Mon2 (p = 0.0007) were clearly raised in patients developing extracardiac complications (n = 35). Using multiple logistic regression analyses, patient’s age, ICU days, number of blood transfusions and elevated post-surgery Mon2 independently predicted extracardiac complications. Our findings demonstrate that elevated Mon2 after cardiac surgery are associated with an increased risk for extracardiac complications. These findings might improve the risk estimation after cardiac operations and the role of Mon2 for inflammation in cardiac surgery. Besides typical cardiac  and infectious complications , extracardiac non-infectious complications like acute kidney injury  and delirium (up to 26–52%) are frequent in patients after cardiac surgery3. It has been speculated that a large part of these complications could be explained by excessive inflammation caused by extracorporeal cardio-pulmonary bypass (CPB), hypothermia, myocardial ischemia and reperfusion and tissue damage due to the surgical procedure5. The influence of distinct immune cells and their subpopulations on the occurrence of post-operative complications is still ambiguous and needs further investigations. Particularly monocytes seems to play in important role within this context7.Despite advances in surgical and anesthetic techniques as well as enhanced postoperative care, cardiac surgery is still associated with a high risk of postoperative complications9. Using the surface markers CD14 (LPS receptor) and CD16 (FcγRIII), blood monocytes can be subdivided into three subpopulations, CD14++CD16− “classical” monocytes (Mon1), CD14++CD16+ “intermediate” (Mon2) and CD14–16+ “non-classical” (Mon3) monocytes11. Monocytes are a key player in cardiovascular disease and atherosclerosis, and elevated CD14−CD16+ non-classical monocytes have been correlated with endothelial dysfunction and vascular oxidative stress13. Several smaller studies (comprising 10–20 patients) have reported alterations in monocyte populations after cardiac surgery14, but the pathogenic involvement and the clinical relevance of these findings have remained elusive. In order to identify associations between monocyte subpopulations and complications after cardiac surgery, we conducted a prospective observational trial in 104 consecutive cardiac surgery patients, in which we obtained detailed circulating immune cell characteristics by multi-color flow cytometry before and sequentially after surgery. We thereby demonstrate that elevated post-operative CD14++CD16+ intermediate monocytes are associated with an increased risk for extracardiac complications, which may indicate a novel role of Mon2 in the context of inflammation related to cardiac surgery.Circulating monocyte subsets represent a continuum of differentiation stages15.A total of 107 patients  were consecutively enrolled in this prospective, observational study after approval of the local institutional review board and after obtaining informed consent . Ethical permission has been obtained from the Research Ethics Committee of RWTH-University Aachen, Germany (EK 151/09). This study has been performed in accordance with the ethical standards in the Declaration of Helsinki. The cohort is a sub-study from our previously published study aiming at identifying novel predictive biomarkers for acute renal failure in this setting17. Patient data, clinical information and blood samples were collected prospectively immediately before (pre) surgery, after (post) surgery as well as 1 day and 4 days after cardiac surgery. Serum samples and whole blood sufficient for multicolor fluorescence activated cell sorting (FACS) analysis were available before surgery in 104/104, directly after surgery in 96/104, at day 1 in 95/104 and at day 4 in 78/104 patients. The clinical course of patients was observed during the follow-up period by directly contacting the patients, their relatives or their primary care physician. Survival was assessed at 30 and 90 days.Patients were included over a pre-specified time period from June1, 2014, to November 31, 2015 on  two pre-defined days of the week. Exclusion criteria were pregnancy, emergency operations and patients aged less than 18 years. Out of these 107 patients, 3 patients received “off-pump” cardiac surgery; these three patients were excluded from the analysis due a potentially different activation of immune cells that had not been exposed to extracorporeal circulation18. After surgery, all patients were transferred to the intensive care unit (ICU), and the following post-operative treatment was standardized according to our institutional guidelines. The surgical procedure with use of conventional cardiopulmonary bypass  was performed in accordance to local clinical standards. The extracorporeal circulation was performed with a non-pulsatile pump flow of 2.2 l/min/m2, targeting a blood pressure between 50 and 70 mmHg.Anesthesia was performed as described before regarding to our institutional routinePostoperative complication within the first four postoperative days were subdivided in infectious complications , cardiac complications  and extracardiac complications (delirium and acute kidney injury (AKI)). Arrhythmia was defined as a novel incidence of atrial fibrillation or flutter, ventricular tachycardia and fibrillation within the first four days after surgery. For extracardiac complications, delirium was defined via the Confusion Assessment Method for the ICU (CAM-ICU) score, acute kidney injury was based on creatinine increase (KDIGO criteria). Other extracardiac complications like ARDS (acute respiratory distress syndrome) and severe liver failure were also recorded, but were so infrequent (with only one case) that they were not further included in the analyses.To understand the potential influence of different immune cell populations on the development of the postoperative complications, we compared immune cell populations and subpopulations of patients with and without the pre-specified complications. Additionally, we analyzed immune cells of 20 control patients without known cardiac, extracardiac or infectious diseases to assess potential changes in immune cell populations in patients requiring cardiac surgery.19. Cytokines concentrations were measured, because they might allow conclusions about systemic inflammatory responses as well as the activation of different immune cell populations.Blood samples for serum analyses were immediately placed on ice and stored at −80 °C after centrifugation. Cytokine concentrations of IL1ß, IL2, IL4, IL6, IL8, IL10, IL12(p70) and IL17 were measured using the Bio-Plex Assay (Bio-Rad Laboratories), following the manufacturer’s protocol. 20 ml whole blood for immune cell analyzes was subjected to red cell lysis by Pharmlyse (BD) and stained with fluorochrome-conjugated antibodies for FACS analysis20.After obtaining the blood samples, peripheral blood mononuclear cells (PBMCs) were isolated and immediately analyzed, as described beforePBMCs were stained with fluorescence-conjugated antibodies  and incubated light protected for 30 min. After washing and centrifugation, cells were measured using a FACS Canto-II  and analyzed by using FlowJo Software . Cell populations were determined after exclusion of doublets and by forward and side scatter  and further by positivity for specific surface markers. For monocytes subpopulations, we selected the monocyte population in the forward and side scatter and excluded in the next step CD56+ cells, followed by subsequent analysis of CD14 and CD16. Subpopulations were divided into Mon1 (CD14++CD16−), Mon2 (CD14++ CD16+) and Mon3 (CD14−CD16+). Absolute cell numbers were calculated using the relative proportions of the subsets (from FACS) and the automated differential white blood cell counts.15. Patient data are displayed as median and range, providing the skewed distribution of most parameters. For testing differences between the control group and the patients, we used the χ2 test and Mann–Whitney U test. Differences in longitudinal samples were tested by Wilcoxon test, correlations were assessed by Spearman rank correlation analysis. Univariate logistic regression analyses were performed for all monocytes subsets and cell populations, interleukin levels and clinical data regarding to different outcome parameters . Multivariate logistic regression analysis was performed with significant parameters from univariate logistic regression analysis followed by backward elimination. All patients measured at the specific time point were includedin uni- and multivariate analysis. Only Box plot graphics illustrate comparisons between subgroups. They display a statistical summary of the median, quartiles and range. Receiver operating characteristic curves were generated by plotting sensitivity against 1-specificity. All values have been included in statistical analyses.All statistical analyses were performed using GraphPad Prism 5.0  and SPSS 23 , as described beforeClinical trials gov. identification: NCT02488876. Ethics committee RWTH-University, Aachen, Germany: EK 151/09.The baseline clinical characteristics of 104 patients participating in our study are displayed in Table Immune cells gained from peripheral blood were analyzed before operation, immediately after cardiac surgery (at admission to the ICU) and at day one and four after cardiac surgery. Compared to healthy controls, patients undergoing cardiac surgery showed similar levels of leukocytes, neutrophils and monocytes before operation (at baseline), while lymphocytes were significantly reduced in patients undergoing cardiac surgery compared to healthy controls Fig. . Post-suComparing patients developing cardiac complications  to patients without cardiac complications, immune cells did not differ within the two groups at the various time points Fig. .Figure 2Comparing patients with and without extracardiac complications, leukocytes and neutrophils were significantly elevated at every time point in patients with complications, while monocytes were higher at the time point immediately after surgery Fig. . Among t21. Moreover, Mon1 were also correlated with surgery time and time of CPB  curve analyses Fig. . SpecifiSignificantly elevated Mon2 after cardiac surgery in patients developing extracardiac complications and the correlation of this monocyte subpopulation at baseline to days of hospitalization and to IL6 led us to hypothesize that circulating monocyte populations might predict extracardiac complications after cardiac surgery. In order to analyze the predictive value of Mon2 for post-operative extracardiac complications, we conducted uni- and multivariate logistic regression analyses. By univariate analysis, several clinical characteristics and immune cell populations, particularly neutrophils and monocytes, at different time-points were associated with the occurrence of extracardiac complications Table . By mult4. While we did not identify such associations with infectious or cardiac complications, manifold inflammatory and immune pathways appeared dysregulated before or immediately after surgery between patients with or without extracardiac complications. Among these complex alterations, post-operatively measured circulating Mon2 independently predicted extracardiac complications within the first four days after surgery.In this study, we comprehensively evaluated the kinetics of circulating immune cell populations and inflammatory cytokines before and during the ICU course in a large, prospectively enrolled cohort of patients undergoing elective cardiac surgery in order to identify dysregulated immunological and inflammatory pathways related to the development of clinical complications23. The latter aspect appears particularly relevant for the healing phase after myocardial injury in the heart25. Novel technologies like single-cell RNA sequencing provided further in-depth insights into the heterogeneity of monocytes in human blood26. Three major human monocyte subpopulations have been described27, of which the Mon1  are relatively immature and preferentially recruited to injured tissue, while Mon2 (intermediate) and Mon3  have a higher capacity to secrete cytokines upon activation. Interestingly, in several specific patient populations with atrial fibrillation, heart failure and chronic kidney disease, Mon2 were an independent predictor for cardiovascular events30. Elevated Mon2 have been reported before in patients undergoing cardiac surgery14. Another study emphasized the relevance of CD16 expression of circulating monocytes by measuring the mean fluorescence index (MFI) of CD16 in patients undergoing cardiac surgery pre-operation and at day 5 and 90. In agreement with our data, elevated MFI of CD16 was observed at day 5 after operation6. This small study (n = 17 patients), however, did not identify an association between CD16 and outcome6. In a larger study, Mon3 have been associated with advanced vascular dysfunction in patients undergoing cardiopulmonary bypass13.Monocytes are involved in many pathogenic processes, ranging from the acute response to injury and infections to modulation of chronic inflammation  to wound healing31. The blockade of CCR2 and thereby the reduced recruitment of inflammatory monocytes was also found to be protective in models of renal inflammation in mice33. Mon2 (intermediate) monocytes were described to be a predictor for cardiovascular disease events in CKD patients with or without hemodialysis34. Regarding to non-dialysis patients there were no elevated Mon2 compared to patients with preserved renal function28. Patients with dialysis had higher numbers of Mon 2 as part of inflammatory monocytes compared to patients without renal dysfunction34.Regarding extracardiac complications, inflammatory monocytes have indeed been implicated in the pathogenesis of delirium and acute kidney injury. Data from mice with sepsis-associated encephalopathy, an established model of delirium, demonstrated reduced neuroinflammation accompanied with reduced signs of cognitive impairment by prevention of CCR2 + monocyte recruitment35. On the other hand, IL6 mediated neutrophil activation is one of the central mechanisms in AKI36. Mon2 produce IL6 under stimulation, which may possibly explain the association of elevated Mon2 with extracardiac complications such as delirium and AKI after cardiac surgery. In this regard, it is unexpected that Mon2 were not associated with infectious complications. This could be potentially related to the low number of infections in our cohort (only 15 patients) as well as to the sampling time (up to 4 days after surgery might be still dominated by the procedure-related inflammatory reaction).In our analyses, Mon1 and Mon2 correlated with IL6 levels before surgery. Other studies could show that patients with delirium hat significant higher blood cytokine levels (including IL6) comparing to those without deliriumUnivariate logistic regression analyses showed that elevated Mon 2 before and after surgery were predictive for extracardiac complications within the first four days after cardiac surgery. In would be of high interest, if measurement of Mon2 in the context of cardiac surgery might be an opportunity to prevent delirium and AKI by modulating immune cells and or eliminating certain cytokines. Until now it is ambiguous at which time point  Mon2 levels might have the best predictive value.37. Our study indicates that even patients undergoing elective cardiac surgery might benefit from implementing immune-related markers in risk stratification.Altogether, our data as well as findings from other studies support the hypothesis that circulating monocyte populations are indicative of activated immunological and inflammatory pathways, thereby supporting their potential as a biomarker as well as a potential therapeutic target. Many efforts currently focus on reversing “immune cell dysfunction” in patients with critical illness and/or sepsis; these approaches include the administration of immune modulators , removal of inflammatory cytokines  or cell-based therapies Our study was a single center study with a limited number of patients and was designed to assess 90-day mortality, but no longer follow-up periods. Due to the very low 90 day-mortality rate (3%), the sample size did not allow to assess the prognostic impact of leukocyte populations on survival. It can be anticipated that larger prospective multicenter studies may allow to better define the predictive value of elevated Mon2 monocytes after surgery and subdivide the extracardiac complications in distinct subgroups of AKI and delirium. To understand the effects of Mon2 monocytes as a pathophysiological component for the development of extracardiac complications such as AKI and delirium, the interaction between these cells and the surfaces of extracorporal circulation has to be analyzed in detail.Based on a prospectively enrolled cohort of 104 patients with sequential, detailed immune phenotyping analyses, our study identifies post-operatively elevated Mon2  monocytes as a predictive marker for extracardiac complications in patients undergoing elective cardiac surgery. Our data support the hypothesis that immunological alterations during and after surgery are related to the subsequent clinical course of surgical patients and that analyzing monocyte subpopulations might be a promising biomarker for the development of post-operative complications. Despite their different pathophysiological background, we focused on acute kidney injury and delirium, because these are the most common non-infectious extracardiac complications after cardiac surgery. Larger multi-center studies are warranted to develop new strategies involving this marker in individual risk prediction for specific (possibly even more rare) extracardiac complications after elective heart surgery and for exploring novel therapeutic targets.