As sulfur‐containing organic molecules thioamides and their isomers are conceivable intermediates in prebiotic chemistry, for example, in the formation of amino acids and thiazoles and resemble viable candidates for detection in interstellar media. Here, we report the characterization of parent thioformamide in the solid state via single‐crystal X‐ray diffraction and its photochemical interconversion to its hitherto unreported higher energy tautomer thiolimine in inert argon and dinitrogen matrices. Upon photogeneration, four conformers of thiolimine form, whose ratio depends on the employed wavelength. One of these conformers interconverts due to quantum mechanical tunneling with a half‐life of 30–45 min in both matrix materials at 3 and 20 K. A spontaneous reverse reaction from thiolimine to thioformamide is not observed. To support our experimental findings, we explored the potential energy surface of the system at the AE‐CCSD(T)/aug‐cc‐pCVTZ level of theory and computed tunneling half‐lives with the CVT/SCT approach applying DFT methods. Keep it simple! Thioamides resemble viable prebiotic building blocks for biologically relevant molecules. However, only a handful of experimental reports on parent thioformamide exist. X‐ray diffraction data of thioformamide are reported and its tautomerization induced by UV irradiation under cryogenic conditions is presented. Four conformers of the corresponding thiolimine form, whose (tunneling) reactivity is investigated. In nature as well as in synthetic chemistry4), a unit that is present in various biologically important molecules  and drugs.3).2S,2S or thiophosphate (5).2O, which has been investigated computationally recently.2S to nitriles is expected to proceed via a thiolimine , which is the thermodynamically disfavored tautomer of 1. To the best of our knowledge, no data exist for 2, which prompted us to study the tautomerization between 1 and 2 in cryogenic argon (Ar) and dinitrogen (N2) matrices to provide first‐hand evidence for the existence and spectral identity of 2. We also report the first single‐crystal X‐ray diffraction structure of 1.Although thioformamide (H)‐pyridinethione,H)‐pyrimidinethione and 3(2H)‐pyridazinethione,H)‐quinolinethione,Various thioamide derivatives have been isolated under cryogenic conditions and characterized by infrared (IR) spectroscopy during the last decades. Several studies focused on the UV‐induced thioamide→thiolimine tautomerization, which has been reported for 2 reports on experimental data of are rare.27, 28The mechanism of the UV‐induced tautomerization of thioamides has been discussed. Excited state intramolecular proton transfer (ESIPT) has been ruled out by Lapinski et al. due to the lack of intramolecular hydrogen bonds in these systems.2 potential energy surface with density functional theory  as well as with coupled cluster methods  in energy. The order of stability is 2 cc<2 tt<2 ct<2 tc at every computational level applied here. The conformers can interconvert through C−S bond rotations or inversion of the C=N−H angle. The latter requires overcoming high lying Cs symmetric transition states.We computed the most significant part of the HC(S)NH1 in ethyl acetate and solved their structure by X‐ray diffraction and refined the data with the independent atom model and Hirshfeld atom refinement  computations predict that the analogous Ch2 dimer of 1 containing two N−H⋅⋅⋅S contacts is 2.0 kcal mol−1 lower in energy than a Cs dimer as depicted in Figure 1 and ethyl acetate (N−H⋅⋅⋅O contact of only 1.82 Å) is presumably the reason for the difference from the crystal structure in Figure We obtained single crystals of 1 Scheme  with the−1. We also recorded an IR spectrum of 1 in an N2 matrix. For the complete assignments see the Supporting Information. Our results are in good agreement with the experimental data by Davies and JonesAfter deposition of a sample of freshly synthesized thioformamide (see Supporting Information for details) together with a large excess of Ar on the cold (3 K) matrix window, we measured IR and UV/Vis spectra of all matrix isolated species. The IR spectrum displayed in Figure f=0.2027) and 225 nm (f=0.1517 nm). After UV irradiation (vide infra) a small new feature at ca. 225 nm appears. This is in accord with the computational result that all thiolimine tautomers show absorptions between 210 nm and 215 nm (0.05<f<0.15). For an analogous spectrum obtained in N2 matrices as well as details of the nature of the transitions see the Supporting Information.The recorded matrix UV/Vis spectrum Figure  shows a 2. The four conceivable conformers of this species can all be observed in these spectra. For ease of identification, we computed anharmonic IR spectra of 1 and 2 at B3LYP/6–311++G. DFT in conjunction with a large Pople basis set generally reproduces matrix IR spectra very reasonably; recent precedence is provided in a study on thiotropolone.Irradiation of the matrix with UV light led to a decrease of the thioformamide bands. New bands appear in the IR spectra that can be assigned to 2 are very low. The remainder of the simulated spectra is similar for all four conformers. The intensity pattern, especially in the C−H and N−H bending regions, turns out to be very useful to distinguish between the two groups 2 tc/2 cc and 2 ct/2 tt. In the CN torsion region, the bands of 2 ct and 2 tt have nearly the same computed value. On the other hand, 2 tc and 2 cc display large shifts, such that this region is the most prominent for assignment  and the second to 2 tc . The band at 1059 cm−1 only has a computed counterpart in conformers 2 ct and 2 tt. As discussed below both conformers are present in the matrix and their IR bands can only be resolved in difference spectra  serve to resolve these two groups further and enables a confident assignment of each observed band to one conformer.A study by Lapinski et al. demonstrated that the thioamide to thiolimine interconversion of thioacetamide yields different ratios of thiolimine conformers when the matrix is irradiated at different wavelengths.−1 and the NH bending region at around 1170 cm−1 four new bands appear after UV irradiation. Taking the described strategy into account we assign the strongest of these bands at 1346.2 cm−1 to 2 ct . With the same strategy the band at 1349.3 cm−1 is assigned to 2 cc , the band at 1352.7 cm−1 to 2 tc , and the band at 1358.0 cm−1 to 2 tt . The different shifts and intensities throughout the spectrum allow us to draw the conclusion that indeed 2 ct and 2 tt are both present. In the N−H bending region the band at 1162.2 cm−1 can be assigned to 2 cc, the strong bands at 1171.8 cm−1 and 1173.2 cm−1 to 2 ct, and the band at 1176.9 cm−1 to 2 tt. The computed values are 1149.7, 1161.3, and 1161.9 cm−1 for 2 cc, 2 ct, and 2 tt, respectively, which reproduce the experimental spectra in good agreement.In the CH stretching region at around 1350 cm2 tt to 2 ct and the rotamerization of 2 tc to 2 cc (vide infra). In the 670–750 cm−1 range, all conformers are computed to show several characteristic bands as well. The assignment is possible by combining the above strategies. Analogously, we observed all four thiolimine conformers in N2 matrices. Spectra and assignments are available in the Supporting Information.Our assignment is further supported by the observation of a tunneling process from −1. These bands cannot be confidently assigned to the decomposition products H2S, HCN, HNC, CS, NH3, H2, HSCN, or HCNS because in the present mixture significant shifts of the predicted bands of the pure compounds might occur due to the formation of complexes. Bands at 1624.1, 1553.0, and 1408.1 cm−1 remain unchanged during UV irradiation and can thus be assigned to unknown impurities in our sample. The highest concentration of the thiolimine tautomers is observed after 10 min of irradiation when 1 is still among the main components.After prolonged UV irradiation the new bands assigned to the thiolimine tautomers vanished. The strongest bands after irradiation for 1 h are at 3361.8, 3202.3, 2091.2, 2026.2, 1233.4, 647.2, and 638.9 cm2 tt and a concomitant increase of those assigned to 2 ct. We measured the kinetics of this process by collecting IR spectra in 5 min intervals. Figure −1) cut‐off filter leads to the formation of 2 cc from 2 tc. This can be rationalized by the low activation barrier of only 6.1 kcal mol−1. We notice that wavelengths >4.5 μm  would in principle suffice to initiate this process, however, we observe that when using such a cut‐off filter only the reaction 2 tt→2 ct occurs. It is important to stress that even after longer times (up to 48 h) the intensities of bands of 2 tt do not reach zero. This might on the one hand be due to the IR Globar irradiation from the spectrometer . On the other hand, such phenomena have been assigned to matrix effects before, namely that specific matrix environments (or matrix sites) inhibit QMT.N,N‐dideuterated isotopologue of thioformamide dark reactions of the thiolimine tautomers were not observed when installing the cut‐off filter. This provides another hint that the reaction 2 tt→2 ct is due to QMT and resembles only the second example of conformational QMT leading to a rotation of a C−S bond.After keeping the matrix for 2.5 h in the dark we observed a decay of bands belonging to 2 tt→2 ct by following the decay (increase) of the strongest bands of 2 tt (2 ct). A first‐order kinetic model with the concentrations of 2 ct and 2 tt , the pre‐exponential factor A  and the time t as in Figure B needs to be included because the bands of 2 tt will not reach the asymptotical value of zero.We obtained the experimental tunneling half‐life of the reaction 2 are rather small making an exact analysis difficult. However, changing the temperature from 3 to 20 K or placing the cut‐off filter between the spectrometer and the matrix window does not change the results significantly neither in Ar nor in N2 (see the Supporting Information for details). The half‐life remains between 25 and 45 min for both matrix materials.Especially when measuring the spectra with the 4.5 μm cut‐off filter the band intensities of 2 tt→2 ct is computed to be the fastest reaction. Even though according to the computations 2 cc→2 tc should be observable at laboratory time scales, we did not observe such a process. This becomes apparent when applying a scaling factor (219.5) to the computed half‐lives accounting for the deviation of the experimental (ca. 30 min) and the computed value of the reaction 2 tt→2 ct. A somewhat more realistic value for 2 cc→2 tc results (1361 d), far outside reasonable experimental time scales. Similar results were recently discussed for matrix isolated thioacetamide.2 tt→2 tc and 2 cc→2 ct the computed tunneling half‐lives are much longer, due to the greater widths of the corresponding reaction barriers.We computed the tunneling half‐lives for all conceivable reactions between the thiolimine conformers with canonical variational theory (CVT) in conjunction with small‐curvature tunneling (SCT). This method has often been demonstrated to yield reliable results for matrix‐isolated species.1 was observed after keeping the matrix for several days in the dark. Substitution apparently has a strong effect on the tunneling half‐lives in thioamides. In this respect it is worth mentioning the different reactivities of amides, thioamides, and selenoamides. Such studies were carried out by Nowak et al. on thioureaH‐shifts in hydroxycarbenes.Substituent effects have been shown to affect tunneling rates before, most prominently in systematic studies on carboxylic acids60, 612 matrices and generated four conceivable conformers of the energetically higher lying thiolimine tautomer by UV excitation. One of these conformers (2 tt) vanished with a half‐life of ca. 30 min to form 2 ct. This process can be ascribed to QMT and is analogous to the findings reported recently on thioacetamide.We isolated thioformamide in Ar and NIn contrast to thiourea a tunneling reaction back to the thioamide tautomer has not been observed. This illustrates that (remote) substitution can enhance or inhibit QMT as it has already been shown in previous studies. As the effect of the substituent on QMT is intricate, further research is planned in our laboratories to fine‐tune QMT and further establish its role as the ‘third reactivity paradigm’.N‐deuterated isotopologues were prepared according to the procedure by Willstätter and Wirth.Thioformamide and its 2033186, and 2032761 contain the supplementary crystallographic data for this paper. These data are provided free of charge by the joint Cambridge Crystallographic Data Centre and Fachinformationszentrum Karlsruhe Access Structures service.Deposition numbers The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Alzheimer’s disease (AD) is a neurodegenerative disease that is characterized by amyloid plaque deposits, neuronal cell loss, and memory impairment. Granulocyte-colony stimulating factor (G-CSF) is a growth factor associated with AD improvement. Stromal cell-derived factor-1 (SDF-1) mediates therapeutic effects of G-CSF. This study investigated the effect of combination treatment of G-CSF and SDF-1 on amyloid plaque deposits, apoptosis, and behavior of AD rats.Intracerebroventricular amyloid-beta [Aβ(1-42)] peptide was used to induce AD in Aβ rats. There were six groups including naive control, sham-operated, Aβ, Aβ + G-CSF, Aβ + SDF-1, and Aβ + G-CSF + SDF-1. SDF-1 intra-cerebroventricular (ICV), G-CSF Subcutaneous (SC), or a combination of them were administered to Aβ rats weekly for 2 months. The cognition and memory were assessed using the novel object recognition, passive avoidance, and Morris water maze tests. Next, rat brains were removed and the amyloid plaque and apoptosis were detected in the brain and hippocampus using immunohistochemistry and TUNEL assay, respectively.P < 0.05).The amyloid-beta and apoptotic cell levels dropped in groups receiving SDF-1 and G-CSF combination compared to the Aβ group. Also, number of microglial cells increased significantly in the combination group compared to other treatment groups. Moreover, learning and memory were significantly improved in the combination group compared to the Aβ groups (SDF-1 and G-CSF combination therapy can offer a promising strategy for AD. Alzheimer’s disease (AD) is one of the progressive neurodegenerative diseases . AD is cThere is extensive research on the effect of stem cell transplantation for the treatment of lost or defective cells in AD . Non-neuStromal cell-derived factor-1 (SDF-1) is a secreted protein that binds chemokine receptor type 4 (CXCR4), a seven-pass G protein-coupled membrane receptor . SDF-1 hOn the other hand, the first cytokine identified and rapidly transitioned into clinical medicine was granulocyte colony-stimulating factor (G-CSF) . G-CSF iThe G-CSF is another way to mobilize stem cells . This faAccording to the above findings, it seems that both SDF-1 and G-CSF stimulate stem cell mobilization to the injury area. However, the combined treatment effects of SDF-1 and G-CSF have not been investigated in AD. The simultaneous use of SDF-1 and G-CSF strengthens the anti-apoptotic effect and decreases the amyloid plaque in the brain. In light of the findings, the aim of this study was to investigate the simultaneous effect of SDF-1 and G-CSF in the rat model of AD.ad libitum access to food and water for 1 week for acclimatization to the new environment. All procedures were performed in accordance with ethical codes stipulated by the Ethical Committee of Hamadan University of Medical Sciences (IR.UMSH.REC.1394.471) as well as the institutional and national guidelines regarding animal care and use.Forty-eight male Wistar rats weighing 250–300 g were procured from the Medical Faculty of Hamadan University of Medical Sciences. Rats were placed under standard lighting conditions (12-h light-dark cycles), temperature (22°C), and humidity (55–65%). Animals had Ketamine was purchased from Rotex Medical, Germany. Xylazine was bought from Alfasan, Woerden, Holland, and Aβ(1-42) was obtained from Tocris Bioscience, United Kingdom.Stromal cell-derived factor-1 was purchased from . SDF-1 powder (10 μg) was dissolved in 1 ml of phosphate buffered saline (PBS). Then, the stock solution was diluted 1:20 to obtain a solution of 500 ng/ml and was injected intraventricular once a week for 2 months .Human G-CSF manufactured by pharmacy . The standard dose of G-CSF was injected SC once a week for 2 months at a concentration of 100 g/kg .The surgery was performed as described in previous studies . BrieflyThere are several methods for inducing memory impairment in animal models, including intracerebroventricular injections of beta-amyloid. In the present study, for induction of memory impairment model, the animals were first anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) and transferred to a stereotaxic device. After observing the ocular reflex, stereotaxic rods were placed inside the animal’s ear, and the head was fixed in the device in the middle area. Then, the shaved part of the scalp is removed with scissors in the desired location. Following this, Bregma and Lambda regions were identified based on the Paxinos atlas and the distance between these two points was determined. Then, the following coordinates were recorded: AP: 0.9 mm (Anterior posterior), ML: 1.5 mm , DV: 3.2 mm (Ventricle Depth). Then, the right side of the brain was pierced using a dental drill. Following that, a 23-gauge cannula fixture (12-millimeter length) was lowered to a depth of 3 mm from the surface of the head bone and then attached to the head using dental cement and a screw. Finally, through the fixed cannula on the head, beta-amyloid or SDF-1 were injected using the Hamilton syringe.n = 8) before the operation. The control group did not receive any treatment or surgery. The sham group underwent surgery without Aβ injection. Aβ group received Aβ-peptide without any treatment. The SDF-1 group received SDF-1 through intravenous administration 2 weeks after Aβ-peptide injection for a period of 2 months. Also, G-CSF group received G-CSF via subcutaneous administration 2 weeks after Aβ-peptide injection for a period of 2 months. G-CSF and SDF-1 group received both SDF-1 and G-CSF administration. The SDF-1 and G-CSF were administrated every week for 2 months. At the end of treatment, the memory of animals was assessed by behavioral tests and histological studies, which were performed after sacrificing animals (Intra-ventricular injection of Aβ(1-42) was used to induce AD. Animals were randomized into six groups  with low ambient lighting. The rats were first placed in the open field, and allowed to explore the surrounding area for 10 min. The velocity and total distance traveled were recorded by a camera .A wooden open box (35 × 43 × 40 cm) was used as the apparatus of a novel object recognition test. The animals were habituated in the empty box for 5 min. After 2 h, two similar objects were presented to rats for 5 min in a learning trial. On the next day, the testing phase was performed with one of familiar objects and a novel object for 5 min. The ratio of time spent on the novel object to the total time spent on each object was calculated as the distinction index. After each session, all areas and objects were cleaned and any residual odors was removed by 70% ethanol.The spatial memory was evaluated by Morris water maze (MWM) test. Briefly, a black circular pool  was filled with water (22 ± 1°C) to a depth of 40 cm. The hidden black escape platform was placed 2 cm under the water surface on the northern quadrant of the pool. The shapes on the walls served as visual cues to rats in order to find the hidden platform. The learning trials consisted of two blocks of four trials per day for four consecutive days. The time needed to reach the hidden platform was recorded during the learning phase. One day after the last session, the retention (probe) test was carried out without the escape platform. The rats were placed into water and the duration of swimming in the target quadrant was recorded for 60 s.The transparent plastic apparatus (20 cm × 20 cm × 30 cm dimensions) consisted of light and dark compartments with a stainless-steel rod (3 mm in diameter) on the floor. An opaque guillotine door (6 cm × 8 cm) was placed between the two compartments. The electricity in the dark compartment floor was induced a shock generator .Initially, the apparatus was presented to each rat in two trials. To do so, the rats were placed in the light compartment away from the door for 5 s. Then, the guillotine door was raised. The rats headed for the dark environment instinctively. When they moved into the dark compartment, the guillotine door was closed and the rats remained in this compartment for 30 s. This trial was repeated with 30 min intervals. In another scenario, after inserting rats in the dark compartment, we measured the delay in entering the dark compartment or step-through latency (STLa). Following the spontaneous entry of rats into the dark compartment, the guillotine door was shut for 30 s and an electrical shock was applied . The procedure was repeated after 2 min. The training was halted when animals managed to stay in the light compartment for 120 consecutive seconds. We recorded the number of trials that involved entering the dark compartment.For the purpose of the retention test, the rats were placed inside the light compartment for 5 s. Then, the guillotine door was shut and the step-through latencies in retention (STLr) was recorded in the retention trial. The amount of time spent in the dark compartment (TDC) was up to 5 min. The retention test was terminated when a rat failed to enter the dark chamber within 3 s .The animals were sacrificed 69 days after the study, and brain tissues were stained by fluorescent antibodies against Aβ(1-42), and Ionized calcium-binding adaptor molecule 1 (IBA1). Animals were deeply anesthetized with ketamine and xylazine and perfused with 150–200 ml PBS and 4% paraformaldehyde. Brain tissues were then harvested and embedded in paraffin  and 5 μm paraffin sections were microtomed. A total of three brain were selected to count beta-amyloid plaques and microglia cells. From each animal, five sections were analyzed with the conditions mentioned. A section with a thickness of five microns was prepared from every 20 tissue sections to examine beta-amyloid and microglia.For immunohistochemistry, the paraffin sections were gradually dewaxed into the water, immersed in 0.01 mol/L citrate buffers and microwaved for the antigen retrieval. Afterward, the sections were incubated with antibodies against Aβ(1-42) (ORB10087) and Iba1 (ORB336635) overnight at 4°C. Sections were then washed with PBS and incubated with the secondary fluorescent antibodies  for 2 h at room temperature. Sections were washed with PBS, and nuclei were counterstained with DAPI (the chromogen) for 45 min. Images were captured by a fluorescence microscope (Leica AF 6000). In this study, fluorescent microscopy and ImageJ software were used to observe Beta amyloid plaques and Iba1 cells directly. Counting beta-amyloid plaques and Iba1 cells were done after merging their nuclei with DAPI.Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) is a known method of detecting DNA fragments that is used to identify apoptotic cells. After the behavioral test, the rats were intracardially perfused with saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH = 7.4), and their brains were fixed. To measure apoptosis, first three animals were selected, and then five tissues with a thickness of 5 microns from each animal were prepared.In the next step, the fixed brains were serially sectioned into 5-μm coronal sections by a microtome , hydrated, and prepared for the detection of DNA fragmentation and apoptotic cells using the TUNEL kit (Roche), as described earlier. Following the permeabilization of samples (citrate buffer pH: 6), the sections were incubated using the TUNEL reaction mixture for 60 min at 37°C. The converter-peroxidase (30 min) and 3,3′-diaminobenzidine substrates (10 min) were added to samples. The sections were counterstained with hematoxylin and analyzed under a light microscope . A cell with dark brown particles in the nucleus was defined as an apoptotic hippocampal neuron. For each animal, the mean number of apoptotic cells was obtained by counting five coronal sections.post hoc test was used to compare the mean of all treatments to the mean of other treatments. P-values ≤ 0.05 was considered as statistically significant.Data obtained from the open field and immunostaining tests were analyzed by one-way analysis of variance (ANOVA) test. Also, the Tukey’s P < 0.001). Also, the groups were not significantly different in the movement of locomotion in the open field box (P < 0.01)  .P < 0.05). The combined treatment with G-CSF and SDF-1 increased the discrimination index compared to the G-CSF and SDF-1 group (P < 0.01).The result of NOR test and total discrimination index are shown in P < 0.05). The combined treatment with G-CSF and SDF-1 decreased the time required to find the platform compared to G-CSF, SDF-1, and Aβ groups (P < 0.05). P < 0.05). The animals treated with a combination of G-CSF and SDF-1 spent more time than Aβ group in the target quadrant (P < 0.05).The results of the spatial learning phase of MWM are shown in P < 0.05; Statistical analysis by ANOVA revealed that the two groups were not different in STLa . The numP < 0.05). The STLr time in the combined treatment with G-CSF and SDF-1 groups was higher than G-CSF and other single groups (P < 0.05). The one-way ANOVA showed differences between groups in TDC on the test day. The Aβ group spent more time on TDC than control and sham groups did (P < 0.05). The combined administration of SDF-1 and G-CSF decreased TDC time in treated rats compared to the Aβ group (P < 0.05).A significant difference in STLr time was revealed by ANOVA . The STLoups did  (P < 0.0P < 0.05).The combination of SDF-1 and G-CSF significantly decreased amyloid plaque deposition in comparison with G-CSF and SDF-1 groups , 7A (P <P < 0.05).Number of Microglial cells significantly increased in the group treated with a combination of SDF-1 and G-CSF compared to other treatment groups , 8 (P < post hoc test showed that the number of apoptotic cells in the Aβ group was significantly higher than in the control and sham groups (P < 0.001). The number of apoptotic cells was significantly lower in the combined treatment with SDF-1 and G-CSF group than in the Aβ group (P < 0.05). There was, however, no significant difference in the number of apoptotic cells between the control and sham groups.This study investigated the combined treatment effects of SDF-1 and G-CSF on rats receiving ICV injection of beta-amyloid (1-42) peptide over 2 months. The results of behavioral tests show that Aβ provoked learning deficiency whilst the combined treatment with SDF-1 and G-CSF resolved this impairment. In addition, the SDF-1 and G-CSF treatment improved reacquisition and memory in the combination group. These findings suggest that both spatial and avoidance memory impairment induced by Aβ peptide could be resolved by combined administration of SDF-1 and G-CSF. In the present study, 96 days after Aβ injection, the amount of beta-amyloid deposition, microglia, and cell death was assessed in the brains of rats in all groups using the immunohistochemical method. The results showed that combined injection of SDF-1 and G-CSF decreased the beta-amyloid accumulations and apoptosis in the hippocampus. Also, the combined treatment of SDF-1 and G-CSF increased microglia in the brain.+ cells, such as cell growth, proliferation, angiogenesis, and cell survival .AKo and ZG designed and managed the study. SS and NH-F performed the experiments, collected the data, and wrote the manuscript. ZR and AKe assisted in experiments. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Chronic rejection and immunosuppression-related toxicity severely affect long-term outcomes of kidney transplantation. The induction of transplantation tolerance – the lack of destructive immune responses to a transplanted organ in the absence of immunosuppression – could potentially overcome these limitations. Immune tolerance to kidney allografts from living donors has been successfully achieved in humans through clinical protocols based on chimerism induction with hematopoietic cell transplantation after non-myeloablative conditioning. Notably, two of these protocols have led to immune tolerance in a significant fraction of HLA-mismatched donor-recipient combinations, which represent the large majority of cases in clinical practice. Studies in mice and large animals have been critical in dissecting tolerance mechanisms and in selecting the most promising approaches for human translation. However, there are several key differences in tolerance induction between these models and humans, including the rate of success and stability of donor chimerism, as well as the relative contribution of different mechanisms in inducing donor-specific unresponsiveness. Kidney allograft tolerance achieved through durable full-donor chimerism may be due to central deletion of graft-reactive donor T cells, even though mechanistic data from patient series are lacking. On the other hand, immune tolerance attained with transient mixed chimerism-based protocols initially relies on Treg-mediated suppression, followed by peripheral deletion of donor-reactive recipient T-cell clones under antigenic pressure from the graft. These conclusions were supported by data deriving from novel high-throughput T-cell receptor sequencing approaches that allowed tracking of alloreactive repertoires over time. In this review, we summarize the most important mechanistic studies on tolerance induction with combined kidney-bone marrow transplantation in humans, discussing open issues that still need to be addressed and focusing on techniques developed in recent years to efficiently monitor the alloresponse in tolerance trials. These cutting-edge methods will be instrumental for the development of immune tolerance protocols with improved efficacy and to identify patients amenable to safe immunosuppression withdrawal. Renal transplantation is the established treatment of choice for kidney failure, as it confers both the highest survival and the best quality of life compared to other renal replacement therapies . DespiteThe induction of tolerance, i.e. the lack of destructive immune responses to a transplanted organ in the absence of immuno-suppression, could potentially overcome both of these limitations. Tolerance in kidney transplantation can be functionally defined by stable renal function and absence of histologic, immune and molecular signs of rejection on a kidney biopsy obtained after complete withdrawal of immunosuppression for at least one year. Spontaneous tolerance is unfortunately a rare and unpredictable event that has been described in a small minority among the patients who choose to discontinue their immunosuppression, who retained graft function despite complete withdrawal of immunosuppression .Among the different methods used to induce tolerance in animal models of kidney transplantation, few have been successfully translated to clinical application. Those protocols that have succeeded in patients entail combined kidney and bone marrow transplantation (CKBMT) as a strategy to induce chimerism, a state wherein donor hematopoietic cells engraft into the recipient bone marrow at a level sufficient to be detected by conventional (as opposed to sensitive PCR-based) methods.Three centers have developed clinical CKBMT protocols, one of which has so far succeeded in achieving tolerance only in the HLA-identical transplant setting . InvestiOnly two strategies have succeeded in effectively inducing operational tolerance across HLA barriers so far. As HLA mismatches are commonly present in solid organ transplantation, in this review we will discuss the features of these regimens and the novel mechanistic insights offered by recent studies in the field.Animal Studies. More than 60 years ago, Main and Prehn used bone marrow infusion following administration of high-dose, lethal total body irradiation (TBI) to achieve skin allograft tolerance in recipient mice. In this experimental setting, semiallogeneic but not isogenic bone marrow infusion consistently permitted donor-specific skin graft acceptance  and expand IL-10-producing Tr1 cells  in mice treated with high TBI doses. Murine FC include a population of CD8αr1 cells –17. Thesr1 cells  and haveClinical Protocols. Investigators from Northwestern University utilized a non-myeloablative conditioning regimen that achieved durable full donor chimerism in humans, attempting to exploit the engraftment-promoting and immunosuppressive effect of FC  and were successfully weaned off immunosuppression after one year from transplant. These subjects showed significantly better kidney function compared to matched controls receiving conventional immunosuppression. Two graft losses due to opportunistic infections were recorded in the first year after transplantation, and one tolerant recipient died due to sepsis. Studies in mice have highlighted that full donor chimeras are somewhat immunoincompetent , 23 due Animal Studies. Mixed chimerism defines a state wherein, unlike full donor chimerism, the host hematopoietic system is not completely destroyed and replaced by the donor’s, and hematopoietic cells of both the recipient and the donor coexist in the bone marrow.via intra-thymic negative selection of alloreactive T cell clones, mediated by both donor- and recipient-derived antigen-presenting cells  . This meng cells , 29 and ng cells . Durableng cells –33. Pering cells –38 and ang cells , 39. Theng cells , 41. Notng cells , 43  in patients with and without hematologic malignancies. Differences between these regimens have been reviewed in detail elsewhere  experienced chronic rejection or glomerulonephritis recurrence, which led to reintroduction of immunosuppressive drugs and ultimately resulted in graft loss more than 10 years after transplantation. Of note, these patients were successfully retransplanted with conventional immunosuppression, and there were no significant opportunistic infections in any of them.Initial studies used a non-myeloablative conditioning regimen that included cyclophosphamide, the anti-CD2 T cell-depleting monoclonal antibody siplizumab and thymic irradiation (TI) Table 1 In parallel with early host T cell recovery, 9 patients unexpectedly developed severe acute kidney injury. Renal histology was consistent with engraftment syndrome, entailing capillary endothelial injury with vascular leak and lympho-monocytic infiltrating cells in peritubular and glomerular capillaries. Renal function normalized in all but 2 recipients, one of whom experienced graft loss due to acute humoral rejection as a consequence of preformed DSA that were undetectable on a pre-transplant ELISA, but were subsequently confirmed by Luminex. In the other patient, acute kidney injury was initially misdiagnosed as rejection and was treated with higher doses of tacrolimus, which triggered thrombotic microangiopathy. Finally, one patient developed severe cellular rejection after a pyelonephritis episode following immunosuppression withdrawal. Protocol biopsies (at 2-8 years) in tolerant subjects showed either completely normal histology or minimal alterations, including focal glomerular basement membrane duplication and mild podocyte foot process effacement .An additional protocol was tested at MGH based on further observations from studies conducted in non-human primates , 51. ComInvestigators at the Samsung Medical Center initially used a nearly identical protocol to those outlined above, but the anti-CD2 monoclonal antibody siplizumab was substituted with ATG due to local unavailability , 53. To The mechanism that underlies tolerance to kidney allografts associated with full-donor chimerism hypothetically involves central tolerance of donor T cells to donor antigens, with donor progenitor cells migrating to the recipient thymus, differentiating into antigen-presenting cells and finally mediating negative selection of “self”-reactive donor T cell clones. Bulk functional assays, including mixed-lymphocyte reactions (MLR) and cell-mediated lympholysis (CML), demonstrated donor-specific hyporesponsiveness in tolerant patients. However, the same effect was observed in recipients who exhibited only transient chimerism and developed rejection after immunosuppression withdrawal , suggestde novo GVH tolerance might be characterized by a combination of clonal deletion, anergy and regulatory T cell-mediated mechanisms. However, GVH tolerance has not been demonstrated in these patients and the inclusion in the infused product of mature donor T cells that eliminate host hematopoiesis suggests that an ongoing GVH reaction may occur, which has culminated in GVHD in several patients. Whether or not GVH reactions in patients without overt GVHD results in thymic injury and failure to negatively select host-reactive T cells, as reported in murine models  and on a minimal fold-expansion , while computational methods were used to account for sorting errors  with evidence of peripheral proliferation, possibly recent thymic emigration and, in one patient, conversion from conventional T cells , Immune Tolerance Network contract (N01 AI15416) and Immune Tolerance Network Award (UM1 AI109565).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The combination of dual extraction with ultra-performance liquid chromatography-electrospray ionization quadrupole-time-of-flight mass spectrometry (UPLC-ESI-qToF-MS/MS) allowed for the monitoring of 257 lipid species across all samples. Complementary multivariate and univariate data analysis identified that glycerolipids , saturated and mono/polyunsaturated fatty acids, primary fatty amides, glycerophospholipids , sphingolipids , N-acylethanolamines and sterol lipids  were significantly increased in the CSF of PD compared to the control group. Interestingly, CSF lipid dyshomeostasis differed depending on neuropathological staging and disease duration. These results, despite the limitation of being obtained in a small population, suggest extensive CSF lipid remodeling in PD, shedding new light on the deployment of CSF lipidomics as a promising tool to identify potential lipid markers as well as discriminatory lipid species between PD and other atypical parkinsonisms.Lipid metabolism is clearly associated to Parkinson’s disease (PD). Although lipid homeostasis has been widely studied in multiple animal and cellular models, as well as in blood derived from PD individuals, the cerebrospinal fluid (CSF) lipidomic profile in PD remains largely unexplored. In this study, we characterized the post-mortem CSF lipidomic imbalance between neurologically intact controls ( SREBF1, ASAH1, SMPD1, PLA2G6, amongst others) have been linked to PD. Moreover, lipids not only influence in the aggregation potential of alpha-synuclein in vitro and in vivo [Due to the lipid heterogeneity, it has been estimated that the human lipidome may be composed by 100,000 different lipid species . Lipids  in vivo , but the in vivo . All theLipidomics is emerging as a powerful approach that complements protein and gene-centric workflows in the biomarker search to evaluate the neurodegenerative risk or the neurodegenerative progression . AlthougInternal standard (IS) compounds, nonadecanoid acid, dehydrocholic acid and tryptophan-(indole-d5), were purchased from Sigma-Aldrich-Merck KGaA . 1-tridecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (13:0 Lyso PC), N-hexanoyl-D-erythro-sphingosylphosphorylcholine SM (d18:1/6:0), 1,2-diheptadecanoyl-sn-glycero-3-phosphoethanolamine (17:0 PE), 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (19:0 PC) and N-heptadecanoyl-D-erythro-sphingosine Ceramide (d18:1/17:0) were purchased from Avanti Polar Lipids . Tritridecanoylglycerol (13:0 TG), Triheptadecanoylglycerol (17:0 TG) and Cholesteryl Laurate ChoE (12:0) were purchased from Larodan Fine Chemicals . All chemicals and solvents  were of analytical, HPLC or HPLC-MS grade. See n = 10; mean age: 77.7 years; 4F/6M) and PD  post-mortem CSF samples were obtained from the Parkinson’s UK Brain Bank funded by Parkinson’s UK, a charity registered in England and Wales (258197) and in Scotland (SC037554)  and 650 µL of supernatant were collected for each platform. Lipidomics platform 1:650 μL of supernatant were dried at 40 °C in a vacuum concentrator and reconstituted in 50 μL methanol with agitation for 20 min at RT. After centrifugation  to precipitate any particles, supernatants were transferred to a plate for UPLC®-MS analysis. Lipidomics platform 2:650 μL supernatant were mixed with 50 μL of H2O and vortexed for a few seconds. After centrifugation , 400 μL of the lower organic phase were dried at 40 °C in a vacuum concentrator. Dried samples were reconstituted in 50 μL of acetonitrile:isopropanol 1:1 and shaken vigorously at RT for 10 min. A centrifugation step  was performed to precipitate any particles, and supernatants were transferred to a plate for ultra-performance liquid chromatography UPLC®-MS analysis.Control (C037554) . During Lipidomic profiling was carried out by OWL Metabolomics S.L. . Briefly, chromatographic separation and mass spectrometric detection conditions employed for each UHPLC-ToF-MS-based platform are indicated in m/z, mass-to-charge ratio pairs, retention time) were incorporated into the program. Using a mass tolerance window of 0.05 Da and after peak detection and noise reduction (at LC and MS levels), only true metabolite related features were processed by the software. For each sample injection, a list of chromatographic peak areas was generated. Data normalization was performed following the procedure described by Barr et al. [t-test = 0.1031) between the total intensities used for normalization of the sample groups compared in the study.TargetLynx application manager for MassLynx 4.1 software  was used for data processing. A set of parameters associated to metabolites included in the analysis  for the comparison between both experimental groups. To help in the interpretation of lipid changes in a biologically meaningful context, OWLStatApp was used (http://rstudio.owlmetabolomics.com:8031/OwlStatApp).Once normalized, the dimensionality of the complex data set was reduced to enable easy visualization of any metabolic clustering of the different groups of samples. This was achieved by multivariate data analysis, including the non-supervised principal components analysis (PCA) and/or supervised orthogonal partial least-squares to latent structures (OPLS) approaches. Univariate statistical analyses were also performed, calculating group percentage changes and unpaired Student’s During a neurodegenerative process, different types of molecules could be released and finally diffused into the CSF circuit, being considered as potential cerebrospinal fluid (CSF) biomarkers. Because cell membrane breakdown is a characteristic feature of a neurodegenerative process in brain syndromes, the deep characterization of CSF metabolomic profiles could reveal specific lipid molecules released by damaged neuronal or glial cell populations, establishing novel molecular panels to help us in the characterization of neurodegenerative diseases. In the current study, we have focused our attention on the metabolic profile of CSF lipids in PD.Due to the wide concentration range of lipids and their extensive chemical diversity , it is nThe 257 detected lipid features were analyzed across all CSF samples. Once normalized, the dimensionality of the complex dataset was reduced to enable easy visualisation of any metabolic clustering of the PD and control CSFs. The quality of the global experiment was assessed see .A supervised OPLS model was also calculated in order to achieve the maximum separation between both experimental groups. t-test p-value  for the PD vs. control comparison. As mentioned in t-test of each individual metabolite and of each metabolic class for both untransformed and sqrt transformed data are included in t-test obtained using the square root (Sqrt) transformation of the data.Univariate data analysis was also performed, calculating group percentage changes and unpaired Student’s According to disease duration, the deregulated lipid classes were highly similar between both groups, except for the phosphatidylcholines and sphingomyelins profiles that were most significantly deregulated in PD subjects with a disease duration of ≥10 years . AccordiA volcano plot was generated highlighting the most significant metabolites considered individually for the PD vs. control comparison .Lipid classes were also calculated as the sum of the normalized areas of all the lipid metabolites with the same chemical characteristics . InteresIn order to help in the interpretation of the potential origin of the lipidomic changes in a biologically meaningful context, pathway analysis was performed, mapping the deregulated lipid species as well as the lipid metabolic enzymes involved in lipid biosynthetic routes .Brain lipids act as the major source of energy, provide insulation to cells and structural integrity to membranes and can be rapidly converted to signaling molecules or to inflammatory intermediates . Thus, cIt has been speculated that SFA could exacerbate PD pathology . MoreoveIn spite of CSF alterations in several cholesteryl esters (ChoE) and steroids (ST), little is known about the impact of sterols in PD pathogenesis. In general, sterols are known to play a role in immunity, membrane fluidity and serve as signaling mediators ,48. In PChronic neuroinflammation is a landmark of PD . SphingoIn addition to the CSF Cer increment detected in PD, an increment in specific N-acylethanolamines and primary fatty amides (FAA) were also observed. N-acylethanolamines play an important role in various processes, from anti-inflammatory activities ,85 to neIt is important to note that our data obtained at the level of CSF partially corroborate previous associations between PD and the levels of fatty acyls, glycerolipids, glycerophospholipids, sphingolipids and sterols. Moreover, our pilot study established novel links between primary fatty amides (FAA) and N-acyl ethanolamines (NAE) with PD. However, although our untargeted lipidomic work has uncovered many intricacies in the CSF lipidomic homeostasis in the context of PD, there are potential limitations of our study that warrant discussion. First, due to the technological approach used, we failed to accurately monitor many lipid species present at low levels that might also participate in PD pathophysiology. Second, and based on the current knowledge, it is unclear whether the CSF lipid imbalance observed reflected pathological or compensatory mechanisms. Third, our study did not consider the effect of variables such as sex, age, PD etiology and/or mutational profiles.Comparing our data with previously published works using early clinical PD biofluid samples, alteration of glycerophospholipid and sphingolipid metabolism was also observed at the plasma level . Howevern = 30) detected 257 metabolic features by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS). A supervised OPLS model showed a clear separation between control and PD subjects, indicating that the lipids responsible for this separation were mainly glycerolipids , fatty acids , primary fatty amides, glycerophospholipids  and sphingolipids , which were increased in the PD group. Univariate data analysis also revealed a general increase in the CSF lipid metabolic profile in PD. Overall, these results suggest that: (i) multiple CSF lipid species tend to be increased in PD compared to control subjects and (ii) the dyshomeostasis observed in the parkinsonian CSF lipid profile varies depending on the disease duration and the neuropathological staging.A CSF lipidomic approach performed in PD and control subjects (


GRN) haploinsufficiency?What is the safety, tolerability, pharmacodynamic, and pharmacokinetic profile of the histone deacetylase inhibitor FRM-0334 in participants with progranulin gene  gene variations.Histone deacetylase inhibitors have been repeatedly shown to elevate progranulin levels in preclinical models. This report describes the first randomized clinical trial of a histone deacetylase inhibitor in frontotemporal dementia (FTD) resulting from progranulin –positron emission tomography (PET), in individuals with GRN and was conducted at multiple centers in North America, the UK, and the European Union. Data were analyzed from June 9, 2019, to May 13, 2021.In this randomized, double-blind, placebo-controlled, dose-escalating, phase 2a safety, tolerability, and pharmacodynamic clinical study, 2 doses of a histone deacetylase inhibitor (FRM-0334) were administered to participants with prodromal to moderate FTD with granulin variations. Participants were recruited from January 13, 2015, to April 13, 2016. The study included 27 participants with prodromal (n = 8) or mild-to-moderate symptoms of FTD (n = 19) and heterozygous pathogenic variations in Daily oral placebo (n = 5), 300 mg of FRM-0334 (n = 11), or 500 mg of FRM-0334 (n = 11) was administered for 28 days.Primary outcomes were safety and tolerability of FRM-0334 and its peripheral pharmacodynamic effect on plasma progranulin. Secondary outcomes were the plasma pharmacokinetic profile of FRM-0334 and its pharmacodynamic effect on cerebrospinal fluid progranulin. Exploratory outcomes were FDG-PET, FTD clinical severity, and cerebrospinal fluid biomarkers .GRN variations were randomized and completed treatment. FRM-0334 was safe and well tolerated but did not affect plasma progranulin , cerebrospinal fluid progranulin , or exploratory pharmacodynamic measures. Plasma FRM-0334 exposure did not increase proportionally with dose. Brain FDG-PET data were available in 26 of 27 randomized participants. In a cross-sectional analysis of 26 individuals, bifrontal cortical FDG hypometabolism was associated with worse Clinical Dementia Rating (CDR) plus National Alzheimer’s Coordinating Center frontotemporal lobar degeneration sum of boxes score , high cerebrospinal fluid NfL , and high CSF t-tau .A total of 27 participants  with GRN haploinsufficiency. International multicenter clinical trials of FTD-GRN are feasible.In this randomized clinical trial, the current formulation of FRM-0334 did not elevate PRGN levels, which could reflect a lack of efficacy at attained exposures, low bioavailability, or some combination of the 2 factors. Bifrontal FDG-PET is a sensitive measure of symptomatic NCT02149160ClinicalTrials.gov Identifier:  GRN (OMIM 138945)6 are among the most common causes of genetic frontotemporal degeneration (FTD).7 Pathogenic GRN variations result in haploinsufficiency,10 with a variable, roughly 50% reduction in secreted PGRN protein in the plasma12 and cerebrospinal fluid (CSF).11 The potential to restore or replace PGRN has spurred the development of a number of new GRN therapies that are entering human clinical trials, with 1 agent having recently entered a phase 3 trial.13 Clinical development programs have been aided by the ability to use blood and CSF PGRN concentrations as pharmacodynamics biomarkers in GRN haploinsufficiency clinical trials. However, the rarity of trial-ready participants and lack of established end points are barriers to successful development of new therapies.Autosomal dominant variations in the progranulin (PGRN) gene 14 In preclinical studies, a similar HDAC inhibitor, suberoylanilide hydroxamic acid, enhanced PGRN transcription to near-normal levels in human-derived GRN-/+(R493X) cells (−/+ denotes that the cells have heterozygous loss of function of the GRN gene) and prevented cytosolic TAR DNA-binding protein 43 accumulation in PGRN-deficient lymphoblasts.16 Although suberoylanilide hydroxamic acid is approved for treatment of cutaneous lymphoma,17 its poor central nervous system availability18 prioritized the exploration of FRM-0334 in GRN haploinsufficiency. In subsequent unpublished preclinical studies, FRM-0334 boosted GRN mRNA and PGRN in a dose-dependent manner in human -/+GRN lymphoblasts and in the cortex of mice . Additionally, single and multiple doses for FRM-0334, ranging from 10 mg to 400 mg daily, were safe, well tolerated, and associated with dose-proportional plasma exposure in an unpublished, phase 1, placebo-controlled clinical trial of FRM-0334 in 87 healthy volunteers aged 18 to 65 years. We investigated the safety, tolerability, and plasma pharmacodynamic (PD) effects (change in PGRN) of FRM-0334 in a multinational clinical trial completed in PRGN GRN haploinsufficiency. We also characterized the peripheral pharmacokinetic (PK) profile and central PD effects (change in CSF PGRN) of FRM-0334 in GRN haploinsufficiency. Additionally, this trial’s inclusion of a variety of exploratory PD end points allowed for novel analyses comparing cortical fluorodeoxyglucose positron emission tomography (FDG-PET), clinical measures of functional FTD severity, and candidate CSF biomarkers of neuronal injury, including neurofilament light chain (NfL), amyloid β 1-42 (Aβ1-42), phosphorylated tau 181 (p-tau181), and total tau (t-tau). We present data from the first trial implementing an international, multisite protocol as a mechanism for patient recruitment in the rare familial FTD space.The histone deacetylase (HDAC) inhibitor FRM-0334, formerly EVP-0334, was designed as a potent, orally available drug with brain-to-plasma concentration ratios of 3 to 6 in rodents.GRN variations, including prodromal and mild-to-moderate 20 FTD stages. Race and ethnicity information was recorded; participants self-identified their race and ethnicity. All participants were recruited and randomized from January 13, 2015, to April 13, 2016, at 11 clinical sites in the US, UK, France, Italy, the Netherlands, and Belgium  was also exclusionary, though these criteria were not cited as the basis for exclusion of any participants recruited for screening  reporting guideline, and local regulations.This randomized clinical trial included participants aged 27 to 75 years with Clinical Laboratory Improvement Amendments–confirmed pathogenic 21 generated randomization assignments in 2 sequential cohorts. Cohort 1 was randomized to placebo or low-dose (300 mg daily) FRM-0334. Cohort 2 began after review of cohort 1 safety data and included randomization to placebo or high-dose (500 mg daily) FRM-0334. Overall, 5 patients were randomized to placebo, 11 to low-dose FRM-0334, and 11 to high-dose FRM-0334. The study drug  and placebo  were presented as identical, opaque, white gelatin capsules and taken daily for 28 days. A central pharmacy was used to distribute packaged drugs to blinded site staff. Plasma collection occurred at screening visit 1 (8-30 days before the first dose), at screening visit 2 (1-7 days before the first dose), on study day 1 (first day of dose), in weekly intervals during dosing , and at 10 days after the final dose (day 38). Lumbar punctures for CSF collection were performed using standard clinical practice, aligned with Alzheimer’s Disease Neuroimaging Initiative procedures,22 on screening day 2 and study day 28 . Cerebrospinal fluid was collected in the afternoon using a gravity drip into a polypropylene collection tube. Magnetic resonance imaging was performed on screening day 1 for safety and diagnostic purposes using scanners, field strengths, and protocols employed for clinical use at each site  and secondary outcome of change in CSF PGRN (screening day 2 and study day 28) were measured via enzyme-linked immunoassay. The cosecondary outcome of FRM-0334 plasma PK properties  were assessed via liquid chromatography and tandem mass spectrometry using day 1  and day 7  plasma. Exploratory CSF biomarkers  and clinical measures  were assessed at screening and on study day 28. Biomarker assay procedures are detailed in the eMethods in The primary outcome of safety and tolerability was assessed via treatment-emergent adverse events (AEs), serum chemistry, hematology, urinalysis, vital signs (including orthostatic measurements), 12-lead electrocardiogram, physical examination, and suicidality assessments.t test, 2-tailed). Longitudinal changes in plasma PGRN, CSF biomarkers, clinical severity, and bifrontal FDG-PET standardized uptake value ratio (FDG-SUVR) were modeled with linear mixed-effects models (controlling for age and sex), allowing each patient’s outcome measures a random slope and intercept with time. In follow-up sensitivity analyses, we modeled interactions between FRM-0334  and time in determining patient outcome measures. The plasma FRM-0334 standard AUC from 0 to the maximum observed time was calculated using cubic splines.26 Linear regression models were used to assess for baseline relationships between baseline PK values and age, sex, FTLD clinical severity , and follow-up PK values (day 1 vs day 7). Linear regression models (controlling for age and sex) were used to investigate baseline cross-sectional linear relationships between bifrontal FDG-SUVR, plasma PGRN, CSF biomarker concentrations, and clinical severity outcome measures. Linear regression models (controlling for age and sex) were also used to investigate linear relationships between magnitude of change in PGRN (plasma or CSF) and magnitude of change in FDG-SUVR, CSF biomarker concentrations, and clinical severity measures after treatment with FRM-0334. Univariate regression models were also used to compare baseline PK values by age and sex. The Shapiro-Wilks W test was used to confirm assumptions of normality , and the Cameron and Trivedi decomposition of IM-test was used to confirm assumptions of residual homoscedasticity. Non-normally distributed dependent variables were log converted before inclusion in our models. Data were analyzed from June 9, 2019, to May 13, 2021.All statistical analyses were performed in STATA, version 14.2 (StataCorp LLC). A description of the original trial protocol statistical analysis plan may be found in the eMethods in P < .001 uncorrected for multiple comparisons, cluster-level familywise error [FWE]–corrected P < .05), converted to Pearson correlation coefficient r maps using the Computational Anatomy Toolbox, version 12 , and rendered on a 3-dimensional brain surface using BrainNet Viewer.27 A more stringent primary threshold of P < .05 FWE-corrected for multiple comparisons was also used for supplemental sensitivity analyses. Based on our initial voxelwise results  we further investigated potential linear relationships between FDG metabolism in a bifrontal region of interest (ROI) and clinical measures or fluid biomarkers. Baseline bifrontal macro ROI FDG-SUVR values were calculated from weighted averages of constituent bilateral frontal SPM12 using ROI definitions from the Neuromorphometrics Atlas28 and compared (as the dependent variable) with clinical measures and fluid biomarkers using linear regression models controlling for age and sex. The included ROIs were anterior, lateral, medial, and posterior orbital gyri, middle and superior frontal gyri, orbital and triangular part of the inferior frontal gyri, and the frontal pole. Change in bifrontal FDG-SUVR over time was also assessed via linear mixed-effects models as a follow-up sensitivity analysis, though the heterogeneity of PET sites and short duration of follow-up limited the potential interpretability of these analyses. The methods of FDG-PET collection and processing (including estimation of W-maps) are further detailed in the eMethods in Baseline FDG-PET data were only available for 26 of 27 individuals randomized to the study drug. Baseline FDG-PET data for the remaining participant could not be recovered after conclusion of the trial, and this participant was excluded from our imaging analyses. A summary of scanner types and PET acquisition parameters is detailed in eTable 1 in Magnetic resonance imaging processing methods and scanner characteristics are further detailed in the eMethods and eTable 1 in GRN variations were randomized and completed treatment . One patient randomized to 500 mg FRM-0334 experienced a serious AE , which was not felt to be related to the study drug in the opinion of the site investigator. Patients randomized to FRM-0334 experienced higher incidences of cardiac, constitutional, dermatologic, infectious, psychiatric, and respiratory symptoms and a lower incidence of gastrointestinal symptoms relative to placebo-treated participants  with gent AEs  was simiicipants .P = .56), CSF PGRN   after 28 days of treatment  A and B, reatment . AdditioP = .18) or Cmax  values on day 1. Compared with the low- dose cohort, the high-dose cohort also did not consistently experience higher AUC  or Cmax  values on day 7. Older age  predicted higher baseline AUC  and Cmax  , although day 1 and day 7 Cmax was not . In patients who received FRM-0334, change in PGRN (plasma and CSF) was not linearly related to change in bifrontal FDG-SUVR, CSF NfL, CSF Aβ1-42, CSF total tau, or measures of clinical severity, although an increase in CSF PGRN was associated with a decrease in p-tau181  , was associated with frontal FDG hypometabolism relative to controls , with additional left-predominant hypometabolism noted in lateral parietal, lateral temporal, posterior cingulate, caudate, and thalamic regions , greater Clinical Global Impressions Scale severity , lower Frontotemporal Dementia Rating Scale score , elevated CSF NfL , and high CSF t-tau , but was not linearly related to plasma PGRN, CSF PGRN, CSF Aβ1-42, or CSF p-tau181 . Greater clinical severity (CDR plus NACC FTLD sum of boxes) was linearly related to high CSF NfL, but not plasma PGRN, CSF PGRN, CSF Aβ1-42, CSF t-tau, or CSF p-tau181 . Moreover, the observed variance in FRM-0334 Cmax did not predict change in PGRN (plasma and CSF) or other exploratory PD measures, further suggesting inadequate target engagement. Additionally, given the unreliable relationship between FRM-0334 dose level, plasma Cmax, and plasma AUC, it is unknown whether alternative regimens (including higher or more frequent doses) of the current formulation could reliably improve the bioavailability of FRM-0334 in future trials. Given the relationship between FRM-0334 exposure and age, future early phase trials of other orally available HDAC inhibitors may require design considerations that account for possible unique, age-related differences in drug oral bioavailability in GRN haploinsufficiency. Given the wide distribution of GRN expression,29 particularly in the liver, it is possible that GRN haploinsufficiency could give rise to a wide array of subtle phenotypic differences in a variety of cell types, including peripheral cells mediating drug absorption or metabolism.The PD and PK profiles of oral FRM-0334 firmly suggest that further development of the tested formulation should not be pursued for 30 and anatomic descriptions of frontal hypometabolism31 in GRN haploinsufficiency but reveal novel fluid-biomarker relationships to FDG-PET data. Cerebrospinal fluid NfL is interpreted to be a biomarker of axonal injury and neuronal degeneration32 and correlates with disease severity34 and aggressiveness in sporadic FTLD as well as in GRN haploinsufficiency.35 The observed relationship with NfL level may attest to the synaptic loss and neuronal metabolism documented on FDG-PET (via astrocyte-neuron metabolic coupling36). Additionally, although GRN haploinsufficiency is not typified by tau deposition on autopsy, our findings support previous observations of elevated CSF t-tau in symptomatic disease.37 The relationship of FDG-PET to t-tau may reflect the lysosomal role of PGRN in supporting autophagy pathways,38 which may impact tau clearance.39 Fluorodeoxyglucose hypometabolism was not associated with CSF p-tau181, therefore, comorbid Alzheimer pathology is unlikely to have mediated its relationship to t-tau. It is unclear how much of our FDG-PET fluid-biomarker correlations were mediated by brain atrophy alone, given our lack of complementary harmonized magnetic resonance images. Our sensitivity analyses, accounting for whole-brain parenchymal volume, did provide some evidence that FDG hypometabolism tracks clinical GRN deficiency severity even after the effect of volume is considered.Our FDG-PET analyses were consistent with previous clinical correlations41 With regard to our FDG-PET analyses, the heterogeneity of our data  may have also limited the quality and resolution of our observations. Moreover, the short duration of our trial provided a relatively short interval to validly discern a true trajectory of change within our heterogeneous data set. Despite these limitations, our novel observations of FDG hypometabolism support further exploration of FDG-PET as an imaging biomarker of FTLD biological severity in future GRN haploinsufficiency studies .Our study had several limitations affecting its interpretability. The inconsistent bioavailability of FRM-0334 made it difficult to reliably extrapolate the broader effect of consistent HDAC inhibition in PGRN deficiency or whether higher exposure levels of FRM-0334 may have affected plasma PGRN. The small sample size of patients randomized to placebo and the relatively short duration of this trial also limited our ability to detect modest deflections in biomarker trajectory. However, recent publicly presented open-label trial data do suggest that a similar duration and smaller sample size may be sufficient to demonstrate biomarker evidence of target engagement from alternative PGRN boosting strategies.GRN haploinsufficiency, this trial demonstrates that international multicenter studies are feasible in this indication.6 Such multicenter trials provide a unique opportunity to collect valuable observational data in rare familial forms of FTD.In conclusion, this randomized clinical trial demonstrated that FRM-0334 did not have a PD effect on CSF or plasma PGRN concentration. However, this study provided useful data to inform future clinical trial methodology in similar cohorts. Given the rarity of patients with


Embora a elevação não isquêmica da troponina seja frequentemente observada em pacientes admitidos no pronto-socorro (PS), não há consenso quanto ao seu manejo.Este estudo teve como objetivo caracterizar os pacientes admitidos no PS com elevação da troponina não-isquêmica e identificar potenciais preditores de mortalidade nessa população.Este estudo observacional retrospectivo incluiu pacientes do PS com resultado positivo no teste da troponina entre junho e julho de 2015. Pacientes com diagnóstico clínico de síndrome coronariana aguda (SCA) foram excluídos. Os dados demográficos dos pacientes e as variáveis clínicas e laboratoriais foram extraídos dos prontuários médicos. Os dados do seguimento foram obtidos por 16 meses ou até a ocorrência de morte. O nível de significância estatística foi de 5%.hazard ratio = 0,43 ; hospitalização, tratamento antiplaquetário anterior e idade permaneceram independentemente associados à mortalidade.A elevação da troponina sem SCA foi encontrada em 153 pacientes no PS. A mediana (IIQ) de idade dos pacientes foi de 78 (19) anos, 80  eram do sexo feminino e 59  morreram durante o seguimento. A mediana do período de seguimento (IIQ) foi de 477 (316) dias. Os sobreviventes eram significativamente mais jovens 76 (24) vs. 84 (13) anos; p=0,004) e apresentaram uma maior proporção de elevação da troponina isolada (sem elevação da creatina quinase ou mioglobina) em duas avaliações consecutivas: 48  vs. 8 , p<0,001. Os sobreviventes também apresentaram menor taxa de tratamento antiplaquetário e internação no mesmo dia. Na regressão logística multivariada com ajuste para variáveis significativas na análise univariada, a elevação isolada da troponina em duas avaliações consecutivas mostrou A elevação isolada da troponina em duas medidas consecutivas foi um forte preditor de sobrevida em pacientes no PS com elevação da troponina, mas sem SCA. A troponina cardíaca (Tnc) acima do ponto de corte do percentil 99, com padrão crescente ou decrescente, é o biomarcador de lesão miocárdica,A Tnc é uma proteína distribuída no citoplasma e sarcômero de um miócito cardíaco, principalmente no retículo sarcoplasmático. Três subunidades constituem o complexo da troponina, um componente inibitório (troponina I), componente de ligação à tropomiosina (troponina T) e componente de ligação de cálcio (troponina C).As subunidades T e I  são específicas do músculo cardíaco e, portanto, podem atuar como marcadores adequados de lesão cardíaca. A TncT apresenta uma descarga dupla, primeiro o componente citoplasmático e depois o componente de ligação.,,Antes do advento da troponina, os MNM anteriores utilizados eram a creatina quinase-isoenzima muscular/cerebral (CK-MB) e a mioglobina, que eram menos sensíveis e não específicas para IM.,turnover celular do miócito, liberação celular de produtos de degradação e aumento da permeabilidade da parede celular.Embora as subunidades Tnc sejam fortemente específicas para miócitos cardíacos, elas podem ser liberadas sob um amplo espectro de condições patológicas não cardíacas, como sepse, doença renal crônica, emergências hipertensivas, sangramento gastrointestinal, acidente vascular cerebral e rabdomiólise.,–O uso generalizado de ensaios de troponina no pronto-socorro (PS) pode representar um desafio diagnóstico difícil quando o teste é anormal em paciente sem SCA.Este estudo teve como objetivo identificar os fatores preditivos de mortalidade/sobrevida em pacientes de PS sem SCA e TncI elevada utilizando as características do paciente, histórico clínico, comorbidades e valores analíticos  medidos no PS.Neste estudo retrospectivo, analisamos os dados laboratoriais de pacientes consecutivos que vieram ao PS de um hospital universitário da comunidade durante o período de 1 mês, de junho a julho de 2015, e selecionamos pacientes que apresentaram elevação da TncI.Todas as informações clínicas foram coletadas, incluindo registros médicos e de enfermagem do PS e hospitalizações, análises e outros exames complementares. O seguimento foi realizado através de registros locais e nacionais para observar as taxas de reinternação cardiovascular e morte.Os dados coletados incluíram dados demográficos, fatores de risco cardiovascular, ensaio de biomarcador miocárdico e resultados da creatinina, sintoma principal no PS, diagnóstico final do PS, hospitalização, mortalidade da hospitalização, taxa de mortalidade em 30 dias e 16 meses e taxa de reinternação cardiovascular.Neste estudo, os critérios para o diagnóstico de infarto agudo do miocárdio foram: aumento ou diminuição da Troponina I com pelo menos um valor anormal acima do limite superior de referência do ensaio e, ao menos um dos seguintes: 1) sintomas de isquemia; 2) novas alterações da onda T / segmento ST ou novo bloqueio de ramo esquerdo; 3) desenvolvimento de ondas Q patológicas na eletrocardiografia; 4) nova perda de miocárdio viável ou anormalidades regionais de movimento da parede na imagem; ou 5) identificação de um trombo intracoronário na imagem.Os registros do PS e a hospitalização de todos os pacientes com TncI elevada foram revisados pelos investigadores, e os pacientes foram divididos em dois grupos: aqueles com diagnóstico de SCA  (grupo A) e aqueles sem SCA com um teste de troponina positivo devido ao desequilíbrio de oferta/demanda de oxigênio ou lesão miocárdica sem sinais ou sintomas de isquemia miocárdica aguda (grupo B). Pacientes com IM tipo 4 ou 5 não foram incluídos neste estudo, pois, por definição, estes não são os pacientes regulares do PS e os pacientes com IM tipo 3 não têm medida de TncI.Pacientes sem SCA foram identificados através de critérios predefinidos que incluíram o seguinte: 1) miocardite/cardiomiopatia: diagnóstico à alta hospitalar ou achados sugestivos de miocardite em teste de imagem ou patológico, cardiomiopatias infiltrativas, como amiloidose ou sarcoidose, uma fração de ejeção ≤30% antes da admissão, ou transplante cardíaco anterior; 2) infecções: condições com impacto sistêmico, como celulite, pneumonia, sepse e pielonefrite; 3) disritmias agudas não relacionadas com a SCA; 4) doença renal crônica ou aguda: doença renal crônica estágio 5, diálise crônica, receptor de transplante renal ou doença renal aguda moderada a grave; 5) patologia do sistema nervoso central: acidente vascular cerebral, convulsão ou hemorragia subaracnóidea; 6) sangramento abdominal ou gastrointestinal agudo; 7) embolia pulmonar; 8) síncope inexplicada; 9) asma ou exacerbação de doença pulmonar obstrutiva crônica; e 10) outros: nível elevado de troponina de etiologia desconhecida que não atende a nenhum dos critérios acima mencionados.endpoint primário para este estudo foi a mortalidade durante a hospitalização, em 30 dias e 16 meses, enquanto o endpoint secundário foi a reinternação por doença cardiovascular durante o seguimento.O O seguimento foi concluído em 16 meses ou em caso de morte. O seguimento foi realizado através de consulta aos prontuários eletrônicos e ao registro nacional de óbitos online. O conselho de revisão institucional aprovou o protocolo do estudo. A exigência de consentimento informado foi dispensada porque os pacientes não receberam qualquer tipo de cuidado diferente por causa do estudo.-O nível de troponina I foi determinado utilizando o mesmo imunoensaio TncI padrão (Troponina I Siemens Dimension EXL)Medidas repetidas dos MNM foram realizadas pelo menos 3 horas após a primeira avaliação.Utilizando a primeira e a segunda avaliação da Tnc, a variação foi calculada da seguinte forma: variação da troponina% = ((segunda troponina × 100)/primeira troponina) × 100%.Todos os dados contínuos foram testados quanto à normalidade com o teste de Shapiro-Wilks; todos apresentaram distribuição não-normal e são apresentados por sua mediana e intervalo interquartil. O teste de Mann-Whitney foi aplicado para comparar variáveis contínuas. As variáveis categóricas foram representadas por sua frequência e comparadas utilizando o teste exato de Fisher ou o teste de qui-quadrado.hazard ratio (HR) com intervalos de confiança de 95% (IC 95%). Para as variáveis independentes preditoras de sobrevida, um gráfico de sobrevida foi obtido utilizando o método de Kaplan-Meier e o teste de log-rank. O nível de significância estatística foi estabelecido com um valor de p<0,05. Todas as análises estatísticas foram realizadas utilizando o software SPSS 23.0 para Mac .A sobrevida foi analisada utilizando modelos de risco proporcional de Cox uni- e multivariados. Os resultados foram expressos como Durante o período de estudo de 1 mês, 10.564 pacientes foram admitidos no PS da nossa instituição. Os pacientes que foram submetidos a avaliações de MNM, incluindo troponina I, CK-MB e mioglobina e sua distribuição de acordo com o status da troponina e o diagnóstico final, estão descritos na Na primeira avaliação do MNM do grupo B, 81 pacientes apresentaram elevação de CK-MB e/ou mioglobina . Na segunda avaliação, 18 apresentaram elevação de ambos e 6 apresentaram elevação isolada do CK-MB e 31 da mioglobina. Em ambas as avaliações, 88 pacientes apresentaram pelo menos uma elevação de CKMB e / ou mioglobina.Os pacientes com resultado positivo no teste da troponina apresentaram um amplo espectro de sintomas clínicos na apresentação . Como esConforme mostrado na Os principais diagnósticos dos pacientes do Grupo B foram miocardite /cardiomiopatia (40 [26%]), seguido de infecção , arritmias agudas , doença renal crônica ou aguda (17 [11%]), doença cerebral , sangramento abdominal ou gastrointestinal agudo , embolia pulmonar , síncope inexplicada , asma ou exacerbação de doença pulmonar obstrutiva crônica  e outros .A mediana do seguimento (IIQ) foi de 477 dias (316). Não foram encontradas diferenças significativas entre os grupos em relação à mortalidade intra-hospitalar , mortalidade em 30 dias  e reinternação cardiovascular no seguimento . Notavelmente, a taxa de mortalidade em longo prazo foi significativamente maior nos pacientes do grupo B , embora as curvas de sobrevivência dos dois grupos não fossem significativamente diferentes . As principais causas de morte do grupo B foram: cardiovasculares em 12 indivíduos (nenhum deles com diagnóstico de infarto agudo do miocárdio), 27 não-cardiovasculares, 13 por causas desconhecidas e 4 mistas de causas cardiovasculares e outras comorbidades.Os preditores de mortalidade dos pacientes do Grupo B estão descritos na log rank, 18,09; p <0,001).A análise de regressão de Cox é apresentada na ,,,Em nosso estudo, 78% dos pacientes com elevação da troponina receberam diagnóstico de não-SCA, o que está de acordo com estudos anteriores.,,Neste estudo, 33,5% dos pacientes com níveis elevados de troponina e diagnóstico de não-SCA tiveram alta do PS sem internação. Esse percentual parece muito alto, mas taxas mais elevadas foram descritas por outros autores.Acreditamos que a alta taxa de mortalidade durante a hospitalização e seguimento está intimamente relacionada à idade avançada e maiores comorbidades (insuficiência cardíaca anterior ou medicação antiplaquetária), conforme relatado por outros autores.,O único biomarcador recomendado para uso no diagnóstico de SCA neste momento é a Tnc, devido à sua sensibilidade e precisão superiores.clearance rápida (liberada a partir de 1h após a lesão e retorna à linha de base em 24-36h), enquanto a CK-MB apresenta uma liberação e clearance mais lentas (liberada a partir de 4-9h após a lesão e clearance em 48-72h),clearance retardada (7–10 dias).Nosso estudo é notável por descobrir que a elevação isolada da TncI em duas análises consecutivas de MNMs é um preditor de sobrevida para pacientes com elevação da TncI e sem SCA, em comparação com a elevação de pelo menos dois MNMs (TncI e CK-MB e/ou mioglobina). Algumas particularidades das diferentes propriedades das moléculas de MNM poderiam explicar esse fato. A mioglobina apresenta liberação precoce e Provavelmente houve uma diferença no mecanismo de liberação de diferentes moléculas de MNM de acordo com o tipo e a gravidade da lesão. Alguns estudos em animais e células humanas sugeriram que a descarga de proteínas miocárdicas, exatamente como a Tnc, pode não implicar em necrose miocárdica.–Em relação às subunidades T e I, a TncI tem um peso molecular de 37 kDa e a TncT tem um peso molecular de 21 kDa, ambas presentes principalmente nos sarcômeros e 4–6% no citoplasma. Após a lesão miocárdica, a troponina citosólica é liberada primeiro; à medida que mais danos ocorrem, a troponina presente no sarcômero é liberada na circulação;A CK-MB também é liberada com necrose tecidual devido ao seu alto peso molecular (86kDa). A mioglobina tem liberação rápida, provavelmente pelo baixo peso molecular (17kDa) e sua localização citoplasmática, podendo ser liberada sob estresse miocárdico sem necrose,,Essa propriedade molecular de diferentes MNMs poderia explicar a incapacidade de uma medida isolada da troponina I em predizer mortalidade no presente estudo e em outro.Nossa instituição segue um protocolo não-restritivo para pedidos de medidas de MNM no PS. Portanto, nossa taxa de pacientes sem SCA estava provavelmente aumentado em comparação com protocolos mais rígidos.Nosso estudo analisou a mortalidade dos pacientes sem considerar que os pacientes foram tratados de forma diferente de acordo com o diagnóstico inicial. Esta pode parecer uma limitação importante, mas deve ser esclarecido que cada processo clínico geralmente tem seu próprio manejo específico que influencia o prognóstico do paciente. Portanto, o prognóstico dos grupos é inerente, de alguma forma, ao manejo fornecido. Por exemplo, os pacientes com SCA são geralmente admitidos para tratamento com agentes antiplaquetários, anticoagulantes, estatinas, revascularização e outras terapias, e essa abordagem tem um prognóstico específico.Os dados foram coletados retrospectivamente, sendo possível que alguns prontuários estivessem incompletos e o histórico clínico subvalorizado.,O ensaio de troponina utilizado anteriormente em nosso hospital era um ensaio contemporâneo denominado ‘troponina sensível’ e não era altamente sensível, ao contrário do ensaio de troponina utilizado atualmente, através do qual espera-se detectar valores positivos de troponina em mais pacientes, como descrito para este ensaio de troponina.Uma alta porcentagem de pacientes com um nível elevado de troponina medido no PS não foi diagnosticada com SCA. Esses pacientes apresentavam perfil clínico de alto risco, ampla heterogeneidade em relação ao diagnóstico principal e prognóstico adverso aos 16 meses. Uma elevação isolada da troponina I em duas determinações consecutivas de MNMs foi um forte preditor de sobrevida em pacientes sem SCA com elevação da troponina. th percentile cutoff point with an increasing or decreasing pattern is the biomarker of myocardial injury,–Clinical myocardial infarction according to the fourth universal definition requires the presence of acute myocardial injury detected by abnormal cardiac biomarkers associated with evidence of acute myocardial ischemia. Cardiac troponin(cTn) above the 99cTn is a protein distributed within the cytoplasm and sarcomere of a cardiac myocyte, mostly in the sarcoplasmic reticulum. Three subunits make up the troponin complex, an inhibitory component (troponin I), tropomyosin-binding component (troponin T) and calcium-binding component (troponin C).The T and I subunits  are specific to cardiac muscle and, thus, can act as suitable markers of cardiac injury. cTnT shows a double discharge, first the cytoplasmic component and later the binding component.,,Before the advent of troponin, the previous MNM used were muscle/brain isoenzyme of creatine kinase (CK-MB) and myoglobin, which were less sensitive and not specific to myocardial infarction.,Although cTn subunits are strongly specific to cardiac myocytes, they can be released under a wide spectrum of non-cardiac pathological conditions, such sepsis, chronic kidney disease, hypertensive emergencies, gastrointestinal bleeding, stroke and rhabdomyolysis.,–The widespread use of troponin assays in the emergency department (ED) may pose a difficult diagnostic challenge when the test is abnormal in patient without an ACS.This study aimed to identify factors predictive of mortality/survival in ED patients without ACS and elevated cTnI using patient characteristics, clinical past history, comorbidities and analytic values  measured in ED.In this retrospective study, we analyzed the laboratory data of consecutive patients that came to the ED of a community teaching hospital during 1-month period from June to July of 2015, and selected patients who presented with cTnI elevation.All clinical information was collected, including medical and nursing records of the ED and hospitalizations, analyses, and other complementary exams. Follow-up was performed through local and national records to observe the rates of cardiovascular re-hospitalization and death.The collected data included demographics, cardiovascular risk factors, myocardial biomarker assay and creatinine results, main symptom at the ED, final ED diagnosis, hospitalization, hospitalization mortality, mortality rate at 30 days and 16 months, and cardiovascular re-hospitalization rate.In this study, the criteria for the diagnosis of acute myocardial infarction were an increase or decrease in Troponin I with at least one abnormal value higher than the upper reference limit of the assay and at least one of the following: 1) symptoms of ischemia; 2) new ST-segment/ T-wave changes or new left bundle-branch block; 3) development of pathological Q waves on electrocardiography; 4) new loss of viable myocardium or regional wall motion abnormalities on imaging test; or 5) identification of an intracoronary thrombus on imaging test.Emergency and hospitalization registries of all patients with elevated cTnI, were revised by the investigators, and patients were divided into two groups: those diagnosed with an ACS,  or type 2 with ischemic signs and symptoms  (group A) and those without an ACS with a positive troponin assay due to oxygen supply/demand imbalance or myocardial injury with no signs or symptoms of acute myocardial ischemia(group B). Patients with type 4 or 5 MI were not included in this study, as by definition these are not the regular ED patients, and type 3 MI patients have no cTnI measure.Non-ACS patients were identified by predefined criteria that included the following: 1) myocarditis/cardiomyopathy: discharge diagnosis or findings suggestive of myocarditis on imaging test or pathology, infiltrative cardiomyopathies such as amyloidosis or sarcoidosis, an ejection fraction ≤30% prior to admission, or prior cardiac transplantation; 2) infections: conditions with systemic impact such as cellulitis, pneumonia, sepsis, and pyelonephritis; 3) acute dysrhythmias unrelated to ACS; 4) chronic or acute kidney disease: stage-5 chronic kidney disease, chronic dialysis, renal transplant recipient, or moderate to severe acute kidney disease; 5) central nervous system pathology: stroke, seizure, or subarachnoid hemorrhage; 6) acute abdominal or gastrointestinal bleeding; 7) pulmonary embolism; 8) unexplained syncope; 9) asthma or chronic obstructive pulmonary disease exacerbation; and 10) others: elevated troponin level of unknown etiology, which does not meet any of the abovementioned criteria.The primary endpoint for this study was mortality during hospitalization, at 30 days and 16 months, while the secondary endpoint was rehospitalization for cardiovascular disease during follow-up.Follow-up was completed at 16 months or at death. Follow-up was performed through electronic medical records and the online national death registry. The institutional review board approved the study protocol. The requirement for informed consent was waived, because the patients did not receive any type of different care because of the study.–The troponin I level was determined using the same standard cTnI immunoassay (Troponin I Siemens Dimension EXL)Repeated MNM measurements were performed at least 3 hours after the first evaluation.Using the first and second evaluation of cardiac troponin, the variation was calculated as follows: variation of troponin % = ((second troponin × 100)/first troponin) × 100%.All continuous data were tested for normality with the Shapiro-Wilks test; all showed non-normal distribution and are presented as median and interquartile range. The Mann-Whitney test was applied to compare continuous variables. Categorical variables were represented by their frequency and compared using Fisher's exact test or the chi-square-test.Survival was analyzed using uni- and multivariate Cox proportional hazard models. The results were expressed as hazard ratio (HR) with 95% confidence intervals (95%CI). For the independent survival predictor variables, a survival plot was obtained using the Kaplan-Meier method and the log-rank test. The statistical significance level was set at p-value<0.05. All statistical analyses were performed using SPSS 23.0 for Mac .During the one-month study period, 10,564 patients were admitted to our institution's ED. Patients who underwent MNM evaluations including troponin I, CK-MB, and myoglobin and their distribution according to troponin status and final diagnosis are described in On the first MNM evaluation of group B, 81 had CKMB and/or myoglobin elevation . On the second evaluation, 18 showed elevation of both and 6 showed isolated elevation of CKMB and 31 of myoglobin. At both evaluations, 88 patients showed at least one elevation of CKMB and/or myoglobin.Patients with a positive troponin test result had a wide spectrum of clinical symptoms at presentation . As expeAs shown on The main diagnoses in Group B patients were myocarditis/cardiomyopathy (40[26%]), followed by infection , acute dysrhythmias (25 [16.6%]), chronic or acute kidney disease (17 [11%]), cerebral disease (13 [8.4%]), acute abdominal or gastrointestinal bleeding (11 [7.1%]), pulmonary embolism (6 [3.9%]), unexplained syncope (4[2.6%]), asthma or chronic obstructive pulmonary disease exacerbation (4 [2.6%]), and others (9 [6.5%]).The median (IQR) follow-up was 477 days (316). No significant intergroup differences were found regarding in-hospital mortality , 30-day mortality  and cardiovascular rehospitalization at follow-up . Remarkably, the long-term mortality rate was significantly higher in group B patients , although the survival curves of the two groups were not significantly different . The main causes of death in group B were cardiovascular in 12 individuals , non-cardiovascular in 27, 13 unknown causes and 4 due to mixed of cardiovascular and other comorbidity.Predictors of mortality in Group B patients are described in Cox regression analysis is shown on ,,,In our study, 78% of patients with troponin elevations received a non-ACS diagnosis, which is in agreement with previous studies.,,In this study, 33.5% of the patients with elevated troponin levels and a non-ACS diagnosis were discharged from the ED without hospitalization. This percentage seems very high, but higher rates have been described by other authors.We believe that the high mortality rate during hospitalization and follow-up is closely related to older age and greater comorbidities (past heart failure or antiplatelet medication), as reported by others.,The only biomarker recommended to diagnose ACS at this time is cardiac troponin, due to its higher sensitivity and accuracy.Our study is remarkable for finding that isolated cTnI elevation in two consecutive analyses of MNMs is a survival predictor for patients with cTnI elevation and non-ACS, compared to the elevation in at least two MNMs (cTnI and CK-MB and/or myoglobin). Some particularities of the different properties of MNM molecules could explain this fact. Myoglobin has an early release and quick clearance (released starting 1h after the injury and return to baseline at 24–36h), while CK-MB shows slower release and clearance (release starting 4–9h after the injury and clearance after 48–72h),Probably, there was a difference in the release mechanism of different MNM molecules according to the injury type and severity. Some studies in animals and human cells suggested that the discharge of myocardial proteins just like cTn may not imply myocardial necrosis.–Regarding the T and I subunits, cTnI has a molecular weight of 37 kDa and cTnT has a molecular weight of 21 kDa; both presenting mainly in the sarcomeres and 4–6% in the cytoplasm. After the myocardial injury, cytosolic troponin is released first; as further damage occurs, the troponin present in the sarcomere is released into the circulation;CK-MB is also released when there is tissue necrosis, because of its high molecular weight (86kDa). Myoglobin has a rapid release, probably because of its low molecular weight (17kDa) and cytoplasmic location, and it could be released under myocardial stress without necrosis,,This molecular property of different MNMs could explain the incapacity of a single troponin I measurement to predict mortality in the present study and another one.Our institution follows a non-restrictive protocol for MNM measurement orders at the ED. Therefore, our rate of Non-ACS probably were increased, when compared with more strict protocols.Our study analyzed patient mortality without considering that patients were managed differently according to the initial diagnosis. This may seem to be an important limitation, but it should be clarified that each clinical process usually has its own specific management, which influences patient prognosis. Therefore, the prognosis of the groups is somehow inherent to the provided management. For instance, patients with ACS are usually admitted for treatment with antiplatelet, anticoagulant agents, statins, revascularization, and other therapies, and this approach has a specific prognosis.Data were retrospectively collected and it is possible that some medical records were incomplete and the clinical history could have been undervalued.,The troponin assay previously used in our hospital was a contemporary assay called ‘sensitive troponin’ and it was not highly sensitive, unlike the currently used troponin assay, which is expected to detect positive troponin values in more patients, as described for this troponin assay.High percentage of patients with an elevated troponin level measured in the ED were not diagnosed with ACS. These patients had a high-risk clinical profile, broad heterogeneity regarding the main diagnosis, and an adverse prognosis at 16 months. An isolated troponin I elevation in two consecutive determinations of MNM was a strong predictor of survival in non-ACS patients with troponin elevation.