Background: The live donor liver transplantation (LDLT) process is circuitous and requires a considerable amount of coordination and matching in multiple aspects that the literature does not completely address. From the coordinators’ perspective, we systematically analyzed the time and risk factors associated with interruptions in the LDLT process. Methods: In this retrospective single center study, we reviewed the medical records of wait-listed hospitalized patients and potential live donors who arrived for evaluation. We analyzed several characteristics of transplant candidates, including landmark time points of accompanied live donation evaluation processes, time of eventual LDLT, and root causes of not implementing LDLT. Results: From January 2014 to January 2021, 417 patients (342 adults and 75 pediatric patients) were enrolled, of which 331 (79.4%) patients completed the live donor evaluation process, and 205 (49.2%) received LDLT. The median time from being wait-listed to the appearance of a potential live donor was 19.0 (interquartile range 4.0–58.0) days, and the median time from the appearance of the donor to an LDLT or a deceased donor liver transplantation was 68.0 (28.0–188.0) days. The 1-year mortality rate for patients on the waiting list was 34.3%. Presence of hepatitis B virus, encephalopathy, and hypertension as well as increased total bilirubin were risk factors associated with not implementing LDLT, and biliary atresia was a positive predictor. The primary barriers to LDLT were a patient’s critical illness, donor’s physical conditions, motivation for live donation, and stable condition while on the waiting list. Conclusions: Transplant candidates with potential live liver donors do not necessarily receive LDLT. The process requires time, and the most common reason for LDLT failure was critical diseases. Aggressive medical support and tailored management policies for these transplantable patients might help reduce their loss during the process. Live donor liver transplantation (LDLT) is the primary treatment option for end-stage liver disease in countries that have organ shortages ,2. LDLT In addition to meticulous surgery for live donors and recipients , carefulThe Institutional Review Board (IRB) of National Taiwan University Hospital (NTUH), Taipei, Taiwan, approved this study (NTUH REC: 201701044RIND and 202004053RINB). Because this was a retrospective study using chart review, the IRB waived the need for informed consent.We retrospectively reviewed the medical records of hospitalized patients who were evaluated for liver transplants and wait-listed on the Taiwan Organ Registry and Sharing Center from January 2014 to January 2021. Candidates whose potential live donors appeared for donation evaluation were included. The index date was the date of approval for pre-claim review (wait-listing). Patients rejected during pre-claim review or without potential live donors were excluded.According to Taiwan Human Organ Transplant Act, live donor candidates should be aged ≥18 years and be a fifth degree relative of the transplant candidates . When paDemographic information, namely sex, age, height, weight, underlying liver diseases and comorbidities (presence of hepatitis B virus (HBV), hepatitis C virus (HCV), alcohol use, hepatocellular carcinoma (HCC), biliary atresia (BA), diabetes mellitus (DM), and hypertension), MELD  or PediaTransplant candidates were excluded from the waiting list when contraindications emerged or the experts deemed the prospect of LDLT as futile. For example, candidates with distant metastases of HCC or profound septic shock were excluded. Transplant panel specialists retained the final decision for waiting list exclusion.The patients were followed-up until their death or August 2021. The event date was either the date of death, liver transplantation, LDLT, or last follow-up. The date of potential live donor appearance was deemed the same date as that of pre-claim approval if the date of the live donor evaluation was earlier than the start date of wait-listing. The primary outcome was implications of LDLT. Secondary outcomes were overall survival and survival for patients on the waiting list.Original medical records for every patient included in the study were jointly analyzed by a senior transplant coordinator (LHY) with a transplant surgeon (HCM). Data were subdivided into three broad categories of barriers: eligible transplant candidates, eligible donors, and matching and pairing. After data consistency and accuracy were reviewed on a case-by-case basis, a synopsis was written and anonymously presented to independent senior transplant surgeons qualified as experts. After evaluating each patient’s data, the expert panel identified the primary barriers to successful live liver donation and categorized transplant candidates using a fishbone diagram.t test, χ2 test, or Fisher’s exact test was used, where appropriate, to compare the variables. Cumulative survival rates, probabilities of donor appearance, and probabilities of successful LDLT were estimated using the Kaplan–Meier method and compared using the log-rank test and post hoc analysis. Logistic regression modelling was employed for univariable and multivariable analyses. Statistical significance was indicated by a two-sided p value of <0.05. Analyses were performed using SPSS version 21.0 .Descriptive statistics are expressed as a mean ± standard deviation or number (percentage) where appropriate. The Student’s During the study period, 968 patients were evaluated for liver transplantation and 784 were wait-listed . After 3In total, 331 candidates (260 adults and 71 children) had at least one potential live donor who completed the second stage of donor evaluation. p < 0.001; p < 0.001; The 1-, 3-, 5-, 7-, and 9-year overall survival rates after placement on the waiting list were 68.2%, 63.4%, 58.2%, 55.7%, and 54.6%, respectively, among the “intend for LDLT” candidates. Overall survival in the pediatric subgroup was superior to that in the adult subgroup (p = 0.034), with post hoc significance in comparison subgroups  days. The probability of intention for liver donation differed for the subgroups who died without having received a transplant, lived without a transplant, and received a transplant (The median time from live donor appearance to either LDLT or DDLT was 68.0 (IQR 28.0–188.0) days. Most (90.2%) of the candidates in this study received LDLT within 138 days of the initiation of live donor evaluation.p = 0.011, p < 0.001, p < 0.001, respectively.Among the 205 patients who eventually received LDLT, the median time from placement on the waiting list to live donor appearance, time from live donor appearance to LDLT, and total waiting time were 65.0 , 24.0 (7.0–61.0), and 101.0 (58.5–170.0) days, respectively. Among the 124 patients who died without having received a transplant, the median time from being placed on the waiting list to live donor appearance, time from live donor appearance to death, and total waiting time were 13.0 , 29.5 (10.5–89.8), and 44.5 (19.3–167.0) days, respectively. The differences of the three duration periods between the LDLT group and the group who died without having received a transplant were significant, with Thirty-five live donors with intention to donate began evaluation before the pre-claim approval as transplant candidates. Among the 35 transplant candidates, 19 received LDLT, one received DDLT, eight died before transplant, five were waiting in a stable condition, one recovered, and one was lost to follow-up after 15 months.According to a univariate analysis, older age, adult patients, HBV carriers, high MELD or PELD scores (≥30), presence of hypertension or encephalopathy, and increased total bilirubin level were significantly associated with successful LDLT . BA was Documented barriers to a successful LDLT process in transplant candidates with “intend to donate” live donors are illustrated in In total, 21 patients who received DDLT were not included. Three patients (two died without having received a transplant) with unfinished potential live donor evaluations for unidentified causes were not included.This study revealed four major findings. First, more than half  of transplant candidates had at least one potential “intend to donate” live liver donor. Most  finished the donor evaluation process, and nearly half  received LDLT. Second, underlying liver diseases (HBV and BA), liver disease severity , and hypertension were associated with receiving LDLT. Among them, BA was the only positive predictor. Third, the median time from being wait-listed to the appearance of a potential live donor was 19.0 (IQR 4.0–58.0) days, and that from live donor appearance to either LDLT or DDLT was 68.0 (IQR 28.0–188.0) days. Finally, the primary barriers to LDLT were, in decreasing frequency, critical transplant candidates, donor physical conditions, motivation of live donation, and stable transplant candidates.Our study indicates that a critical or inappropriate status of transplant candidates was the primary reason for exclusion from the waiting list. This is consistent with a report by Pamecha et al.  Trotter Physical condition and motivation were two major donor-related factors in our study. As advancements in imaging and surgical science are made, the barriers of insufficient graft volume (to maintain enough remnant liver volume) or anatomical variation may have less of an effect ,17,18,19A limitation of this study is that not all of our results may be externally applicable because of the differences in government regulations, sociocultural backgrounds, institutional policies, and operations management. However, we illustrated the difficulty of the LDLT process, addressed the risk factors, and analyzed the root causes of LDLT. Some of our results are consistent with those of previous studies.In conclusion, half of transplant candidates with “intend to donate” live donors failed to receive LDLT. The most common reason for excluding patients from the waiting list was the existence of critical diseases. The median time between placement on the waiting list and the appearance of the donor was 19 days, and the preparation time for the transplant was 68 days. Therefore, additional time is required, and LDLT is not guaranteed for all patients, especially adults with HBV and children without BA. Aggressive medical care is essential for transplantable patients. The current data may serve as a guide for liver allocation policy makers.


Treatment of pregnant cancer patients should adhere as much as possible to standard treatment protocols in order to safeguard maternal prognosis. The use of Granulocyte colony-stimulating factor (G-CSF) can be indicated for dose dense chemotherapy in high risk breast cancer patients or for the treatment or prevention of neutropenic fever, which can be an important threat for maternal and fetal survival. However, as evidence is still scarce, physicians are still reluctant to the use of G-CSF during pregnancy. In this series, the International Network of Cancer, Infertility and Pregnancy (INCIP) reports on 42 pregnant patients who received G-CSF during oncological treatment. Reported maternal and neonatal complications are acceptable; however, a continuous evaluation of clinical practice is necessary as the limited data in numbers and follow-up do not allow robust conclusions.Data on the use of Granulocyte colony-stimulating factor (G-CSF) in pregnant cancer patients are scarce. The International Network of Cancer, Infertility and Pregnancy (INCIP) reviewed data of pregnant patients treated with chemotherapy and G-CSF, and their offspring. Among 2083 registered patients, 42 pregnant patients received G-CSF for the following indications: recent chemotherapy induced febrile neutropenia , dose dense chemotherapy , poly chemotherapy , or prevention of neutropenia at delivery . Among 24 women receiving dose dense chemotherapy, three (13%) patients recovered from asymptomatic neutropenia within 5 days. One patient developed pancytopenia following polychemotherapy after which the pregnancy was complicated by chorioamnionitis and intrauterine death. Nineteen singleton livebirths (49%) were born preterm. Sixteen neonates (41%) were admitted to the Neonatal Intensive care Unit (NICU). No neonatal neutropenia occurred. Two neonates had congenital malformations. Out of 21 children in follow-up, there were four children with a motor development delay and two premature infants had a delay in cognitive development. In conclusion, the rate of maternal and neonatal complications are similar to those described in (pregnant) women treated with chemotherapy. Due to small numbers and limited follow-up, rare or delayed effects among offspring exposed to G-CSF in utero cannot be ruled out yet. The co-occurrence of cancer and pregnancy, estimated to affect 1 in 1000 pregnancies, is expected to rise due to increasing maternal age and incidental findings at the occasion of the non-invasive prenatal testing (NIPT) . Breast 9/L) and 4 (ANC < 0.5 × 109/L) neutropenia or febrile neutropenia (grade 3–4 neutropenia with fever) in patients receiving chemotherapy [G-CSF supports the clonal growth of progenitors of neutrophils and regulates the proliferation and differentiation of hematopoietic stem cells. Both in vitro and in vivo studies confirmed transplacental passage of this glycoprotein ,7. In onotherapy ,9. In pa ANC < 0. × 109/L As available data are still limited to case reports and small case series, use of antenatal G-CSF is still debated ,12. HencWomen with cancer and treated with G-CSF during pregnancy were identified in the INCIP database. The INCIP study has been approved by the ethical committee of the university hospitals of Leuven in Belgium (S25470) and the Erasmus Medical Center in the Netherlands (NL4354607813). The international multicenter study is registered on ClinicalTrials.gov, number NCT00330447. The registry contains both retro- as prospectively collected obstetric and oncological data of women (and their offspring) with a cancer diagnosis in association with pregnancy.9/L—lower limit of normal (LLN) or ANC 1.0–1.5 × 109/L, respectively) and grade 3–4 neutropenia . Febrile neutropenia was defined as an ANC below 1.0 × 109/L and fever (=>38 °C). Grade 3–4 leukopenia was defined as a white blood cell count (WBC) below 1.0 × 109/L), thrombocytopenia was divided in grade 1–2 (mild) [platelets count (PC) < 150 × 109/L) and grade 3–4 (severe) (PC < 50 × 109/L) [The INCIP database was reviewed for oncological, obstetrical, neonatal, and pediatric data, and missing data were requested from participating hospitals. Pregnancy dating was confirmed in all patients by early ultrasound. To define the efficacy of G-CSF in prevention of chemotherapy-induced neutropenia (dose dense regimen or polychemotherapy regimens with a high risk of neutropenia), results of all available maternal blood samples taken before and during chemotherapy until two weeks after the last administration of G-CSF were retrospectively extracted from patient files. The incidences of maternal neutropenia, leukopenia, anemia, and thrombocytopenia were assessed. Grading of neutropenia, leukopenia, anemia, and thrombocytopenia was defined according to the Common Terminology Criteria for Adverse Events (CTCAE version 5.0) . Neutropfever =>3 °C. Grad9/L, leukopenia as white blood cell count (WBC) < 5.0 × 109/L, thrombocytopenia as PC < 15 × 109/L, and anemia as Hb less than 14 g/dL [The neonatal blood samples were taken within 48 h after delivery. Neonatal neutropenia was defined as absolute neutrophil count (ANC) < 1.0 × 10 14 g/dL ,17. Cust 14 g/dL . NeonateIn addition, available data of children included in the long-term prospective follow-up study of the INCIP project were collected. In this follow-up study, children underwent a general physical examination by a pediatrician , cognitive  tests by a psychologist , followed by non-Hodgkin lymphoma , Ewing sarcoma , and acute lymphocytic leukemia (ALL)   .-n = 28; 67%).Long-acting G-CSF was mostly given as part of a dose dense schedule (-Five patients (12%) developed grade 3–4 neutropenia (including one patient with neutropenic fever) after one to three cycles of 3-weekly chemotherapy (without G-CSF). Two of them received filgastrim during the acute episode of neutropenia and all five patients had long-acting G-CSF with the subsequent chemotherapy (without treatment delay) administrations in prevention of febrile neutropenia or dose delays.-n = 4), Ewing sarcoma (n = 1) or ALL (n = 1).Seven women (7%) received long-acting G-CSF following ‘high risk’ polychemotherapy for Non Hodgkin lymphoma (-Long-acting G-CSF (pegfilgastrim) was given prophylactically after the last chemotherapy before delivery in two women (5%).The median GA at the start of chemotherapy was 22 weeks (range 11–36). One patient started chemotherapy for stage 3 breast cancer at 11 weeks of gestation and delivered of twins without malformations, in all other patients chemotherapy was initiated after 13 weeks of gestation. Both short-acting (filgrastim) and long-acting G-CSF  were administered for the following indications:Blood results of 24 women who received dose dense chemotherapy were registered . Uncompl9/L) following vincristine, ifosfamide, doxorubicin, etoposide (VIDE) chemotherapy. Two patients received rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone (R-CHOP) with pegfilgastrim and did not develop hematological toxicities, except for mild anemia in one patient.Three patients with available blood counts received long-acting G-CSF as part of intense polychemotherapy with a high risk for neutropenia. One patient suffered from a pancytopenia severe anemia, severe thrombocytopenia, and WBC < 0.1 × 10The obstetric outcomes of 40 singleton pregnancies are described in Nineteen singleton livebirths were born preterm . Of all singleton neonates, 11 (28%) were SGA. Thirteen neonates (41%) were admitted to the NICU, mainly because of prematurity (81%) . There wOne women delivered of healthy twins following induced labor at 37 weeks after dose dense epirubicin, cyclophosphamide (EC) and paclitaxel weekly. Another twin pregnancy ended in a spontaneous delivery at 29 weeks after dose dense EC and paclitaxel weekly.n = 1) and 18 months (n = 3). One child had a hip dysplasia and one child had a preferential posture, which both required physiotherapy. Eventually, all four children had a normal motor development at 18 months and 36 months of follow-up according to standardized and clinical measures of development. In four children (19%), delayed cognitive development was identified; one child born at a GA of 29 weeks and 2 days, was assessed at 18 months of age and had an appropriate cognitive development, but delayed language development. The child was referred to a speech-language therapist. Two children (10%), both born after GA of 37 weeks, had a delay in cognitive development at 18 months of age, but cognitive development was appropriate at 3 and 6 years of age according to standardized and clinical measures of neurocognitive development. Another child, prenatally exposed to nicotine, cannabis and chemotherapy, was born at a GA of 35 weeks and 4 days with a birthweight of 2060 g (P 5.1). The child had an appropriate cognitive development at 18 months of follow-up, but cognitive and language development was delayed at 3 years of age. At the age of 3 years, the child also had behavioral and emotional problems and bodyweight was above the 97th percentile. The child and parents were referred to a specialized center for childcare.Twenty-one children participated in the follow-up study of the INCIP. The median follow-up was 18 months (range 2 months–9 years) . No neurn = 3; 13%). There was one stillbirth following maternal pancytopenia and chorio-amnionitis after VIDE chemotherapy, despite administration of G-CSF to prevent hematological toxicity. Two out of 39 singletons had a congenital malformation (5%). Neonatal neutropenia did not occur and there were no major abnormalities reported in the clinical follow-up of 21 children.In this manuscript, we report the maternal and neonatal outcomes of 42 patients with chemotherapy and G-CSF treatment during pregnancy. In the 24 patients who received dose dense chemotherapy supported with G-CSF, febrile neutropenia did not occur and grade 3–4 maternal neutropenia could often be prevented  [Neonates born from 12 mothers who received G-CSF just before delivery were shown to have increased neutrophil counts compared to a control group . La Nasactively) . Using tctively) .The incidence of SGA in this population was 28% and 41% of neonates were admitted to the NICU, mostly because of prematurity, which is comparable to large cohort studies on pregnant women with cancer  . In thisThe rate of severe neutropenia following dose dense chemotherapy was 13% (3/24), which is comparable with the reported rates in the non-pregnant population (14.9%) . HoweverMajor neurologic or functional cardiac abnormalities were not found during follow-up of 21 children. Earlier studies showed that prematurity is a predictor for worse cognitive outcome rather than prenatal exposure to cancer treatment . In addiThe maternal and neonatal outcomes in this series are in line with previously published cohort studies of 10 and 34 pregnant women that received G-CSF during oncological treatment ,12. A stSince we did not observe any marked increase in perinatal complications and the outcomes of this series are in line with available literature, we conclude with caution that the use of G-CSF during pregnancy can be considered when this is clinically indicated for maternal oncological treatment. However, our study was not powered for perinatal complications with low incidence, such as thrombosis, or delayed long-term effects, including secondary malignancies. G-CSF should therefore continue to be administered with caution, and only for indications which provide proven benefit for survival and cancer prognosis, and preferably in the context of ongoing registration studies. These data further contribute to the policy to treat pregnant cancer patients as much as possible as non-pregnant cancer patients in order to safeguard cancer outcomes.


Anti-citrullinated protein antibodies (ACPAs) are highly specific for rheumatoid arthritis (RA). In vivo, ACPAs target peptidyl-citrulline epitopes (cit-) in a variety of proteins (cit-prot-ACPAs) and derived peptides (cit-pept-ACPAs) generated via the peptidylarginine deiminase (PAD) isoenzymes. We aimed to identify a cell line with self-citrullination capacity, to describe its autoantigenic citrullinome, and to test it as a source of autocitrullinated proteins and peptides.Human cell lines were screened for cit-proteins by Western blot. PAD isoenzymes were identified by RT-PCR. Autocitrullination of ECV304 was optimized, and the ECV304 autocitrullinomes immunoprecipitated by sera from three RA patients were characterized by mass spectrometry. Cit-pept-ACPAs were detected using anti-CCP2 ELISA and cit-prot-ACPAs, by an auto-cit-prot-ECV304 ELISA. Sera from 177 RA patients, 59 non-RA rheumatic disease patients and 25 non-disease controls were tested.Of the seven cell lines studied, only ECV304 simultaneously overexpressed PAD2 and PAD3 and its extracts reproducibly autocitrullinated self and non-self-proteins. Proteomic analysis of the cit-ECV304 products immunoprecipitated by RA sera, identified novel cit-targets: calreticulin, profilin 1, vinculin, new 14–3-3 protein family members, chaperones, and mitochondrial enzymes. The auto-cit-prot-ECV304 ELISA had a sensitivity of 50% and a specificity of 95% for RA diagnosis.ECV304 cells overexpress two of the PAD isoenzymes capable of citrullinating self-proteins. These autocitrullinated cells constitute a basic and clinical research tool that enable the detection of cit-prot-ACPAs with high diagnostic specificity and allow the identification of the specific cit-proteins targeted by individual RA sera.The online version contains supplementary material available at 10.1186/s13075-021-02698-2. Rheumatoid arthritis (RA) is a chronic polyarthritis where ACPAs define a major subset 50 to 75%) of patients, with an increased risk of severe disease course –3. ACPAs0 to 75% Several cit-prot-autoantigens with potential pathophysiologic roles have been proposed , 11. HowA convenience sample of seven human cell lines from American Type Culture Collection (ATCC) were tested: ECV304 (ATCC® CRL-1998™ derivative of T24), T24 , BJ , HCA-2 , HEK293T , PC-3 , and HeLa . To prevent PAD activation, cells collected mechanically from monolayers were washed with TEN solution . Cell lysates were prepared by three freeze–thaw cycles. Post-centrifugation, the protein concentration in the cell lysates supernatant, hereafter ‘cell lysate’, was measured , aliquoted and stored at -80˚C.2] [Cell lysates [0—50 µg] were incubated in citrullination buffer   at 37˚or2] .3, 2.3 M H2SO4, 1.5 M H3PO4, 0.25% diacetyl monoxime, 0.125% antipyrine, and 0.25% acetic acid) at 37 °C , 59 patients with non-RA autoimmune rheumatic diseases: ANCA-positive vasculitis (p < 0.001), RF + , presence of erosions , and older age [each year increased the probability to be ECV304 + (OD 1.02 p = 0.036)]. There was a trend that the presence of subcutaneous nodules  was associated with ECV304 positivity. In multiple logistic regression models, only CCP +  and RF +  were associated with ECV304 positivity.In univariate logistic regression, CIT-ECV304 ELISA positivity was associated with CCP + ; mitochondrial enzymes (triosephosphate isomerase and α-enolase 1); and chaperones . The 14–3-3 protein family has seven isoforms , of which both native and cit-14–3-3η (not present in ECV304) are recognized RA autoantigens associated with accelerated joint damage . We deteFour cit-autoantigen candidates need to be discussed in relation to our findings in the ECV304 autocitrullinome: cit-fibrin, cit-collagen II, cit-histones, and cit-vimentin. The first two are not present in ECV304 cells. Pept-histones and cit-pept-histones were detected in both CTR and CIT-ECV304 lysates respectively, suggesting that native histones are also antigenic in RA as in other connective tissue diseases. Cit-vimentin, the Sa antigen was originally found in placenta and pannus lysates . VimentiOther identified CIT-ECV304 ACPA targets are novel citrullinated targets of RA. Cathepsin D mRNA, for example, is differentially expressed in RA and OA synovial tissues . ChloridThe CIT-ECV304 ELISA is a multi-auto-cit-prot-ACPA ELISA and was used to identify relevant “private” epitope-carrying-proteins. It has a diagnostic specificity in RA comparable to anti-CCP2 and slightly superior to RF . Its sensitivity is lower than anti-CCP2 and RF . This could support the hypothesis that each RA patient’s serum may favour a limited set of “private” cit-protein epitopes, but reacts more with CCP, a “promiscuous” widely cross-reactive cit-peptide. It would also be consistent with RF, being a prevalent secondary autoantibody system in a disease where many currently undetected autoimmune systems exist.Future research should aim at documenting the coexistence of the two types of ACPAs: the less pathogenic promiscuous cit-pept-ACPAs currently used in the clinic and the more pathogenic private cit-prot-ACPAs . Even ifWe validated that ECV304 has several autoantigens, including cit-antigens recognized by RA and that these can be identified post-citrullination of the lysate utilizing IP with RA sera and MS analysis. The cit-antigens were produced by PAD2 and PAD3; however, evaluating novel antigens produced by the other PAD isotypes would be of interest. Several cell lines express one or more of the PAD isotypes in abundance. Therefore, the same approach used here could be applied to other cell lines with high levels of PAD1, PAD2, PAD3, PAD4 or PAD6.The different proteins identified by RA sera highlight the complexity of the cit-prot-ACPAs. Our work provides a platform to facilitate understanding why different cit-autoantigens and ACPAs occur in individual patients, and document the role of cit-proteins-ACPAs in the course of RA .ECV304 cells overexpress PAD2 and PAD3 capable to citrullinate auto-proteins that are recognized by RA sera, informing novel, and known private cit-protein targets. A cit-ECV304 ELISA could be used to screen for ACPAs to many private cit-protein epitopes without the need of having an ELISA for each cit-protein. In selected patients, during the pre-RA to clinical RA phase, using proteomics on RA immunoprecipitates of the autocitrullinome of ECV304 might allow the specific identification of potentially critical private cit-epitopes, thus providing new insights into the putative role of ACPAs in RA pathogenesis. The ECV304 cell line is a promising tool to study citrullination and its inhibition/modulation by anti-rheumatic drugs.Additional file 1: Fig. S1. Time course of PAD activity in ECV304 cells by WB. ECV304 lysates (2µg of proteins/lane) were incubated at 37˚C in the presence of calcium (10 mM). Citrullinated proteins were detected by anti-citrulline (modified) detection kit. The reaction was stopped by adding EDTA (100 mM). PAD activity from ECV304 lysate can generate cit-proteins in vitro. No cit-proteins were present at time point 0.Additional file 2: Fig. S2. Detection of vimentin and citrullinated vimentin by WB. (A) WB using anti-vimentin monoclonal clone V-9 , showing the presence of vimentin in placenta lysate and ECV304 cell lysate (5µg protein/well). (B) WB using RA serum, showing the presence of citrullinated proteins in ECV304 cell lysate containing proteases inhibitors. The ECV304 containing protease arrest, calpain inhibitor and acetyl-calpastatin  showed the presence of citrullinated proteins. The inhibitors were added before the citrullination. Inhibitors: 1 – Protein Arrest reagent (1X) ; 2 – Calpain inhibitor VI (1 µM) (Sigma-Aldrich) and 3 – Acetyl-Calpastatin (1 µM) (Sigma-Aldrich).Additional file 3: Table S1. Primers used for identification of PAD isoenzymes.Additional file 4: Table S2. Authentication of the ECV304 (ATCC CRL-1998) and T24 (ATC HTB-4) cell lines by Short Tandem Repeated DNA profiling at ATCC.Additional file 5: Table S3. Proteins unique to CIT-ECV304 immunoprecipitates.


Here, we suggest that the elusive local hyperosmotic compartment resides within the membrane transport proteins themselves. In this manner, the battery of plasma membrane transporters expressed in choroid plexus are proposed to sustain the choroidal CSF secretion independently of the prevailing bulk osmotic gradient.Cerebrospinal fluid (CSF) envelops the brain and fills the central ventricles. This fluid is continuously replenished by net fluid extraction from the vasculature by the secretory action of the choroid plexus epithelium residing in each of the four ventricles. We have known about these processes for more than a century, and yet the molecular mechanisms supporting this fluid secretion remain unresolved. The choroid plexus epithelium secretes its fluid in the absence of a trans-epithelial osmotic gradient, and, in addition, has an inherent ability to secrete CSF against an osmotic gradient. This paradoxical feature is shared with other ‘leaky’ epithelia. The assumptions underlying the classical  Brain fluid homeostasis is critical in health and disease. Normal brain function is highly reliant on a spatial connectivity that may be altered with pathological brain fluid accumulation. Conditions such as stroke or traumatic brain injury may associate with elevated intracranial pressure, which, if left untreated, may lead to subsequent brain herniation and death. In addition, brain fluid imbalance occurs in hydrocephalus patients, in which a mismatch between cerebrospinal fluid (CSF) production and absorption may result in dilated CSF spaces with potentially devastating consequences. Although CSF secretion is a crucial component in brain fluid homeostasis, there is still uncertainty regarding the molecular mechanisms underlying its secretion. An understanding of those mechanisms is essential for development of novel therapeutic approaches. This review assesses evidence related to CSF secretion and posits that transporters expressed in the CSF-secreting tissue, the choroid plexus, play a central role in CSF secretion by their inherent ability to mediate transporter-mediated water transport.The mammalian brain consists of approximately 80% water, which is the main constituent of the cerebrospinal fluid (CSF). The CSF is interconnected with the interstitial fluid (ISF) –4 that sThe ventricular walls are lined by a layer of ciliated columnar ependymal cells, which serve to separate the ventricular cavities from the brain tissue . Notably2, Fig. +/K+-ATPase α1 subunit   , 106, anhe cell) .As an alternative, the inter-microvillar space at the apical surface of the choroid plexus was proposed to represent a smaller space of a similar function to that of the lateral intercellular spaces in the ‘forward’ epithelia Fig. F. As suc−/−) display no overt phenotype o, Fig. +-induced onset of inwardly-directed cation-Cl− cotransporter activity. Inhibition of the KCC  or the NKCC1 (rodent) abolished the K+-induced cell swelling and permitted the tissue to respond as a passive osmometer, as with mannitol and NaCl as the osmolyte, Fig. The ability of cotransporters to mediate water transport in a manner independent of an osmotic gradient has been demonstrated in ex vivo preparations of choroid plexus from salamander (culosus) , 124 andculosus) , 146 by +/K+-ATPase, NKCC1, and the HCO3− transporters are key to CSF secretion  or pCO2, etc. ISF/[K+]CSF), and the extracellular K+ concentration ([K+]o) is therefore tightly regulated CSF (and plasma K+ concentration ([K+]plasma)) in neonates and fetuses [+]o rises quickly with anoxia in these age groups CSF management appeared gradually with development. Only after postnatal day 10, did the K+ concentration in the CSF remain stable with variable plasma [K+] in the rat [+/K+-ATPase abundance and activity [+ clearance increased as a function of ventricular [K+]CSF and was nearly abolished by the Na+/K+-ATPase inhibitor ouabain in vivo CSF fluctuations.Adult mammals have a remarkable ability to sustain their ventricular and interstitial fluid Krkalemia , 195. Thrkalemia , but arorkalemia , likely activity . In cont the rat , which aactivity , 198. Th in vivo  and ex v in vivo . The Na+d plexus , 200. In+ uptake , and of + uptake . These r+ influx into the choroid plexus epithelial cells across the luminal membrane is conducted by the Na+/K+-ATPase, a smaller ouabain-insensitive K+ influx was observed in rats [+ uptake was stable with age but increased with elevated [K+]o [+ uptake was not functionally assigned in the young rats [+]CSF [+]i CSF management, and thus proposed as a novel CSF drainage pathway in early development CSF , 163 or CSF [+]i . The NKCpression , 198. Baelopment .+ (86Rb+ flux) and Na+ (Na+-sensitive fluorescent dye) i, potentially following enzymatic and mechanical isolation of choroid plexus epithelial cells followed by hours of culturing [+]CSF in neonate mice CSF . With thmembrane , one maymembrane , Fig. 6.The past century of research on the ventricular system and the molecular mechanisms of CSF secretion peaked in the 1960s-1980s with a flurry of solid publications describing technically challenging in vivo experimentation on various animal models, performed by highly skilled and insightful physiologists. Although divergent viewpoints have emerged, direct experimental evidence supports that the choroid plexus is the dominant source of the CSF continuously secreted into the brain. CSF secretion occurs independently of conventional osmotic forces and readily continues in the face of an oppositely-directed osmotic gradients. This ability to transport water against an osmotic gradient does not arise from local osmotic forces, as those exemplified by the standing gradient hypothesis. This model lacks experimental support and is, in addition, poorly adaptable to the ‘reverse’ choroidal epithelium with its lateral intercellular spaces located on the ‘wrong’ epithelial aspect. Instead, emerging evidence suggests that it is membrane transport mechanisms with an inherent ability to couple fluid transport to their solute translocation that are the molecular motors driving CSF secretion. Future research aimed at identifying key transporters in the basolateral membrane of the choroid plexus and quantifying the contribution of the various choroidal transporters and their regulation in health and disease is envisioned to contribute to closing one part of the large knowledge gap that pertains to most aspects of brain fluid dynamics, i.e. CSF secretion, circulation, and drainage. Hopefully, such future knowledge will contribute new approaches towards regulation of brain fluid dynamics in pathological conditions such as hydrocephalus.


Although cardiac troponins are considered the most specific biomarkers for the diagnosis of acute myocardial infarction (AMI), their diagnostic consideration goes far beyond the detection of this dangerous disease. The mechanisms of cardiac troponin elevation are extremely numerous and not limited to ischemic necrosis of cardiac myocytes. Practitioners should be well aware of the underlying pathological and physiological conditions that can lead to elevated serum levels of cardiac troponins to avoid differential diagnostic errors, which will be greatly increased if clinicians rely on laboratory data alone. This article presents a classification of the main causes of an elevation in cardiac troponins and discusses in detail the mechanisms of such elevation and the diagnostic consideration of cardiac troponins in some conditions not associated with AMI, such as physical exertion, inflammatory heart diseases (myocarditis and endocarditis), pulmonary embolism (PE), renal failure, and systemic inflammation (sepsis). The troponin complex is the most important regulator of the contraction/relaxation of striated muscle tissues  and consists of three troponin isoforms (subunits): troponin T, troponin I, and troponin C. Each of these proteins performs a specific function. Troponin T, or a tropomyosin-binding subunit, interacts with tropomyosin and attaches the troponin complex to actin filaments. Troponin I, or an inhibitory subunit, inhibits interaction of actin filaments with myosin heads in the absence of calcium ions in the cardiac diastolic phase. Troponin C, or a calcium-binding subunit, binds itself to calcium ions in the cardiac systole phase initiating conformational changes in the troponin complex to enable muscle contraction. The amino acid structure of troponin T and troponin I is different in cardiac and skeletal muscles, while the amino acid composition of troponin C is exactly the same in cardiac and skeletal muscles. Therefore, troponin C does not possess the required specificity to diagnose acute myocardial infarction (AMI) ,2.The cardiac isoforms of troponins T and I, due to their specificity, are the most common markers for the diagnosis of myocardial infarction ,2,3. An Elucidation of all the causes and mechanisms of an increase in cardiac troponins has both theoretical and practical consideration and is necessary to improve the differential diagnosis of myocardial infarction from other conditions accompanied by non-ischemic alteration of cardiomyocytes ,11,12.Based on the literature data, the most significant causes of elevation of cardiac troponins T and I, complicating differential diagnosis, include heavy and prolonged physical activity, inflammation of the heart wall layers , cardiomyopathies, heart failure, pulmonary embolism (PE), systemic inflammation (sepsis), acute and chronic kidney disease, cardiotoxicity of some therapeutic agents and heart surgeries, and skeletal muscle damage ,4,12 Fi. False-pNotable that improving the sensitivity of troponin immunoassays not only has positive aspects (accelerating diagnosis and improving the prognosis of patients)  but alsoMedical institutions in many countries are gradually switching to the use of highly and ultrasensitive troponin test systems. Anand A. et al. conducted a large study to estimate the global prevalence of highly sensitive assays. The authors, using a questionnaire, collected information from 1902 institutions distributed in 23 countries all over the world. In total, 96% of medical centers used cardiac troponins as the main biomarkers of myocardial infarction, of which 41% employed highly sensitive analyses and an accelerated algorithm for diagnosing myocardial infarction (0 → 3 h) .An official document, the fourth universal definition of myocardial infarction (UDMI) from 2018, regulates the use of highly sensitive immunoassays with accelerated diagnostic algorithms for the early detection of myocardial infarction: the determination of troponin concentration immediately upon admission and then within 1–3 h . Moreover, an earlier diagnosis contributes to the early onset of correct therapy and a significant improvement in the prognosis of admitted patients [The guidelines of the European Society of Cardiology (ESC) emphasize the high efficiency of using early algorithms (0 → 1 h and 0 → 2 h) in order to exclude or confirm myocardial infarction without ST-segment elevation (NSTEMI) . These eIf the level of high-sensitivity cardiac troponins measured at admission is very low or low and there is no significant increase thereof in 1 (or 2) hours, then NSTEMI diagnosis is excluded, and such patients are recommended for early release and ambulant or elective treatment. If the level of high-sensitivity cardiac troponins measured at admission is above the thresholds or upon repeated estimation in 1 (or 2) hours there is a significant increase thereof, then the probability to diagnose NSTEMI is high, and such patients are recommended to be hospitalized. The timing of an invasive strategy is secondary to risk stratification .However, it is important to realize that the increase in cardiac troponin level is not always a sign of AMI (’ischemic necrosis of cardiomyocyte’) but can be indicative of any type of damage to the cardiomyocytes  and can be observed in many, not related to AMI, physiological  and pathological conditions . This rep < 0.01). According to EchoCG data, the ejection fraction of athletes after the finish decreased by an average of 24% in comparison with the indicators before the start (p < 0.002). The results obtained prompted the suggestion that such a heavy load is undesirable, as it damages the myocardium [p < 0.001). Multiple regression analysis showed that higher levels of cardiac troponins were found in older runners with less athletic experience. In 24 h after the race, there was a decrease in troponin levels, but they were still 3–4 times higher than the initial concentrations [Physical activity is one of the widespread reasons for an increase in the concentration of cardiac troponins in the blood serum. Rifai N. et al. were among the first to study cardiac troponins T and I (cTnT and cTnI) in athletes (n = 23) participating in the Ironman triathlon in Hawaii (swimming 3.9 km → cycling 180.2 km → running 42.2 km). The athletes’ concentrations of troponins were measured several days before the start and immediately after the finish, parallel with electrocardiography (ECG) and echocardiography (EchoCG). After the triathlon, increased troponin values were recorded in 26% of the athletes. The highest concentrations of cTnT were observed in athletes who finished in prize-winning places. Moreover, there was a significant correlation between finish and cTnT values . The researchers also noted that the magnitude of the increase in hs-cTnT is associated with the intensity of physical activity against the respiratory threshold, heart rate (HR), and maximum oxygen consumption [Scherr J. et al. studied the longer term kinetics of many inflammatory, cardiac biomarkers, including hs-cTnT, in marathon runners (n = 102). The authors measured the concentration before the start, immediately after the finish, then 24 and 72 h after the race. The concentration of hs-cTnT after the marathon increased by about 10 times in comparison with the indicators before the start (from 0.00361 ng/mL to 0.031 ng/mL), and the complete normalization of hs-cTnT levels was reached 72 h after the finish . A recensumption .Lazzarino A. et al. first noted the relationship between neurosis (the concentration of cortisol) and increased levels of troponin T measured by a high-sensitivity test system (RocheDiagnostics). The authors examined 508 persons without signs of coronary artery disease. According to scientists, the further establishment of the exact role of stress in the pathophysiology of cardiomyocyte damage is an urgent direction .Discussions are ongoing regarding the mechanisms of troponin elevation during physical activity. Some researchers believe that prolonged and heavy loads cause subclinical  necrosis of contractile cardiomyocytes, and more pronounced hypertrophy of the remaining cardiomyocytes (compensation), further increasing the risk of cardiosclerosis (decompensation), which is clinically manifested by heart failure. Perhaps the frequency and intensity of exercise are associated with the risk of heart failure in athletes. Most researchers think that heavy loads lead to transient myocardial ischemia and reversible damage to cardiomyocytes ,26,27,28Transient ischemia during prolonged or heavy physical exertion is probably due to an imbalance between the demand for oxygen in cardiomyocytes, substrates for the energy supply of the myocardium, and their delivery through the coronary arteries. Myocardial tissue is known to be very sensitive to hypo-oxygenation due to the low reserves of myoglobin, glycogen, and other energy resources. Therefore, against the background of increased demand for the myocardium, mediated by tachycardia, the blood supply to the myocardium is reduced due to vasospasm. The main part here belongs to catecholamines and cortisol, which lead to the temporary disruption of myocardial metabolism and reversible damage to cardiomyocytes ,33,34.It is believed that one of the mechanisms for the release of troponins through the intact membrane of a cardiomyocyte is blebbing vesicles (membrane vesicles) with cardiomyocyte proteins inside them. Schwartz P. et al. for the first time observed membrane vesicles in an in vitro experiment using electron microscopy. In this case, the number of blebbing vesicles on the surface of the cardiomyocyte increases respectively to the duration of ischemia, and when it is eliminated, the vesicles are reabsorbed back into the cytoplasm. However, there is still no reliable evidence in favor of the functioning of this mechanism in vivo .Stress echocardiography with mechanical stress  or drugs , as shown by the recommendations ,36, is cSamaha E. et al. in a prospective study studied the kinetics of hs-cTnT concentration (Roche Diagnostics Elecsys 2010) in patients (n = 48) after stress echocardiography. Thirty-three patients underwent stress echocardiography with exercise, and fifteen patients underwent stress echocardiography with pharmacological (dobutamine) stress. Hs-cTnT levels were measured before and 30 min, 1–2, and 4–6 h after completion of stress echocardiography. The maximum increase in hs-cTnT concentration was observed 4–6 h after stress echocardiography. At the same time, the increase in hs-cTnT concentrations was higher after stress echocardiography with dobutamine  than after stress echocardiography with exercise . However, these researchers found no association between elevated troponin levels and stress-induced myocardial ischemia as measured by echocardiography .The studies that showed a direct relationship between the stage of IHD and elevated levels of cardiac troponins in the serum and oral fluid ,39, as wManjunath L. described a case of overestimated troponin I (0.123 ng/mL at a rate of <0.055 ng/mL) in a young patient who was admitted to the emergency department with chest discomfort. Doctors suspected myocardial infarction, additionally supported by unfavorable family history and the presence of hypercholesterolemia. However, ECG, Echo-KG, and coronary angiography did not reveal any signs of myocardial ischemia. The studied anamnesis clarified that the young man was actively involved in sports, and the day before admission, he ran several miles preparing for a marathon .Thus, the elevation of the levels of cardiac troponins is a common effect during heavy physical exertion, while highly sensitive methods register the troponin level diagnostic thresholds crossing much more often than moderately sensitive ones. Such conditions may pose issues in differential diagnosis, but a thorough history taking , as well as taking into account the dynamics of concentration (faster decrease), and the use of additional methods, allow avoiding overdiagnosis and subsequent unnecessary treatment and hospitalization costs. It is also noted that the use of highly sensitive methods for the determination of troponins has great prospects in the diagnosis and prediction of IHD concealed forms using stress tests.An increase in cardiomarkers in myocarditis is due to the direct damaging (cytotoxic) effect of infectious agents , toxins, and autoantibodies on cardiomyocytes. At the same time, troponin concentrations vary widely. Thus, in several studies, troponins, determined by moderately sensitive methods for the diagnosis of myocarditis, had a sensitivity of 34–71% and a specificity of 86–94% ,43,44,45p < 0.0001), and with patients with no inflammation (19.5 pg/mL (13.8–50.7); p < 0.0001). The concentration of hs-cTnT ≥ 50 pg/mL had high sensitivity and specificity in the diagnosis of acute myocarditis. At the same time, the levels of copeptin and NT-proBNP did not significantly differ between these groups and, accordingly, were ineffective in the diagnosis of myocarditis [High-sensitivity troponins are much more likely to increase in myocarditis and have certain advantages over other methods and biomarkers. Thus, Ukena C. and colleagues, for the first time, studied the concentration of hs-cTnT, copeptin, and the N-terminal precursor of natriuretic hormone (NT-proBNP) in patients with suspected myocarditis (n = 70). According to endomyocardial biopsy, which is considered the “gold standard” in detecting myocarditis, patients were conditionally divided into three groups: (1) acute myocarditis was diagnosed in 6 patients, (2) chronic myocarditis in 36, and (3) absence of inflammation in 28 patients. In acute myocarditis, the highest concentrations of hs-cTnT were observed (262.9 pg/mL (61.4–884.2)), compared with patients with chronic myocarditis (20.4 pg/mL (15.6–20.4); carditis .p = 0.042) in patients with the detected viral genome (37.4 pg/mL (21.9–163.6)) compared with those patients in whom viral nucleic acid particles were absent (20 pg/mL (14–44.4)) [According to accumulated information, endomyocardial biopsy has shortcomings in the detection of viral myocarditis since it does not reflect the presence of the viral genome in cardiomyocytes and, accordingly, will not “see” inflammation in the case of a weak course of chronic viral myocarditis . The pat4–44.4)) . This inDue to the high mortality rate of newborn infants and children from myocarditis, averaging 75 and 25%, respectively, a timely diagnosis and severity-adequate therapy are of paramount importance . Since tAccording to Soongswang J. et al., the concentration of cTnT > 0.052 ng/ml has a sensitivity of 71% and a specificity of 86% for the diagnosis of myocarditis in children ,43. A rep = 0.0074). Apart from troponin I, other factors that predisposed to poor outcomes were hypotension, natriuretic peptide (BNP) levels, and decreased ejection fraction [Abrar S. et al. conducted an observational study of pediatric patients with suspected myocarditis. The cTnI concentrations and echocardiographic data contributed to the correct diagnosis, with the mean cTnI levels (0.958 ± 1.13 ng/mL) being higher in the group of non-surviving patients  in ischemic myocardial damage, not such effective markers for the diagnosis of myocarditis? This is likely due to the diagnostic characteristics (primarily sensitivity) of the test systems used. Beyond that, it is assumed that this is due to analytical interferences arising from the presence of autoantibodies to cardiac troponins in the blood serum of patients with myocarditis. Japanese researchers Matsumori A. et al.  revealedAlthough endocardial and pericardial cells do not contain cardiac troponins, endocarditis and pericarditis are often accompanied by elevated troponin levels. This is likely due to the involvement of myocardial tissue in the inflammatory process, because of the tight location of the heart wall layers. Researchers recorded an increase in cTnI concentration in 11 out of 15 (73%) patients with confirmed infective endocarditis. None of the troponin-negative patients died, while two troponin-positive patients passed, indicating a worse prognosis for patients with high cTnI. At the same time, there was no correlation between elevated cTnI concentrations and the need for valve replacement in these patients. The authors also noted that all patients with staphylococcal endocarditis had elevated cTnI levels, while among those with streptococcal endocarditis, only 50% were marked with such elevation . It is pp < 0.005). As a result of hemoculture, Staphylococcus aureus was detected in 71% of troponin-positive individuals [Tsenovoy R. and colleagues studied a larger number of patients with infective endocarditis: an increase in troponin levels above the reference (>0.4 ng/mL) was observed in 35 out of 62 people (57%). It is noteworthy that among troponin-positive patients, in-hospital mortality or the necessity for valve replacement surgery occurred more frequently  [Purcell J.B. et al. in a prospective study concluded that elevated cardiac troponin I levels in patients with myocarditis are associated with a combination of adverse events, such as death, myocardial abscess, and central nervous system damage (< 0.001) .p < 0.001), male sex (p = 0.007), ST-segment elevation (p < 0.001), and pericardial effusion (p = 0.007). At the same time, cTnI was not a negative prognostic marker [In a large study that included 118 patients with viral or idiopathic viral pericarditis, cTnI concentrations exceeded threshold values (>1.5 ng/mL) in 38 people (32.2%). Positive troponin I was significantly associated with younger age (c marker .p = 0.03). Fatal cases were not recorded in the hospital, which indicates that cTnT is not a negative prognostic marker of acute pericarditis [In another study by Gamaza-Chulian S. et al., an increase in cardiac troponin T was recorded in 64 of 105 patients with pericarditis (60.9%). Only young age was associated with higher cTnT levels in a multivariate analysis .Cardiac troponins, according to the clinical guidelines of the European Society of Cardiology (ESC) for the diagnosis and treatment of acute pulmonary embolism, are one of the main biomarkers for predicting and stratifying the risk of pulmonary embolism ,57,58.According to Giannitis E. et al., an increase in cardiac troponins depends on the severity of the thrombotic occlusion of the vascular bed of the lungs: with massive PE on average in 50% of patients; with submassive in about 35%; and with non-massive PE, it practically does not occur. Inpatient mortality, cardiogenic shock, and the need for resuscitation were more common in patients with elevated cTnT values. Troponin-positive patients more often required inotropic support and mechanical ventilation (MV) .p < 0.001, respectively. High levels of cTnI were recorded in 80% of patients with massive PE, 56.25% with submassive PE, and 38.4% with non-massive pulmonary embolism. It is also calculated that a high level of cTnI in the diagnosis of pulmonary embolism has a sensitivity of 50.8% and a specificity of 88.3%. When analyzing a combination of elevated levels of cTnI and D-dimer, the sensitivity in the diagnosis of PE increased to 93.5%, but the specificity decreased to 54.5% [Turkish researchers led by Kilinc G. determined the concentration of cTnI using a moderately sensitive immunoassay (Beckman Coulter). Of 106 patients admitted to the hospital with suspected PE, the diagnosis was confirmed in 63 people. At the same time, increased cTnI levels were found much more often in patients with confirmed PE compared with patients in whom the diagnosis of PE was not confirmed: 50.8% (n = 32) versus 11.6% (n = 5); to 54.5% .Becattini S. et al. performed the most detailed meta-analysis on the predictive value of moderately sensitive troponins in PE. The analysis included 20 studies (1985 patients). The predictive value of cTnI and cTnT was approximately the same. Among 618 patients with elevated levels of cardiac troponins, 122 died ). In contrast, among 1367 subjects with reference troponin values, the deaths were much less frequent—51 . High troponin concentrations are significantly associated with short-term patient mortality (odds ratio (OR) 5.24; 95% CI 3.28–8.38). In the subgroup of hemodynamically stable patients, elevated troponins were also associated with high mortality . Taking this information into account, the authors made an important conclusion: increased concentrations of cardiac troponins in PE allow the identification of patients with a high risk of short-term death and unfavorable outcomes [The kinetics of cTnT concentration in PE and ACS is different, which may indicate different mechanisms of troponin release. In a study, 56% of patients with acute PE were cTnT positive on admission. After 10 h, cTnT concentrations reached peak values (median 0.48 μg/L), and the duration of circulation at the increased concentrations was 30–40 h. In myocardial microinfarction, the peak values were in the range of 0.22–0.41 μg/L, the concentration curves had several repetitive up and down slopes, and the duration of cTnT circulation was more than 120 h .p = 0.029 for the log-rank test). Moreover, according to Cox regression, hs-cTnT was the only biomarker predicting an increased risk of death in the long term. In turn, approximately 50% of patients with poor prognosis had normal levels of moderately sensitive cTnT and, accordingly, could be mistakenly included in the group of patients with a favorable prognosis. This study demonstrates a significant advantage of hs-cTnT over the conventional cTnT test for assessing the prognosis of patients with PE [Lankeit M. et al. prospectively investigated the predictive value of moderately and highly sensitive troponin T in 156 patients with a confirmed diagnosis of PE. In 64% of patients (n = 100), hs-cTnT values exceeded the 99th percentile threshold (>14 ng/mL). Patients with elevated hs-cTnT concentrations had a reduced likelihood of long-term survival  of patients with myocardial infarction, and the concentration of cTnI was increased in 38.6% of patients with PE. The threshold values for differentiating PE and myocardial infarction made 1.12 mg/L for D-dimer  and 0.72 ng/mL for cTnI . Moreover, when using the value of the ratio D-dimers/cTnI > 1.82 in the differential diagnosis of PE from myocardial infarction, the sensitivity and specificity were significantly higher than the diagnostic value of individual markers and amounted to 93.3 and 86.6%, respectively. The authors believe that by using this ratio, an invasive coronary angiography procedure can be avoided .The mechanisms of increasing cardiac troponins in non-massive PE are most likely associated with the release of the cytosolic pool through the membrane of the right ventricular cardiomyocytes, due to increased afterload, which is similar to how it occurs during intense physical exertion ,68,69. IFollowing modern concepts, when using the latest (highly sensitive and ultrasensitive) immunoassays, cardiac troponins can be considered as products of normal myocardial metabolism (“there are no more troponin-negative patients”) ,72,73,74However, it is also important to understand that the concentration of cardiac troponin isoforms in the blood serum depends not only on the mechanisms of troponin release from cardiomyocytes but also on the mechanisms of their elimination from the bloodstream. The most well-known methods for removing many proteins, in particular troponins, include cleavage by specific proteases inside the cells of the reticuloendothelial system; extracellular cleavage by proteases, such as thrombin; and renal filtration ,78,79,80The most controversial mechanism for the elimination of troponins is glomerular filtration. Some researchers denied this mechanism of troponin removal since it was impossible to determine troponins in urine . An indip < 0.05). The authors believe that the determination of the level of hs-cTnI in urine can be used in the diagnosis and monitoring of hypertension [p < 0.001) [p < 0.001 [The work of Croatian researchers led by Pervan P. provides direct evidence for the involvement of the kidneys in the processes of troponin I elimination. Researchers found hs-cTnI by a highly sensitive method (Abbott Architect i1000SR) in all examined patients: in the collected morning urine of normotensive patients, hs-cTnI concentrations were 14.95 pg/mL compared to urinary levels of hs-cTnI = 26.59 pg/mL in patients with high blood pressure (rtension . Given t< 0.001) . In a st < 0.001 . These dNevertheless, not all researchers succeeded in confirming the relationship between the fall in GFR and the increased concentrations of cardiac troponins, as well as detecting troponin in urine ,85. It iTwo more hypotheses intend to explain the increase in cardiac troponins in chronic kidney disease: (1) the “skeletal hypothesis” ,87 and  occurs, followed by reparative regeneration processes, during which, as some scientists believe, the expression of cardiac troponin isoforms occurs. Ricchiutti V. et al. reported the detection of messenger RNA of cardiac troponin T in skeletal muscle biopsies in 50% of patients on hemodialysis due to chronic kidney disease . Haller A long course of chronic kidney disease leads to overload and gradually increasing compensatory myocardial hypertrophy due to an increase in the volume of circulating blood. In conditions of sharp hypertrophy, narrowing of the subepicardial branches of the coronary arteries may happen as a result of their compression, which leads to the release of troponins due to the death of cardiomyocytes and the development of cardiosclerosis. It proves the fact that in some people who died from chronic kidney disease, postmortem examination revealed foci of microinfarction and cardiosclerosis ,90. ThusThe mechanisms of increasing cardiac troponins in sepsis are diverse. One of them is myocardial ischemia, which occurs due to an imbalance between the need for oxygen in cardiomyocytes and its delivery. The imbalance between the demand and the need for myocardial oxygen is due to a variety of pathophysiological pathways. The most significant of them are fever, hypotension, respiratory failure (respiratory hypoxia), and impaired acid-base and water-electrolyte balances, as well as microcirculation disorders, which lead to a decrease in the hemoperfusion of all organs, including the myocardium. Therefore, against the background of fever and hypotension, tachycardia increases in conditions in which cardiomyocytes consume oxygen faster, but its delivery through the coronary arteries decreases. Acid-base disorders, in turn, are accompanied by a disruption in the work of enzyme proteins that provide energy processes in the myocardium . The gradually forced (due to lack of oxygen) transition of the myocardium to anaerobic glycolysis leads to the additional production of lactate, the progression of acidosis, general hypoxia, and metabolic disorders, thereby closing the vicious pathogenetic circle. Under such conditions, reversible or irreversible damage (death) of myocardial cells leads to the release of troponins ,91,92,93Some researchers believe that the main role in damage to cardiomyocytes in sepsis is played by inflammatory mediators, such as tumor necrosis factor-alpha (TNF-ɑ), interleukins , and bacterial exo- and endo-toxins, which have a direct cytotoxic effect on cardiomyocytes ,93,94. KAdditional reasons for the increase in cardiac troponins in patients with sepsis may include microthrombosis of myocardial vessels and increased apoptosis of cardiomyocytes ,97,98. IThus, the mechanisms of increasing cardiac troponins in sepsis can be diverse; their complex participation is very likely with the predominance of one of them in a particular situation.A large meta-analysis by Bessiere F. et al., combining 13 original works with 1227 patients with sepsis, found that an increased level of troponin was significantly associated with an increased risk of death . The prevalence of elevated cardiac troponin concentrations in patients with sepsis was 61% (95% CI 58–64%) .p < 0.001). Nevertheless, despite significantly lower hs-cTnT concentrations, it was higher than the 99th percentile in 51.6% of patients with uncomplicated sepsis and 34.5% of patients without renal insufficiency. Moreover, this also indicates the contribution of additional mechanisms  in the increase in the levels of cardiac troponins in sepsis. The concentration of troponin hs-cTnT correlated with inflammatory markers  and procalcitonin ), serum creatinine , and patient severity scale APACHE II . Hs-cTnT showed good predictive value and means; deceased patients had higher hs-cTnT levels than survivors  [Wilhelm J. et al. determined the concentration of hs-TnT in patients (n = 313) admitted to the emergency department with sepsis. Elevated hs-TnT were found in 197 patients (62.9%). Higher hs-TnT values were recorded in patients with severe sepsis (52.6 pg/mL) and septic shock (65.1 pg/mL) compared with uncomplicated sepsis  .p < 0.001), multisystemic disorder , and creatinine concentration . The median hs-cTnT level was higher in the group of deceased patients than in the group of survivors (0.054 (0.022–0.227) versus 0.035 (0.015–0.111) μg/L, p = 0.047)), while moderately sensitive troponin T did not differ significantly in these groups (p = 0.14). The hs-cTnT concentrations in patients with septic shock were significantly higher than those in patients without shock (0.044 (0.024–0.171) versus 0.033 (0.012–0.103) μg/L; p = 0.03), while the levels of cTnT in patients with shock and without shock did not differ [Rosjo N. et al. investigated the concentration of cTnT and hs-cTnT in 207 patients with sepsis. Of these, 166 patients (80%) had hs-cTnT concentrations above the 99th percentile, while moderately sensitive troponin (cTnT) levels were elevated only in 86 people (42%). There was a correlation between hs-cTnT levels with the severity of the disease , pulmonary embolism, sepsis, and renal failure are very common causes of elevated cardiac troponins. Modern highly sensitive analyses, despite their advantage over moderately sensitive analyses , have a significant disadvantage (reduced specificity). This leads to a frequent increase in troponins in the above conditions, which should, by all means, be considered by clinicians when interpreting the results. High-sensitivity troponins are promising markers for detecting latent  coronary heart disease, especially in combination with physical and pharmacological stress tests. Cardiac troponins in sepsis, pulmonary embolism, endocarditis, and myocarditis can be used as prognostic markers, while highly sensitive analyses are much better suited for this role.


The metabolic processes of endo- and exogenous compounds play an important role in diagnosing and treating patients since many metabolites are laboratory biomarkers and/or targets for therapeutic agents. Cardiac troponins are one of the most critical biomarkers to diagnose cardiovascular diseases, including acute myocardial infarction. The study of troponin metabolism is of great interest as it opens up new possibilities for optimizing laboratory diagnostics. This article discusses in detail the key stages of the cardiac troponins metabolism, in particular the mechanisms of release from a healthy myocardium, mechanisms of circulation in the bloodstream, possible mechanisms of troponin penetration into other biological fluids , mechanisms of elimination of cardiac troponins from the blood, and daily changes in the levels of troponins in the blood. Considering these aspects of cardiac troponin metabolism, attention is focused on the potential value for clinical practice. The authors also noted that in 30% of patients, elevated cTnT levels were not associated with any of the causes of increased cardiac troponins described in the literature [Cardiac troponin isoforms  are the most important regulatory proteins part of the troponin-tropomyosin complex localized on the actin (thin) myofilaments in cardiac myocytes. The protein molecule cTnI is an inhibitory subunit; it blocks the hydrolysis of adenosine triphosphate and the interaction of actin with myosin in the absence of calcium ions in the diastolic phase. The protein molecule cTnT is a tropomyosin binding subunit; it attaches two other troponin subunits to actin filaments. The protein molecule cTnC is a calcium-binding subunit; it binds calcium ions that enter the cytoplasm during the systolic phase ,2. The iterature . Thus, iterature . The diagnostic value of cTnI and cTnT depends on detection methods that constantly improve and change our understanding of biochemistry and broaden the possibilities for using cTnI and cTnT in clinical practice . AccordiThe levels of cardiac troponins, determined using modern immunological methods, depend on the metabolism of troponin proteins. The metabolism of troponins, like any endo- and exogenous compounds, can be conditionally divided into several stages: (1) the release of troponins from cardiomyocytes into the extracellular fluid and blood; (2) circulation in the bloodstream for a certain time; (3) degradation of troponins intra- and extracellularly into smaller fragments and their elimination; (4) troponin molecules can also be more actively released from myocardial cells at certain times of the day, contributing to the formation of circadian rhythms of cardiac troponins.The relevance of studying the metabolism of troponin proteins is not only due to its great theoretical value but also has great practical value. First of all, this must be considered when interpreting elevated troponin levels in patients without established acute myocardial infarction (AMI), optimizing algorithms for AMI diagnosis, and improving the differential diagnosis of AMI ,19,20,21Troponin molecules can also penetrate other human biological fluids , opening new questions and prospects for their use ,33,34,35With the development of highly sensitive assays and the subsequent detection of troponins in all of the healthy people, the attention of researchers is directed to the study of troponin release pathways from viable (intact) myocardial cells. The most studied mechanisms of troponin release from the myocardium of healthy individuals are the following: processes of regeneration and renewal of myocardial cells, apoptosis of cardiomyocytes, the release of troponin as a part of membrane vesicles, the release of fragments of proteolytic degradation of troponins, increased permeability of cardiomyocyte cell membranes, and small-scale  myocardial necrosis ,39. It s14C radioisotope integrated into the DNA of cardiomyocytes, evidence has been presented for the renewal of myocardial cells, the intensity of which decreases with age. Thus, in the age group up to 25 years old, around 1% of cardiomyocytes are divided every year, and the division activity steadily diminishes until it is just 0.45% by the age of 75. It was calculated that about half of the cardiomyocytes are replaced throughout a lifetime, indicating that the myocardium has a limited regeneration capacity. The calculation of the renewal rate of cardiomyocytes is based on assessing the rate of DNA synthesis produced by determining the rate of accumulation of the radioisotope in cardiomyocytes [Although the absence of a significant regenerative capacity of the myocardium is generally recognized, studies prove the myocardium has a small regenerative potential. Using the labeled myocytes . Presumamyocytes ,42. Accomyocytes . It has myocytes . In addimyocytes ,45.The initiation of apoptosis is accompanied by an increase in the activity of caspase enzymes and intracellular proteinases, which leads to the cleavage of DNA and protein structures, with the relatively preserved cell membrane integrity . The modWeil et al. (2017) experimented on pigs, simulating short-term (10-min) ischemia by balloon occlusion in the pool of the second diagonal branch of the left anterior descending artery. Complete occlusion was confirmed by contrast angiography. After reperfusion, the myocardium was removed post mortem for histological evaluation in some animals. Moreover, in another experiment on (live) animals, a series of measurements of troponin I were carried out after reperfusion using a moderately sensitive method  in blood serum samples obtained from the regional (anterior interventricular vein) and systemic (jugular) blood flow. Histological studies did not reveal signs of ischemic damage and necrosis. Apoptosis was confirmed by a sixfold increase in TUNEL-positive cardiomyocytes in the focus of short-term ischemia compared to the non-ischemic (control) area. The concentrations of cardiac troponin I were slightly increased after 10 min. After 30 min, they reached the values of the 99th percentile (38 ng/L) and continued to increase, reaching a peak at 24 h (1021 ± 574 ng/L). There was a very close and reliable correlation between the values of troponin I in the regional and systemic circulation. Thus, the authors noted that short-term ischemia does not lead to cardiomyonecrosis but leads to apoptosis of the cardiomyocytes accompanied by the release of troponins into the bloodstream . AlthougApoptosis of cardiomyocytes can also occur through mechanisms not associated with myocardial ischemia. Cheng et al. (1995) found an increase in programmed cell death in response to myocardial distension . In thisp = 0.01) and remained elevated after 24 h (1462 ± 1691 ng/L). Pathological analysis showed the presence of apoptosis of myocardial cells , which returned to normal after 24 h  without signs of necrosis [An experimental study in pigs showed that an increase in left ventricular preload leads to apoptosis and troponin I release without ischemia. The animals received phenylephrine (300 μg/min) for 1 h to increase the end-diastolic pressure. The basal release of troponin I was low (16 ± 20 ng/L), but 30 min after the increase in end-diastolic pressure, the concentration of troponin I increased above the 99th percentile, and after 1 h, it was 856 ± 956 ng/L : (1) associated —in the contractile apparatus; (2) cytoplasmic—freely located in the cytoplasm of cardiomyocytes and by volume accounts for approximately 3.5% of the total intracellular mass for troponin I and 7.0% for troponin T ,64,65. Animal hepatocytes and cardiomyocytes studies have revealed that during the early stages of ischemia, in the absence of necrosis, vesicles form on the surface of the cell membrane, containing cytoplasmic proteins, including troponins. The release of troponins occurs when these vesicles rupture on the surface of the cardiomyocyte. Schwartz et al. (1985), for the first time, studied the features of the formation of vesicles on the surface of the membranes of cultured cardiomyocytes using electron microscopy and noted a significant increase in the number of vesicles 30 min after ischemia, compared with the initial state . This hypothesis is consistent with the concept of biphasic troponin release following irreversible damage. The initial phase is associated with the release of the cytoplasmic pool of troponins. In the case of rapid elimination of the causative factor —reversible damage—the vesicles with their contents come back, and everything ends there. However, if the ischemia is more prolonged, for example, in myocardial infarction, then the second phase begins—on the surface of the plasmalemma of the cardiomyocyte, an avalanche-like formation of vesicles occurs with the destruction of the membrane; in parallel to this, there is slow lysis of the sarcomere, which includes a structural pool of troponins, and troponin circulation in the blood takes significantly longer .Thus, it can be suggested that conditions such as prolonged/excessive physical activity, psychoemotional stress, ischemia, and increased load on the myocardium are also accompanied by the release of troponins through membrane vesicles.Myocardial tissue and the liver and kidneys are essential organs that ensure acid-base balance through lactate utilization. Lactic acid is formed in significant quantities by skeletal muscles and is delivered to the liver, where, under the action of lactate dehydrogenase (LDH), it is converted into pyruvic acid and then into glucose—the Cori cycle. The myocardial tissue can use lactate as an energy source, which is also converted to pyruvate due to the action of LDH. Pyruvate is further converted to acetyl-coenzyme A, which enters mitochondria and is metabolized in the Krebs cycle ,69,70. HTwo main mechanisms are proposed: one of the mechanisms may be associated with proteolytic damage to cell membranes during ischemia. The second mechanism for increasing the permeability of biological membranes of cardiomyocytes and the release of troponins may be associated with stretching. A relationship has been established between the increased load on the myocardium and the release of troponins. Troponins are released, predicting adverse cardiac events, in the same way that natriuretic peptides (heart hormones) are released in chronic heart failure due to myocardial stretching . It is bMRI with gadolinium contrast showed no foci of necrotic or sclerotic changes in healthy athletes . DespiteScherr et al. (2011) investigated troponin T by a highly sensitive method (Roche Diagnostics) dynamically in the blood of marathon runners. Immediately after the finish, troponin T levels were 0.031 ng/mL, which was about ten times higher than normal, and the concentrations returned to normal after 72 h. Approximately similar tendencies were shown by cardiac fatty acid-binding protein and N-terminal brain natriuretic peptide (NT-proBNP) . Thus, tp = 0.003). The authors emphasize the need for further research to clarify the role of psychoemotional stress in the pathophysiology of cardiomyocyte damage [Severe psychoemotional stresses, imbalances in the neurohormonal system, and subclinical inflammation of the heart tissues  can be other presumptive causes of minor necrosis ,81. Lazze damage ,83. StreIt is essential to understand that troponins circulate in the blood in the form of a heterogeneous pool: free (single) troponin T and I molecules, combined (binary and triple) troponin complexes, fragments of proteolytic cleavage of troponins, as well as their oxidized, phosphorylated and glycosylated derivatives. The half-life of troponins released into the bloodstream, according to some data, averages 1–2 h .Released into the bloodstream, cardiac troponins and/or their fragments, unlike many biologically active compounds (hormones), do not perform any regulatory functions but circulate for some time, serving as specific biomarkers of myocardial damage, and then are eliminated. Cells of the reticuloendothelial system (mainly macrophages of the spleen) play a significant role in purifying blood from troponin molecules ,89. PresFurthermore, the previously described cleavage of troponins by proteases, which happens in cardiomyocytes under normal conditions but becomes more intense in borderline or pathological conditions, facilitates their release into the blood and subsequent removal.Along with the intracellular cleavage of cardiac troponins , proteolysis of these molecules occurs directly in the vascular bed. It has recently been found that troponins can be specifically cleaved by the enzyme thrombin directly in the bloodstream . MoreoveKatrukha et al. used Western blotting with monoclonal antibodies specific for distinct troponin I epitopes to measure serum samples from patients with acute myocardial infarction (AMI) within 1–36 h of the beginning of chest pain. In addition to free (intact) troponin I, the researchers found 11 more fragments of this molecule with different molecular weights and stability in the bloodstream. When studying the stability of molecules, it is noted that the least stable regions of the troponin I molecule are the N- and C-terminal regions. At the same time, the central fragments are more stable and acquire high resistance to proteases due to their binding to troponin C. The authors draw attention to the extreme importance of studying the processes of fragmentation of troponin molecules and their stability under various conditions for the development and improvement of methods for determining troponins . Zahran et al. examined serum/plasma samples from 29 patients with different types of infarction for proteolytic degradation using several ELISA kits designed to determine the N-terminal, nuclear, and C-terminal fragments of troponin I. Patients with type 1 AMI (with acute atherothrombosis) and ST-segment elevation had the highest degree of proteolytic cleavage of troponin I molecules; patients without ST elevation, as well as patients with type 2 AMI , had the lowest degree of proteolytic cleavage. The lowest degree of proteolytic degradation was in patients after percutaneous coronary intervention. As a result, the authors concluded that the degree of proteolytic degradation is a better indicator of ischemia and AMI than the total level of serum troponin. It was discovered that the degree of troponin proteolytic cleavage corresponds to the severity and prognosis of AMI more than the overall concentration of troponin I in blood serum. In addition, this work indicates that it is possible to assess the quality of treatment based on determining the degree of fragmentation of troponin I . conducting clinical studies for a comparative assessment of the diagnostic effectiveness of already existing highly sensitive immunoassays;searching in blood and other biological fluids for fragments of troponins, which are released first of all during ischemia or AMI;developing antibodies to these (the early released) troponin fragments, which will allow earlier diagnosis of AMI;studying the lifespan/circulation of troponin fragments in the bloodstream and biological fluids to optimize laboratory diagnosticscomparative studies will probably reveal the most specific troponin fragments specifically for AMI, but not for other conditions , which may be accompanied by an increase in troponins when using currently existing test systems and significantly complicate differential diagnosis.Studies on troponin degradation processes explain why commercial antibodies to unstable (rapidly degraded) epitopes at the N- and C-termini of troponin I have a poor correlation with clinical data and patient prognosis when compared to test systems that use antibodies to central (more stable) regions of the molecule. Further research on troponin intra- and extracellular cleavage is needed to improve and standardize troponin immunoassays. In our opinion, the most important goals of such studies should be: The exact mechanisms of eliminating the cTnI and cTnT protein molecules from the bloodstream and the factors that influence the elimination of troponins are not entirely known. The elimination of cTnI and cTnT molecules from the blood is thought to occur in three ways: (1) capture of protein molecules cTnI and cTnT by cells of the reticuloendothelial system and intracellular cleavage ,92,93; ; this, according to the authors, can be used in clinical practice for the diagnosis and monitoring of arterial hypertension [Despite this, due to a lack of direct proof, not all scientists recognize renal filtration as the principal mechanism for eliminating cardiac troponins from the blood ,100,101.rtension . 2 had approximately three times larger highly sensitive troponin T values than those with a GFR greater than 60 mL/min [In the normal range of blood pressure in healthy individuals, the glomerular filtration is not dependent on blood pressure levels because of the Bayliss effect (myogenic mechanism). However, under conditions of strongly pronounced changes in blood pressure, the glomerular filtration rate (GFR) will change: a significant increase in blood pressure leads to an increase in the (GFR), which is a calculated value based on the concentration of the endogenous metabolite creatinine. Considering that an increase in blood pressure increases the troponin concentration in the urine, this validates the direct dependency of serum concentrations of cardiac troponins on GFR. An increase in serum troponin values accompanies a decrease in GFR. This is confirmed by the data of a large study, which involved 2464 patients with chronic renal failure. Participants with a GFR < 30 mL/min/1.73 m0 mL/min . GFR sup2. Moreover, this suggests that the increased values of cardiac troponins in patients with lower GFR may be due to a cause that has nothing to do with ischemic damage to cardiomyocytes, leading to AMI overdiagnosis and unnecessary costs for unnecessary therapeutic and diagnostic manipulations. It is evident that for the most optimal use of highly sensitive analyzes, physicians must necessarily consider GFR [Understanding the characteristics of troponin elimination is critical when utilizing rapid algorithms for the diagnosis or exclusion of AMI, as demonstrated in the study by Kavsak et al. (2018) . Highly ider GFR . It shouIt has been reported that troponin fragments are removed across the blood-salivary barrier. However, the specific process has not been determined. This is most likely accomplished through ultrafiltration of small fragments of troponin proteolytic degradation. In the case of AMI, an increase in troponin concentration in the circulation leads to an increase in troponin concentration in the oral fluid . AnotherSeveral studies have identified cardiac biomarkers, including troponins, in cerebral fluid and pericardial fluid during postmortem examination, which may help estimate the degree of the myocardial injury. The entry of troponins into the pericardial fluid is due to the proximity of the location. Higher concentrations are observed in transmural myocardial infarction. The presence of troponins in the cerebrospinal fluid indicates their ability to cross the blood–brain barrier ,32. r = 0.526; p = 0.003). Erythropoietin is produced in the cells of the juxtaglomerular apparatus of the kidneys in response to hypoxia. The authors believe that troponin detection in amniotic fluid should be performed in pathological pregnancy to diagnose fetal hypoxia, myocardial damage, and malformations [Stefanovic et al. found cardiac troponin T in the amniotic (amniotic) fluid, while its concentration was positively correlated with erythropoietin levels , has a significant daily fluctuation in concentration: the maximum level of highly sensitive troponin T is detected in the morning hours (8:00), then a decrease is observed and reaches its minimum value by about 20:00, after which a gradual increase in concentration begins again, which again reaches a maximum in the morning. The average values in the morning and evening were 16.2 ng/L and 12.1 ng/L. The authors believe that the circadian rhythm of troponin release (according to highly sensitive methods) should also be considered for screening purposes . In some cases, the circadian features of troponins can affect the diagnostic accuracy for AMI. van der Linden et al. (2016) estimated the daily fluctuations in hs-TnT and hs-TnI concentrations in an elderly patient with severe chronic renal failure (GFR = 14 mL/min). Their serum cardiac troponin T concentration had been chronically elevated for many years with the absence of acute coronary events, highlighting the role of the kidney in troponin elimination. Changes in troponin T levels during the day were in line with the previous study, with peak concentrations in the morning and a gradual decline in the evening. During the day, the maximum fluctuations in troponin T concentrations were 50.9 ng/L and up to 20 ng/mL within one hour. Several times a day, one-hour delta-changes in troponin T levels exceeded delta values in the developed one-hour and three-hour algorithms for diagnosing AMI. In other words, in this case, circadian fluctuations in troponin T concentrations mimicked the kinetics of troponin T characteristic of AMI, which could lead to overdiagnosis . Few works are devoted to circadian features, and there is a need for further research, especially for modern highly and ultrasensitive test systems. When significant influences are found, the diagnostic algorithms for AMI should reflect the circadian aspects . When itThe daily concentration variability characteristic of cardiac troponin T corresponds to the circadian organization of the cardiovascular system and the hemostatic system. It has been shown that the heart rate, blood pressure, vascular resistance, prothrombotic tendency, platelet aggregation ability, activity of the renin-angiotensin-aldosterone system, activity of the sympathoadrenal system, and levels of catecholamines and cortisol increase in the morning hours, which have evolved and are necessary for normal functioning in the period of wakefulness. However, this has important implications for the pathophysiology of cardiovascular diseases: the frequency of cardiovascular accidents is significantly higher in the morning hours ,117,118.Taking everything into account, it is possible to conclude that the concentration of cardiac troponins at a specific point when drawing blood from a patient is affected by a variety of metabolic factors . This must be understood to use fast algorithms to diagnose and exclude acute myocardial infarction, based on small concentration rises  during the first hours. It should be understood that some of the above factors can lead to a distortion of the result; therefore, additional fundamental studies of the metabolic characteristics of cardiac troponins are needed to improve the diagnostic process. According to the literature review, the following significant influencing features that can lead to diagnostic errors should be considered: troponin release during stress, transient ischemia, and apoptosis of cardiomyocytes; impaired renal elimination, which can overestimate troponin concentrations in the absence of cardiomyocyte damage; and circadian aspects. Further research of troponin circulation in the bloodstream and troponin lifespan is required to develop novel diagnostic systems . Determining troponins in other biological fluids  opens up new questions and perspectives.


At inflammatory loci, pro-inflammatory activation of macrophages produces large amounts of reactive oxygen species (ROS) that induce DNA breaks and apoptosis. Given that M-CSF and GM-CSF induce two different pathways in macrophages, one for proliferation and the other for survival, in this study we wanted to determine if these growth factors are able to protect against the DNA damage produced during macrophage activation. In macrophages treated with DNA-damaging agents we found that GM-CSF protects better against DNA damage than M-CSF. Treatment with GM-CSF resulted in faster recovery of DNA damage than treatment with M-CSF. The number of apoptotic cells induced after DNA damage was higher in the presence of M-CSF. Protection against DNA damage by GM-CSF is not related to its higher capacity to induce proliferation. GM-CSF induces differentiation markers such as CD11c and MHCII, as well as the pro-survival Bcl-2A1 protein, which make macrophages more resistant to DNA damage. Macrophages play a central role in immune response and tissue homeostasis. These cells have two functions in inflammation, initially being pro-inflammatory by destroying infectious agents and infected tissues and then being anti-inflammatory by repairing the damaged tissues . In the We showed that macrophages have systems to repair DNA breaks when they become activated or injured . FollowiROS can cause several types of DNA damage ranging from base modifications to DNA backbone breaks in human biology and disease . The celHighly proliferative cells are the most sensitive to DNA-damaging agents. Since tumor cells are rapidly dividing cells, many DNA-damaging agents have been used to treat cancer, including methylating agents, cross-linking chemotherapeutics, topoisomerase inhibitors and radiomimetic drugs . In addiWaf1 -thymidine incorporation as described previously -thymidine. After 6 additional hours of incubation, the medium was removed and the cells were fixed in ice-cold 70% methanol. After three washes, the cells were solubilized and their radioactivity was measured. Each experiment was performed in triplicate and the results were expressed as the mean ± SD. For cell counting we used a hemocytometer.Macrophage proliferation was measured by . H. H17]. Hwaf−1p21 and reduces that of Gadd45 and cyclin B1, which explains the arrest of the cell cycle due to DNA damage  and anti-inflammatory (IL-4) agents . In our diseases . GM-CSF is an important factor that induces differentiation . Our resBcl-2A1 was induced by GM-CSF and not by M-CSF, confirming previous results [BCL2-A1 gene is induced and is a direct transcriptional target of nuclear factor NF–κB [BCL2-A1 [Interestingly, the more differentiated macrophages were the more resistant to induction of apoptosis/necrosis. After etoposide treatment and 24 h incubation with GM-CSF, fewer apoptotic cells were observed in relation to those incubated with M-CSF. Fascinatingly, when we tested different anti-apoptotic genes, only  results . Bcl2-A1 results . In respor NF–κB . The tra[BCL2-A1 . BCL2A1 is able to reduce the release of pro-apoptotic cytochrome c from mitochondria and block caspase activation. The interaction of BCL2A1 with pro-apoptotic factors BAX and BAK has been described with conflicting results; however, it seems clear that BCL2A1 interacts with and blocks most of the BH3-only proteins that induce apoptosis through activation of BAX and BAK .high monocytes in an inflammatory locus differentiate into macrophages with a pro-inflammatory profile, releasing a number of molecules  that activate helper T cells and induce the production of GM-CSF [Our results could have implications for our understanding of the inflammatory process. Ly6Cf GM-CSF ,67. Thisf GM-CSF , are cruf GM-CSF  and monof GM-CSF ,70, theyf GM-CSF ,72,73.DNA breaks in the cells of the immune system and, particularly in macrophages, play a key role in immune-senescence . It is iThese observations, together with our results, emphasize the critical role of GM-CSF in protecting against DNA damage and ensuring the survival of macrophages in the homeostasis of the immune response.