Mice with established PDXs received a liquid diet enriched with fish oil or placebo, and fatty acid profiling was performed to measure fatty acid content in whole blood. Moreover, a biobank consisting of tissue and blood samples from patients was established. Histology, immunohistochemistry and in situ hybridization procedures were used for staining of tumor and xenograft tissue slides. Results demonstrate that key histological characteristics of the patients’ tumors were retained in the established PDXs, and the liquid diets were consumed as intended by the mice. Some of the older mice developed lymphomas that originated from human Ki67+, CD45+, and EBV+ lymphoid cells. We present a detailed description of the process and methodology, as well as possible issues that may arise, to refine the method and improve PDX engraftment rate for future studies. The established PDX model for CRC can be used for exploring different cancer treatment regimes, and liquid diets enriched with fish oil may be successfully delivered to the mice through the drinking flasks.Cancer patient-derived xenografts (PDXs) better preserve tumor characteristics and microenvironment than traditional cancer cell line derived xenografts and are becoming a valuable model in translational cancer research and personalized medicine. We have established a PDX model for colorectal cancer (CRC) in CIEA NOG mice with a 50% engraftment rate. Tumor fragments from patients with CRC ( In preclinical studies there has been a tradition for establishing cancer xenografts in immunosuppressed mice using cancer-derived cell lines (CDX). However, the results from CDX studies often do not correlate with the results from clinical studies, partly because cancer cell lines fail to represent the true complexity and heterogeneity of tumors . ThIL2Rg) gene reduces the chance of spontaneous lymphoma development in these animals, which was a known problem in previous NOD/SCID models [The successful establishment of PDXs depends on several factors. The choice of animal model is important, and immunodeficient mice are found to have a higher engraftment rate for xenografts from foreign tissue as their lack of functional immune system would refrain from interfering with the foreign engrafted substance. Immunodeficient NOD/SCID/IL2Rg null (NOG) mice lack functional natural killer cells as well as B and T lymphocytes, and have defective macrophages, complement activity and dendritic cells . This maD models ,10. OtheD models .In this study we aim at describing the procedure for the establishment of a PDX model of colorectal cancer (CRC). The outcome of CRC has improved significantly over the past few decades. However, CRC is still the second and third most common cancer type worldwide among women and men, respectively , and thePatients enrolled in the study were scheduled for cancer surgery at St. Olav’s University Hospital in Trondheim between April and September 2019. Medical records of patients scheduled for consultation at the preoperative clinic at St. Olav’s University Hospital were assessed. Patients were included based on the following criteria; clinically verified colon- or rectal cancer stage 1–4, tumor size exceeding 3 cm in diameter, and age > 40 years with no preoperative treatment. After signing an informed consent form, data regarding gender, age, diagnosis, tumor type, stage of disease, prior cancer treatment, use of lipid modifying medicaments, and intake of fish, cod liver oil, and omega-3 supplements were collected. Five patients who met the inclusion criteria following preoperative evaluation were included in this study. The study was approved by the Regional ethics committee for central Norway . A Data Protection Impact Assessment (DPIA) for the project was performed in cooperation with the Norwegian Centre for Research Data (NSD) and was approved by both St. Olav’s University Hospital and NTNU. Data were securely stored at the HEMIT net at St. Olav’s University Hospital, locked by 2 step authorization by chip and password.® system ) by Biobank1®. To minimize the blood sampling burden of the patients, blood samples for the study was sampled together with the standard clinical blood tests. Blood plasma and serum were frozen at −80 °C. Whole blood was stored at −80 °C for verification of germline mutations that may be found in normal colon tissue of patients and requires follow up and genetic counseling of these patients. Whole blood (2 × 50 µL) was spotted onto Whatman 903 protein saver cards (Whatman products (Cytiva), Little Chalfont, Buckinghamshire, UK), dried for 2 h at room temperature, and frozen at −80 °C for later FA profiling.Blood samples and related information were collected, processed and stored . The CRC tissue fragments for PDX were kept on ice and transported from the clinic to the animal facility, where the mice were prepared for the surgical procedure.Tissue samples of both tumor and healthy colorectal tissue were prepared for exome and RNA sequencing, protein isolation and IHC. A fresh tumor tissue sample for PDX was cut into equally sized fragments , placed The mice were acclimatized for minimum 1 week after arrival, and four mice per cage were housed at a switched 12-h light/dark cycle at the Comparative Medicine Core Facility (CoMed), NTNU. Due to the compromised immune system of the CIEA NOG mice, all water, food, and cages were autoclaved before use. At the time of engraftment, the mice were 11 weeks or older.® (NOG) mice . The size of the tissue fragments from the two first patient tumors were about 4 × 4 × 3 mm (~50 mm3), and for the last three tumors about 3 × 3 × 3 mm (~30 mm3). Tissue fragments from each tumor were engrafted in four mice; in total twenty mice  gas using an anesthetic vaporizer, until they were asleep. Eye gel  was applied, and the mice were placed on a heating pad covered with a surgery tissue and kept anaesthetized using a nose cone with 1.5% isoflurane. The anesthetic level was checked by foot pinch using forceps and the mouse was marked by ear clip. The incision area was shaved and washed with Hibiscrub  and chlorohexidine . Metacam  and Marcaine  were given subcutaneously for systemic and local pain relief, respectively. The tumor fragments were placed in a sterile petri dish and washed briefly with sterile physiological NaCl. A small cut (3–4 mm) was made in the skin in front of the back curve, and the tissue fragment was placed in a small pocket under the skin using forceps. The wound was closed by stitching using surgical sutures , before the mice were placed on clean paper in a new cage.The surgical table and equipment were cleaned with ethanol and the surgical equipment  was autoclaved. The mice were weighed to estimate correct dosing of medications and put into an anesthesia induction chamber provided with 2% isoflurane /2. In addition, tumor growth was recorded regularly by imaging.The mice were observed for two hours after surgery. If any stiches were opened by the mice within two hours, the mice were anesthetized and restitched. Metacam was injected subcutaneously 24 h postoperative for systemic pain relief. The mice were housed in single cages for 1–2 weeks after surgery until the wounds had healed, before placing the mice together in groups of four mice per cage. The “rat and mouse No. 1 Maintenance Autoclavable”  pellet food and water were autoclaved before use. The pellet food was available to the mice at all time. Water was available until the start of treatment regime; see details below. The mice were weighed 1–3 times per week, and observations were noted in a score sheet for each animal . SubcutaBlood samples were taken from vena saphena before treatment was initiated a. The miAt the endpoint, the mice were anesthetized, and blood was collected from the main vein/heart b before When growth of the xenograft was confirmed, mice were given either the nutrition drink Smartfish Remune with fish oil (DHA and EPA (2000 mg/200 mL), Smartfish AS, Oslo, Norway), or placebo containing an equal amount of rapeseed oil (Smartfish AS). The diets were isocaloric and isolipidic, differing only by the type of oil. The liquid diets were aliquoted, frozen at −20 °C, thawed, and freshly provided. Mice were given nutrition drinks five days a week for seven hours a day during their active time period; otherwise fresh water was available. The drinking bottles containing the nutrition drink were weighed before and after the seven-hour feeding period to estimate daily intake. A human dose of 4 g omega-3 FAs/day is equivalent to ~1.6 mL Smartfish Remune daily. Animal equivalent dose (AED) based on body surface area was calculated by the equation: AED (mg/kg) = human dose (mg/kg) *  = (4000 mg/60 kg) * (37/3) = 822.22 . Oral co3, reduced health/weight or a xenograft not growing after 6 months, blood sampling, and euthanization were performed as described above and in 2 for protein- and RNA analysis. The samples were stored at –80 °C. Tumor tissue (both PDX and any secondary tumors) for histology/IHC was collected in neutral buffered formaldehyde . The mice bearing xenografts from the last three patient tumors were necropsied. The abdomen was opened, and the lungs, heart, liver, kidneys, stomach, colon, spleen, and thymus were localized, inspected and collected in NBF for histology analysis. Gross findings were registered on an autopsy card  for eacg, 10 °C). The sample (3 µL) was injected into the GC-FID with an HP 7890A Gas Chromatograph System . FAs were separated on a Supelco 30 m × 250 µm × 0.2 µm column and the results for 11 different FAs were reported as g FAME/100 g FAME. Omega-3 index was calculated by the formula: Omega-3 index = (g DHA FAME + g EPA FAME)/total g FAME * 100.The profiling of FAs in human and mouse whole blood was performed using gas chromatography with flame-ionization detection (GC-FID). Samples were analyzed by Vitas AS, Oslo, Norway. Two punches of human whole blood from the Whatman cards were diluted in sodium methylate . FA methyl esters (FAME) were formed by methylation  and extracted with distilled water (300 µL) and hexane (500 µL) before thorough mixing (5 min) and centrifugation  analyses. Tumor tissue and PDX fragments were collected in tubes with 4% NBF. The Cellular and Molecular Imaging Core Facility (CMIC), Department of Clinical and Molecular Medicine (IKOM), NTNU used a histological routine procedure to process, paraffin embed (FFPE), section (4 µm) and dry (60°) the samples. The slides were stained with hematoxylin, erythrosin and saffron (HES) in the automatic slide stainer Sakura Tissue-Tek © PrismaTM. The slides were dried further in the instrument’s heat chamber, before being deparaffinized through several baths of Tissue Clear and rehydrated through descending grades of ethanol to water. The slides were stained in Haematoxylin followed by bluing in water, before being stained with Erythrosine and rinsed in water for removal of excess dye. Slides were further dehydrated through ascending grades of ethanol and stained in saffron before being rinsed in several baths of absolute ethanol and cleared in Tissue Clear before cover slipping in Sakura Tissue-Tek © GlasTM automatic coverslipper. The sections were dried overnight in a well-ventilated place due to chemical evaporation. Dyes used were: Heamatoxylin , Erythrosine 239  and saffron . Interpretation was performed by experienced pathologists using light microscopy.For IHC analysis FFPE tissue sections (4 µm) were cut onto SuperFrost Plus slides, dried overnight at 37 °C, and then baked for 2 h at 60 °C. The sections were dewaxed in Tissue Clear and rehydrated through graded alcohols to water in an automatic slide stainer (Sakura Tissue-Tek © Prisma™). Next, the sections were pretreated in Target Retrieval Solution, High or Low pH  in PT Link (Dako) for 20 min at 97 °C to facilitate antigen retrieval. Further staining was performed on the Dako Autostainer. Following soaking in wash buffer, endogenous peroxidase activity was quenched by incubation in Peroxydase block (Dako S2023). Sections were washed in wash buffer and incubated with primary antibody for 40 min. Further, the slides were washed in wash buffer before incubating for 30 min in labelled polymer HRP anti-Mouse (Dako K4001) and DAB (Dako K3468) to develop the stain. Tris-buffered saline (TBS) (Dako K8007) was used throughout for the washing steps. In Sakura Tissue-Tek © Prisma™, the slides were lightly counterstained with Hematoxylin, dehydrated through ascending grades of ethanol, cleared in Tissue Clear, and coverslipped. For the tissue studied, appropriate negative controls were performed by omitting the primary antibody. Antibodies used were Ki67 , CD45  and CD20 . Interpretation was performed by experienced pathologists using light microscopy.To detect Epstein–Barr-Virus (EBV)-encoded RNA the Inform EBER (EBV early RNA) probe  was used for in situ hybridization (ISH) of NBF fixated paraffin embedded patient tumor and lymphoma tissue sections. The EBER ISH staining was routinely performed at the Unit of Immunohistochemistry, Department of Pathology, St. Olav’s University Hospital. Paraffin embedded tissue sections were cut at 3 µm onto Superfrost slides . On the slides was also a known positive sample (as control). Each sample was sectioned onto two slides, one for the EBER probe and one for a Negative control probe (REF 800-2847). The section with the negative control probe was used for identification of unspecific staining. The sections were dried at 60 °C for 1 h. The analysis was performed on BenchMark Ultra instrument  using the Ventana ISH IView Blue Detection Kit , ISH protease 3 , RED II counterstain  and INFORM EBER Probe . After the ISH process the slides were dehydrated through ascending grades of ethanol and xylene before cover slipping in Microm Cover- Tech CTM6 (Thermo Scientific) automatic coverslipper. Interpretation was performed by experienced pathologists using light microscopy.p-values for multiple testing within each model as calculated by the ghlt function, p-values were further adjusted based on the number of FAs measured using the Bonferroni correction.All data analyses were performed in R. For the FA profiling, a hierarchical mixed linear model accounting for multiple measurements from the same mice were fitted using the lme function from the nmle package. Overall effects of each FA was assessed using the Anova function, and pairwise differences using the ghlt function to perform post hoc Tukey tests. In addition to the internal adjustments of All invited patients attended the preoperative information meeting and accepted enrollment of the study by signing an informed consent form. n = 20, Fresh tissue fragments from five primary CRC tumors were engrafted in four immunodeficient CIEA NOG mice per tumor  days, respectively  days. Only four PDXs exceeded humane endpoint of 1500 mm3 before euthanization. Of these, two originated from patient tumor 3 and two from patient tumor 11 (engrafted with 50 mm3 fragments).Of the twenty mice engrafted with tumor tissue fragments, ten mice established tumor growth , giving ectively . When enImages and score sheets including animal weight and PDX size are presented in The weight for most mice was stable throughout the study , Table 3Some mice developed growth of secondary tumors or abscesses. Mouse #23 developed a dark colored secondary tumor behind the right foreleg. At necropsy the tumor was found to contain a dark dense liquid, probably due to blood accumulation .Four mice  developed spontaneous rapidly growing secondary tumors located on the front shoulders . NecropsThree of the four mice engrafted with tumor fragments from patient 18 developed abscesses in the surgery area after tissue implantation and were euthanized. However, treatment was initialized before the “tumors” were recognized as abscesses . For mouThe histological architecture of the growing PDXs demonstrated high correlation to the primary tumors as shown by hematoxylin, erythrosine and saffron (HES) staining of tumor tissue slides . All fivThe tumor from patient 3 was described as a typical colorectal adenocarcinoma. The glandular forms found in the HES stained section were also observed in the corresponding xenografts in mouse #15 and 16. Mouse #17 had a growing xenograft, but died due to technical problems during anesthesia and the xenograft was not sampled. Mouse #12 did not establish growth of the xenograft.HES staining of the histology slide from patient 11 showed that the tumor was compact and mainly consisted of signet ring cells. These cells are rare CRC cells with the nucleus placed at one side and a large mucus droplet filling most of the cell. Growth was established for all four PDXs from patient tumor 11. However, mouse #21 was euthanized due to acutely reduced health and the xenograft was not sampled. HES staining confirmed that the corresponding PDXs had a high degree of histopathological similarity to the patient tumor .The tumor from patient 17 had classical CRC histology with glandular forming and mucus producing cells . Growth The tumors from patients 18 and 19 were both confirmed to be typical colorectal adenocarcinoma . HoweverAfter euthanasia, the mice engrafted with tumor tissue from the three last patient tumors were necropsied. To study the histology by HES staining, lungs, heart, spleen, liver and any secondary tumors were sampled.HES stained histology slides of the secondary tumors from mouse #25, 26, 32 and 33 contained malignant looking lymphoid cells (lymphoma). The secondary tumor of mouse #25 was a massive tumor consisting of lymphoid cells, while the spleen, pancreas and lungs appeared healthy . Mouse #Mouse #32 developed lymphoma and was euthanized before growth of the xenograft was established. Lymphoid cancer cells were also found in the lungs and red swollen leg and shoulders of mouse #32. The spleen was enlarged (>2 cm) and contained lymphoid tumor cells . Mouse #Mouse #23 developed a secondary tumor behind the right foreleg . At necrAll four lymphoma cases  were composed of actively proliferating neoplastic lymphoid cells including a high number of mitotic cells . To distWhen PDXs reached “established growth”, mice were given liquid diets; Smartfish Remune Peach supplemented with either omega-3 FAs (fish oil) or rapeseed oil (placebo). The mice were observed to drink from the bottles , and theWhole blood from both patients and mice were spotted on Whatman filter cards to analyze FA content in blood by FA profiling. The results presented in Mouse blood samples were obtained before and after treatment to detect changes in blood FA content during treatment. In mice, the levels of EPA, DHA, and DPA significantly correlated with each other and the Omega-3 index as indicated in The aim of this study was to establish a preclinical PDX model of CRC in immunodeficient mice and give a thorough presentation of the procedure. In addition, we wanted to evaluate administration of omega-3 FAs enriched in a liquid diet in this model.n > 10 would allow detection of a 30% change with a significant level of 0.05 and a power of 0.08. However, we experienced that patient tumors had a high degree of heterogeneity and different tumorigenic levels. Not all tumors gave established PDXs, and if growth was established, the latency time was highly variable. Some mice developed abscesses or secondary tumors that reduced animal health and hence the PDX development time. These are important issues that we will address in future PDX studies to estimate required group sizes.PDXs preserve the biological characteristics of tumors better than CDX models, and therefore serve as a better research model for personalized cancer treatment. PDXs with established growth may be considered first generation xenografts, while several studies have made PDX lines (third generation PDX) stored in a PDX line biobank for future studies . By usin3, in line with the study by Katsiampoura et al. [3 to reduce the size of the wound and possibly the distress to the mice. Other studies have used CRC tumor fragments as small as 1–2 mm3 [Successful PDX establishment relies on several factors, one of them being the animal host. The CIEA NOG mouse has been shown to be a good host for establishment of PDX models due to its severe immunodeficiency . Tissue a et al. . However 1–2 mm3 . The engn = 8) engrafted with tumor fragments were 11–13 weeks old compared to over 6 months old for the last mice (n = 12). The PDXs engrafted in mice at a younger age had a higher engraftment rate compared to the older mice. However, the engraftment rate may also be affected by the size of the tumor fragments, and younger mice were engrafted with larger tumor fragments compared with the older mice. In future PDX studies we will strive to use mice aged 8–12 weeks and use 3.5 × 3.5 mm tumor fragments to increase the PDX engraftment rate. When size of the tumor fragments is reduced, the amount of cancer cells implanted is also reduced, which may affect the growth of the xenografts. Larger fragments will likely represent the heterogeneity of the tumors to a larger extent.The first mice . In thiThe most common CRC tumor type is adenocarcinoma, representing over 90% of all colorectal carcinomas (reviewed in ). All fiWhen establishing PDX models, there is a risk of spontaneously developing mouse tumors. In our study, the secondary tumors were first detected close to the location of the xenograft. Hence, inspection and comparison of the histology and molecular markers from the primary tumors and the corresponding xenografts are necessary to be able to distinguish spontaneously growing tumors from xenografts, and to ensure that key characteristics of the original tumors are maintained in the PDXs.IL2Rg gene [The secondary tumors of mice #25, 26, 32 and 33 were recognized by pathology experts as lymphomas. However, the CIEA NOG mice are reported to have a very low incidence of developing spontaneous lymphomas ,10. Yasu2Rg gene . Fujii e2Rg gene , which w2Rg gene . They re2Rg gene . Some st2Rg gene ,29,30,312Rg gene ,29. They2Rg gene . In our 2Rg gene . The lymThree mice developed rapidly growing abscesses within one week after surgery. This may indicate that the tumor fragments were contaminated during the procedure or that the tumor tissue contained intracellular bacteria. For future studies we will provide the mice with antibiotics in the drinking water for 1 week after engraftment to reduce the risk of infection and the formation of abscesses.Omega-3 PUFAs from fish oil have previously been shown to have a growth inhibitory effect on CRC cells both in vitro ,35, revi. We estiWe performed FA quantification/profiling to investigate whether the patients’ reported intake of fish and omega-3 supplements correlated with their FA profile. Patient 17 stated a daily intake of cod liver oil and had the highest whole blood levels of EPA, DPA and DHA, as well as the Omega-3 index. Patient 17 also had the lowest whole blood level of AA, an omega-6 PUFA known to be partially reduced in membrane phospholipids when omega-3 PUFA intake increases . PatFA profiling showed that the average content of the omega-3 PUFAs EPA, DPA, and DHA increased in both mice given fish oil and placebo compared to untreated animals, but the levels were highest and only significant in the mice provided with fish oil. Changes in the Omega-3 index and the DPA, as well as OA, DGLA, and AA content were statistically significant in the ANOVA analysis. Rapeseed oil is known to be rich in OA (over 50%) , and as Regarding estimation of FA levels, whole blood reflects the content of both plasma and blood cells and is a more easily obtainable approach compared to using blood plasma . Whole bIn this study we established a method for the engraftment of CRC PDXs in CIEA NOG mice with an engraftment rate of 50%. The highest engraftment rate was obtained when engrafting larger tumor fragments in young mice. Max latency time was set to six months; however, this time frame should be extended in future PDX setups in order to increase the engraftment rate. The optimal engraftment site was in front of the back curve of the mice to prevent the mice from opening the wounds. Histological staining confirmed that the established PDXs originated from human CRC adenocarcinoma. Some of the older mice developed abscesses or secondary tumors which originated from human Ki67, CD45, and EBV positive lymphoid cells. These are important findings that researchers should be aware of when planning and performing PDX studies. We have presented a strategy to successfully provide mice with fish oil and placebo by liquid diets. The intake of omega-3 FAs was confirmed by the increased omega-3 ratio in blood. The PDX model described represents a valuable research tool for the assessment of different anticancer treatment strategies. Furthermore, the establishment of a biobank with tissue and blood samples from CRC patients will provide a unique platform for future translational research.


The results of this pilot study show a marked alteration in the systemic metabolic profile of TTS patients, mainly characterized by significant elevation of ketone bodies, 2-hydroxybutyrate, acetyl-L-carnitine, and glutamate levels, in contrast with a decrease of several amino acid levels. The overall metabolic fingerprint reflects a systemic response to oxidative stress caused by the stressor that triggered the syndrome’s onset.Takotsubo syndrome (TTS) presents as an acute coronary syndrome characterized by severe left ventricular (LV) dysfunction and non-obstructive coronary artery disease that typically shows spontaneous recovery within days or weeks. The mechanisms behind TTS are mainly related to beta-adrenergic overstimulation and acute endogenous catecholamine surge, both of which could increase oxidative status that may induce further deterioration of cardiac function. Although several studies reported evidence of inflammation and oxidative stress overload in myocardial tissue of TTS models, systemic biochemical evidence of augmented oxidant activity in patients with TTS is lacking. In this study, serum samples of ten TTS patients and ten controls have been analyzed using  Few diseases show a more creative name and a more emotionally related origin than the Takotsubo syndrome (TTS), a disorder that differentially affects women. They account for 80% to 100% of cases, with a mean age at diagnosis of 61 to 76 years . JapanesTTS is often precipitated by major emotional/physical stress such as grief, fear/panic, anger, anxiety, embarrassment, or even positive emotions. For this reason, it has often been called “stress cardiomyopathy” or “broken heart syndrome”. Until recently, many cases of TTS were misdiagnosed as “myocardial infarction” (MI). A striking increase in apparently MI episodes was noticed in the past, coinciding with natural disasters such as earthquakes  or durinHowever, in the acute phase, patients with TTS have increased concentrations of plasma catecholamines  and stress-related circulating neuropeptides that are several times higher than those in patients with myocardial infarction and remain markedly elevated even a week after the onset of symptoms . AdrenerFrom a metabolic point of view, mental stress increases the concentration of oxidants in the CNS and peripheral organs , leading1H NMR profiling of serum for the personalized diagnosis and prognosis of cardiovascular diseases have been developed. They can boost the development of personalized medicine, with the additional advantage of representing non-invasive methods capable of stratifying the disease risk at a population level [1H-NMR spectroscopy. Each group was formed by ten subjects, which is generally a low number for a clinical study. However, it represents the average number of patients with TTS that a single institution receives in one year [Metabolomics represents an efficient tool to obtain insights into cardiovascular pathophysiology and to help in biomarker discovery, as already proved in the case of heart failure (HF), atherosclerosis, and ischemic and non-ischemic cardiomyopathy . Novel son level . On the on level . We usedone year . We hypog for 10 min in a refrigerated centrifuge. The resulting supernatant (serum) was stored at −80 °C.Blood samples were collected following the ethical guidelines, and patients provided written consent. A 10 mL sample of venous blood was collected from each patient in nonfasting condition upon admission. All patients were medicated with aspirin and/or beta-blockers at baseline. After collection, the whole blood coagulated. The clothing was removed by centrifugation at 3000× g, 4 °C for 20 min), then the samples were centrifuged at 13,800× g, 4 °C for 90 min. To each filtered serum, NMR buffer  was added to reach a final volume of 600 μL.Serum samples were prepared as previously described . Briefly1H-NMR spectra were acquired using a Bruker Avance 700 MHz spectrometer equipped with a triple resonance TXI probe and a SampleXpress Lite autosampler. All spectra were acquired at 25 °C using noesypr1d sequence, 1024 scans, 4 dummy scans, a spectral width of 16 ppm, an acquisition time of 3 s, a relaxation delay of 3 s, and a mixing time of 100 ms.All All the spectra were processed using 0.5 Hz of line-broadening followed by manual phase and baseline correction. Chenomx NMRSuite 8.5  was used to quantify the metabolites. The spectra database in this software allows a manual deconvolution of the different signals and determines the concentration of the compounds that form the mixture. TSP was set as an internal standard, and 53 metabolites were quantified in almost all samples . Concentration values of pyroglutamate were summed to those of glutamine as the cyclization reaction of glutamine to pyroglutamate occurs during protein removal .2Y, predicted percentage of the response; R2X, variation of X explained by the model; and Q2, the goodness of prediction. R2 varies between 0 and 1, Q2 varies between −1 and 1. When the Q2 value is higher than 0.5, the predicted model is good [For univariate and multivariate analyses, metabolite concentrations were normalized according to the method of probabilistic quotients , while f is good . In the n is the minimum sample size per group; σ is the population variance; Zβ is the conventional multiplier for power = 0.80 (0.84); Zα is the conventional multiplier for α = 0.05 (1.96); ∆2 is the difference between the population means.The minimum sample size was calculated using the following equation :(1)n=2σ2This pilot study enrolled one male and nine female patients showing TTS and ten control subjects (CTRL). Their baseline clinical characteristics are shown in 1H-NMR spectra of serum metabolites with the principal signals of the quantified compounds highlighted, both for a control subject and a TTS patient. A total of 53 metabolites were identified and quantified by spectral deconvolution using the software Chenomx as already described [escribed . Their mA multivariate analysis using an OPLS-DA model  showed sWe have then calculated the minimum sample size that can statistically sustain our results, given the magnitude of the observed differences . As a reThe absolute concentrations measured by NMR provide the opportunity to use as control the reference ranges reported in the literature, thereby allowing for an extra validation of the results. The reference values were obtained mainly through the Serum Metabolome server , contain1H-NMR and LVEF%, one of the most used parameters related to the prognosis of cardiovascular diseases, and that was significantly lower for the TTS group. We found a significant linear correlation between LVEF% values and the AcAc/3HB ratio  enhanced lipolysis ,34,35 an+ ratio ,37.Catecholamines stimulate lipolysis in adipose tissue, incrementing the level of circulating free fatty acids (FFAs) . As blooThe KB levels measured in our study are similar to those of a recent study about the circulating KB in humans after ST-segment elevation myocardial infarction (STEMI), which demonstrated for the first time that KB concentrations were >3-fold higher in patients with this condition, reporting values up to 8 mM in 3HB and 1 mM in AcAc . This la•, inhibits inflammatory intermediates synthesis and mitochondrial ROS production, and promotes antioxidant pathways through the regulation of FOXO1, FOXO3, and NRF2 transcription factors [KB represent fundamental substrates for the heart and brain when glucose availability is reduced, and their utilization is directly related to their systemic concentration. In addition to its use as energy fuel, 3HB also exerts signaling roles in maintaining energy homeostasis and promoting oxidative stress resistance ,41. 3HB  factors ,40,41.+ ratio, particularly in the liver and cardiac muscle [An increase in ROS levels causes an intracellular elevation of the NADH/NADc muscle ,37. Excec muscle ,43, explc muscle .Another consequence of the NADH concentration elevation is the inhibition of acetyl-CoA’s entry in the TCA cycle to stop its production ,43. RaisWe noticed an important unbalance in circulating amino acid levels in the metabolic profile of TTS patients. The total amino acid concentrations were significantly lower in the serum of TTS patients compared to the control group. Histidine, arginine, methionine, and alanine were among the amino acids that showed the most significant reductions. On the contrary, glutamate and the Phe/Tyr ratio were increased in TTS patients. All these changes can be related to a systemic response to oxidative stress. A decrease in serum histidine and arginine has been associated with inflammation and oxidative stress in obese women, while histidine was negatively associated with oxidative stress in non-obese controls . MethionIn addition, we can speculate the existence of a link between the opposite changes in circulating alanine and glutamate levels. An elevated serum level of alanine aminotransferase (ALT) was considered a potential marker of inflammation and oxidative stress . ElevatiOur study revealed a significant correlation between the serum level of different metabolites and LVEF, the central measure of left ventricular systolic function. Its value shows a significant reduction during the acute phase of TTS ,57, in lMoreover, the search of the metabolic fingerprint of HF with preserved ejection fraction revealed that six amino acids were elevated with respect to a control group . Finally2O2. A final limitation of this research is that no subjects with other cardiomyopathies other than TTS, particularly STEMI, were included. However, some results could be compared with other studies indicating certain particular features in the metabolic profile of TTS patients, such as the decrease in the amino acid levels. Further studies are thus needed to investigate the specific alterations in global metabolism associated with TTS that can improve our understanding of the disease’s systemic consequences, opening new avenues for specific treatments.Our study had several limitations. The first is the reduced number of subjects enrolled. Increasing this number will imply extending the time of patient recruiting and/or involving several institutes as in other studies . AlthougThe correlation between TTS and oxidative stress at the myocardial level is already well established . Our res


Dipeptidyl peptidase 4 (DPP4) levels are associated to metabolic and cardiovascular diseases in humans; initial evidence reported a relationship between DPP4 and chronic liver diseases. Aim of this study was to investigate hepatic and systemic DPP4 levels/activity in relation to NAFLD/NASH in individuals with and without metabolic disease.We recruited fifty-two obese individuals undergoing bariatric surgery and intra-operative liver biopsy at Sapienza University, Rome, Italy. The association between DPP4 levels/activity and NAFLD was also evaluated in 126 non-obese individuals recruited in the same setting.DPP4 mRNA was not associated to either its circulating levels/activity or NAFLD. In the multivariate logistic regression analysis on all the study participants (n = 178), higher circulating DPP4 activity was associated with NAFLD independently of potential confounders with OR (95% CI): 3.5 (1.2–10.21), p = 0.022.NAFLD patients had significantly higher circulating DPP4 activity than no-NAFLD in both the obese and non-obese cohorts; plasma DPP4 activity and levels linearly correlated with steatosis grade and inflammation at the liver biopsy. Hepatic This study demonstrates the coexistence of increased plasma DPP4 levels and activity in NAFLD. Circulating DPP4 measurement may represent a novel cost-effective strategy for NAFLD/NASH risk stratification and a potential tool for monitoring disease’s progression in established NAFLD. Non-alcoholic fatty liver disease (NAFLD) is a chronic pathological condition characterized by an excessive deposition of triglycerides in the hepatocytes and has become one of the most epidemic hepatic diseases worldwide . NAFLD dAlong with insulin resistance, multiple additional hits are necessary for NAFLD to develop and eventually to progress to steatohepatitis (NASH), cirrhosis and hepatocarcinoma –5. AccumDPP4 is highly expressed in liver , 8; incrTherefore, it is plausible to hypothesize that DPP4 may play a role also in human NAFLD and, in consideration of the characteristics of this enzyme, which is cleaved and released in the bloodstream in relation to its tissue expression, its circulating concentration and activity may reflect the severity of liver impairment in presence of NAFLD and NASH.DPP4 mRNA in the liver, along with the measurement of its plasma concentration and activity is available yet.Studies investigating hepatic or circulating DPP4 levels in relation to the presence of NAFLD in humans, found controversial results , 16, 17;Therefore, aim of this study was to investigate the relationship between hepatic and systemic DPP4 levels/activity and the presence of NAFLD/NASH in individuals with and without obesity and T2DM.n = 126) with or without T2DM and metabolic syndrome (MS), referring to the same outpatient clinics for metabolic evaluations, including upper abdomen ultrasonography (US) for assessing the presence of hepatosteatosis.For this study, we recruited fifty-two consecutive morbid obese individuals undergoing bariatric surgery for clinical indication at Sapienza University of Rome, Italy. The presence of an association between circulating DPP4 and NAFLD/NASH was further explored in an additional cohort of non-obese individuals ; negative tests for the presence of hepatitis B surface antigen and antibody to hepatitis C virus; absence of history of cirrhosis and other causes of liver diseases ; no treatment with drugs known to cause liver steatosis .The study protocol has been reviewed and approved by the local Ethics Committee and conducted in conformance with the Helsinki Declaration. All the study participants signed a written informed consent before undergoing all the study procedures.2) was calculated. Waist circumference (cm) was measured midway between the 12th rib and the iliac crest. Systemic systolic and diastolic blood pressure  were measured after five minutes resting; three measurements were taken and the average of the second and third one was recorded and used in the analyses. Fasting venous blood samples were drawn for routine biochemistry and metabolic profiling. Fasting blood glucose , glycosylated haemoglobin , total cholesterol (mg/dL), high-density lipoprotein cholesterol , triglycerides (mg/dL), aspartate aminotransferase , alanine aminotransferase , gamma-glutamil transpeptidase , creatinine (mg/dL) and conjugated/unconjugated bilirubin (mg/dl) measurement were performed by standard laboratory methods. The FIB-4 score was calculated to estimate liver fibrosis [All study participants underwent medical history collection and clinical work-up. Weight and height were measured light clothes on and without shoes and the body mass index , according to the manufacturer’s instructions.MS was diagnosed according to the modified NCEP ATP-III criteria . Diabeten = 52) underwent intra-operative liver biopsies during sleeve-gastrectomy intervention, following the recommendations by the American Association for the Study of Liver Diseases [Morbid obese patients . GAPDH was used as an internal standard which yielded a predicted amplicon of 171 bp. GAPDH was detected using gene-specific primers and probes labelled with reporter day FAM .TaqMan real-time quantitative PCR was performed on an ABI PRISM 7500 Fast Real-Time PCR System . PCR reactions were carried out in triplicate on 96-well plates with 10 uL per well using 1X TaqMan Master Mix and the results were evaluated at the end of the reaction using the ABI PRISM 7500 software .PCR product of human In the additional study cohort of individuals without severe obesity, NAFLD was diagnosed by clinical criteria in presence of fatty liver at the upper abdomen US examination , which was conducted by the same operator blinded to laboratory values. Liver steatosis was defined on the basis of abnormally intense, high-level echoes arising from the hepatic parenchyma, liver-kidney difference in echo amplitude, echo penetration into the deep portion of the liver and clarity of liver blood vessel structure .For this purposes of our study, the sample size was calculated according to results obtained in a previous research investigating circulating DPP4 activity, set as the primary outcome of our study, in individuals with and without biopsy-proven NASH . Thus, wT test or Mann–Whitney for continuous variables and p value < 0.05 was considered statistically significant, with a confidence interval of 95% (95% CI).Continuous variables are reported as the mean ± standard deviation (SD) and categorical variables as percentages. Skewed variables underwent natural logarithmic transformation before performing the analyses. Comparisons between two groups were performed by Student's Thirty-two out of fifty-two 61%) patients had biopsy-proven NAFLD; characteristics of the study population in relation to the presence of NAFLD are shown in Table % patientp = 0.02) and directly correlated with the severity of hepatic impairment, as expressed by greater steatosis grade  and lobular inflammation  at the biopsy and higher AST , ALT (r = 0.30 p = 0.03), GGT , LDL cholesterol  and FIB-4 .Plasma DPP4 activity was significantly higher in NAFLD than in no-NAFLD patients .The association between higher circulating DPP4 activity and the presence of NAFLD persisted statistically significant, after adjusting for age, sex and T2DM diagnosis (p = 0.06). As observed for circulating DPP4 activity, also plasma DPP4 concentration positively correlated with steatosis grade , lobular inflammation  and ALT levels .NAFLD patients displayed a trend towards higher plasma DPP4 concentration than no-NAFLD, although this difference did not reach the statistical significance , p = 0.35; Mann–Whitney test applied). Hepatic DPP4 levels were not associated with either histological/clinical indexes of liver injury or with the presence of metabolic disease, such as MS and T2DM. Liver DPP4 mRNA expression levels did not correlate with plasma DPP4 concentration and activity (not shown).Hepatic p = 0.006; plasma DPP4 concentration = NAFLD: 533.6 ± 161.9 ng/ml vs. no-NAFLD: 453.7 ± 125.8 ng/ml, p = 0.037). Clinical and metabolic characteristics of this study cohort are described in Table In order to confirm the finding of an association between increased plasma DPP4 activity and NAFLD diagnosis in non-severe obese subjects, we analysed data from an additional cohort of 126 individual with and without MS and/or T2DM undergoing liver US examination to screen NAFLD. Eighty-nine individuals (71%) had US-detected fatty liver, both plasma DPP4 activity and DPP4 concentration were significantly higher in NAFLD in comparison to no-NAFLD individuals , AST , ALT  and GGT  levels and with the presence of NAFLD , MS  and T2DM  , AST , ALT , GGT  and HOMA-IR . As observed for circulating DPP4 activity, plasma DPP4 concentration associated with the presence of NAFLD , MS  and with T2DM diagnosis  .Greater DPP4 activity was associated with the presence of NAFLD in a multivariate logistic model adjusted for sex, age, BMI, HOMA-IR and MS (n = 178), higher plasma DPP4 activity independently associated to the diagnosis of NAFLD with an OR: 3.504  regardless of potential confounders, as shown in the multivariate logistic regression analysis (Table Finally, in the whole study population (The main finding of this study is that individuals with NAFLD have increased plasma DPP4 activity than no-NAFLD subjects, regardless of the presence of obesity and metabolic disease.DPP4 mRNA expression and NAFLD. Moreover, liver DPP4 mRNA was not associated to either circulating DPP4 activity or concentration.In NAFLD patients, both plasma DPP4 activity and concentration linearly correlated with hepatic inflammation and steatosis grade at the liver biopsy, and with clinical markers of hepatic damage. Differently, no relationship was found between hepatic This is the first study investigating contextually circulating DPP4 enzyme activity, as well as its plasma concentration and hepatic expression, in relation to the presence of NAFLD.DPP4 levels were available [Some studies reported altered DPP4 levels in a number of hepatic diseases, such as chronic C hepatitis , hepatocvailable , 29.Williams et al. found an association between circulating DPP4 activity and markers of hepatic fibrosis, but no relationship with hepatic fat content . BalabanDPP4 expression was not influenced by liver damage in the course of NAFLD/NASH.In our research, both DPP4 activity and concentration were associated to more severe steatosis grade and inflammation at the biopsy whereas hepatic DPP4 and NAFLD could not be ruled out [Previously, Baumeier and collaborators demonstrated a correlation between hepatic DPP4 DNA methylation and stages of hepatosteatosis and NASH . Anotheruled out .Another finding of our study is that circulating DPP4 activity and concentration are increased in presence of MS and T2DM. Previous investigations demonstrated altered DPP4 activity/concentration in relation to metabolic diseases such as obesity –32 and TIn our study, DPP4 activity was independently associated with the presence of NAFLD, after adjustment for potential cofounders such as obesity, metabolic disease and insulin resistance. Indeed, our data depict DPP4 as a potential biomarker of hepatic injury in the course of metabolic disturbance.DPP4 and its circulating concentration.Many reports attribute to the adipose tissue a role as a major contributor of DPP4 levels into the bloodstream , 35. ThuDPP4 levels do not seem to be related to either the hepatic fat content or inflammation and fibrosis. Hepatic DPP4 has been shown to be increased in severe liver diseases and cancer [DPP4 and histological signs of liver damage in individuals with more advanced liver impairment, cannot be definitively ruled out.In our NAFLD/NASH patients, liver d cancer , 28. OurMajor strength of this study is the coexistence of data on plasma and tissue DPP4 in a large population of metabolically characterized individuals with different grade of obesity, metabolic impairment and NAFLD/NASH. Moreover, the primary study endpoint was set on a cohort of individuals undergoing liver biopsy, which represents to date the gold standard for NAFLD and NASH diagnosis.DPP4 mRNA expression in liver samples, in relation to the presence of biopsy-proven NAFLD/NASH. Another novel aspect of this study is the replication of the experiments in two parallel cohorts of individuals with different metabolic characteristics, i.e. morbid obese vs. normal-weight individuals, with and without NAFLD and metabolic diseases. Finally, our study is the first one which investigated DPP4 activity and concentration in relation to the FIB-4 score, a broadly validated marker of liver fibrosis which play a central role in the diagnostic algorithm of NAFLD and NASH in high risk populations [This is the first study evaluating contextually both DPP4 concentration and activity, as well as the ulations .A limitation of this study is the cross-sectional design which does not allow to establish a causal nexus between increased plasma DPP4 activity/concentration and NAFLD; longitudinal studies are warranted in order to test the role of DPP4 as a marker of NAFLD development and progression in individuals at high risk, as those with T2DM and metabolic disease.In conclusion, this study demonstrates the coexistence of increased plasma DPP4 concentration and DPP4 enzymatic activity in patients with NAFLD, with and without obesity and metabolic disorders. Circulating DPP4 measurement may represent a novel cost-effective strategy for NAFLD/NASH risk stratification in dysmetabolic populations and a potential tool for monitoring disease’s progression and response to treatment in established NAFLD.


Groups slightly differed with respect to age  and years of education (13.28 ± 2.89 vs. 14.56 ± 2.45). After correcting for age, C50 significantly outperformed C30 in all domains except concentration and verbal fluency. After additionally adjusting for education, C50 significantly outperformed C30 in declarative memory performances and abstract thinking only. Prevalence rates of MCI were 25.2% in C30 and 9.6% in C50 (p < 0.001). Our findings confirm the association between better educational attainment and enhanced cognitive performance in “younger” old individuals. While this association corresponds to the Flynn effect, various life course influences may have also contributed to better performance, including improvements in healthcare provision, medication, and lifestyle factors. Their overall effects may foster cognitive reserve and thus translate into the decline in MCI prevalence reported here.We compared neuropsychological functioning and prevalence of mild cognitive impairment (MCI) in two birth cohorts born 20 years apart when participants had reached the same age, i.e., the mid-60s. The study followed up 500 volunteers born 1930–1932 (C30) and 502 born 1950–1952 (C50). Participants underwent medical, neuropsychological, and psychiatric examinations in 1993–1996 (T1), 1997–2000 (T2), 2005–2008 (T3), and 2014–2016 (T4), including assessment of abstract thinking, memory performance, verbal fluency, visuo-spatial thinking, psychomotor speed, and attention. Healthy participants from C30 at T2 ( Projections of cognitive functioning in ageing are essential for adequate organization of future healthcare provision, including the adaptation of primary and secondary preventive measures. In this respect, an increase in life expectancy may or may not go hand in hand with an increase in functional limitations and disability, including a rise in dementia prevalence. Previous comparisons demonstrated massive IQ gains over time . This fiThe term “intelligence” as a description of general mental abilities is not universally accepted, and different approaches to its conceptualization have been made. According to one of the earliest models, intelligence can be separated into two overlapping albeit distinct categories: fluid intelligence refers to the capacity to solve problems for which previous experience and acquired knowledge is not important, while crystallized intelligence is based on a body of knowledge that an individual has retained over time . AccordiAs demonstrated in a cross-national investigation , the FlyIn the present study, we sought to investigate these findings in a prospective examination of two birth cohorts born 20 years apart. As participants were followed for more than 20 years, both cohorts could be compared in their mid-60s at nearly the same age. We expected to find performance gains in the young vs. the old cohort, independent of educational attainment. This effect should primarily involve components of fluid IQ such as executive functions and abstract thinking. Moreover, we expected that performance gains are linked to a decreased risk of mild cognitive impairment (MCI).Data were drawn from the Interdisciplinary Longitudinal Study of Adult Development and Aging (ILSE) that was launched in 1992. Detailed descriptions of variables and procedures of the study can be found elsewhere . Select n = 1002 subjects (500 participants from C30 and 502 participants from C50). A second examination was conducted between 1997 and 2000 (T2) with n = 896 (449 from C30 and 447 from C50). A third examination took place between 2005 and 2008 (T3) with n = 789 (381 from C30 and 408 from C50), and a fourth examination was conducted between 2014 and 2016 (T4) with n = 574 subjects (253 from C30 and 321 from C50).The ILSE is a prospective longitudinal study of two birth cohorts born 20 years apart . The oldFor the purpose of the current study, cognitive performance of the older cohort at T2 was compared to cognitive performance of the younger cohort at T4. We opted for the second examination wave instead of the first examination wave in order to control for potential effects of repeated examinations due to test sophistication that were absent during the first examination wave.Exclusion criteria were the presence of age-associated cognitive decline (AACD), which can be compared with multiple domain MCI ,10; AlzhFor T4, 31 (9.7%) individuals from C50 were diagnosed with AACD, one individual (0.3%) was diagnosed with AD, 21 individuals (6.5%) suffered from MCD, and no subject was diagnosed with VD. Eight of the remaining subjects were diagnosed with depression, four with alcohol/substance dependence, and one with both depression and alcohol substance dependence, leaving 255 individuals. Fifty participants who did not fully take part in neuropsychological testing were also excluded, leaving 205 subjects for analyses.At each examination wave, physical and mental health was assessed by a trained physician. To assess cognitive capacity, we administered different neuropsychological tests, including the subtests Word List (WL) and Digit Symbol Test (DST) of the Nuremberg Age Inventory (NAI) , the subp-values less than 0.05 were considered significant. For descriptive analyses, ANOVAs were used. In the case of categorical variables, group differences were computed with chi-squared tests. To examine cohort differences in cognitive aging multivariate ANCOVAs were calculated. While the respective cognitive test scores  were dependent, the cohort was the independent variable. To address potential effects of educational achievement and minor age differences between the cohorts, we included years of school education and age as covariates. Higher educational achievement may also involve a greater study adherence. Potential selection effects were addressed by comparing years of formal school education of the entire sample with the subsample considered in the present analysis. To examine the proportion of persons with MCI, we compared the prevalence rates of MCI in C30 at T2 and C50 at T4 using a chi-squared test.All statistical analyses were performed using SPSS (version 27). For significance tests, an alpha of 0.05 was set, such that A comparison of demographic characteristics can be seen in Neuropsychological performance and results of the multivariate ANOVAS and ANCOVAs are presented in 2χ(1) = 24.91, p < 0.001).Prevalence of MCI was 23.6% in the earlier-born (C30) and 9.7% in the later-born (C50) cohort. Chi-squared tests revealed a significantly higher MCI rate for C30 than for C50 . These differences reached the significance level  = 23.83, p < 0.001; C30: F = 18.38, p < 0.001).To identify potential selection effects, we compared school education of the two cohorts under examination with the respective values obtained in the original cohorts at study intake. As to be expected, both cohorts investigated here showed a slightly higher number of educational years when compared with the original cohort at intake (C30The present study yielded two major findings: (i) further support of an increased neuropsychological performance in younger birth cohorts partially due to greater educational achievement as predicted by the Flynn effect, and (ii) an indication that this effect may translate into a lower risk of MCI in the mid-60s.When compared with the old birth cohort, the young cohort was characterized by better educational attainment, as demonstrated by a significantly higher number of years of education. Both cohorts comprised similar proportions of women and men and showed only minor—albeit marginally significant—age differences. Hence, age was included as a covariate in all analyses.Along with that, the young outperformed the old cohort in all neuropsychological domains examined, except concentration and verbal fluency. A significant effect of education was confirmed for all domains except memory . When adjusted for age and education, the differences in declarative memory performance  and abstract thinking (mosaic test) remained solely significant between birth cohorts, indicating that performance gains in these domains cannot be attributed to education alone.On basis of data derived from studies in 14 nations, Flynn  as earlyDifferent conceptualizations of “intelligence” and overlapping features thereof render a clear allocation of the neuropsychological functions investigated in our study to any intelligence framework negligible. However, for the purpose of comparison, our data suggest that general performance gains appear across different facets of “intelligence”, while differences that are independent of age and education (declarative memory and abstract thinking) can be subsumed under the terms “crystallized”  or “lingn = 2262, mean age 93.1 ± 0.3) and 1915 . Members of the later-born cohort performed better than members of the earlier-born cohort in both cognitive functioning and activities of daily living suggesting a clear improvement in cognitive functioning in line with the Flynn effect. This effect applied to both women and men and was illustrated on the Mini Mental Status Examination MMSE [Using data from the German Socio-Economic Panel, Steiber  (p. 1) rion MMSE  revealedIn a seminal paper, Gerstorf and colleagues  comparedIn addition, the present study yielded a significantly reduced risk of MCI in the later- when compared to the earlier-born cohort. A similar finding was reported by Langa et al. , who comMCI is associated with an increased risk of developing dementia and can be thus considered as the consequence of cognitive aging. Hence, the reduced risk of MCI in the later-born cohort may result from less steep rates of cognitive aging described by Gerstorf and colleagues . AccordiSelection effects must be discussed as a potential confounding variable. As to be expected, both cohorts investigated here showed a slightly higher number of educational years when compared with the original cohort at intake. Although these differences reached significance, the selection effect refers to a similar magnitude of the change in both groups . In addition, potential training effects need to be considered when interpreting the results of this study. While the younger cohort did not undergo the entire neuropsychological test battery at T1 and T2, three subtests  were conducted at all four examination waves, while other subtests  were conducted at T1, T3, and T4 for C50. As a result, participants from C50 had undergone the testing procedure three or four times, while C30 had been tested only twice at the relevant examination waves. To address the effect of test sophistication, we opted to consider T2 rather than T1 for the older cohort. Moreover, potential training effects, in terms of an economization of cerebral activation under performance gain consolidation  are rathOthers  have chaThe decreased MCI prevalence in the later-born cohort may either refer to decelerated cognitive aging or a lower MCI risk per se; a question that clearly calls for an additional examination wave. Educational attainment does not only contribute to cognitive reserve but also involves social differences and diverse life course influences that may have also contributed to better performance in the later-born cohort, including improvements in healthcare provision; adequate medication of age-association concomitant conditions ; and factors loosely labelled lifestyle that are known associates of better cognitive aging such as social engagement, physical activity, cognitively effortful occupational and leisure pursuits, and continuing education .These findings add to a rather optimistic picture of cognitive aging in later-born cohorts. This development cannot be solely assigned to the beneficial effects of rising levels of education, but also refers to technological advances with increasing cognitive challenges in both, work, and leisure activities , as wellWe identified enhanced cognitive functioning in a younger compared to an older birth cohort, which could partially be attributed to greater educational achievement and is likely to reflect other lifestyle factors that foster cognitive reserve. In line with this, we found lower rates of MCI in the younger as opposed the older cohort when they had reached their mid-60s. Albeit painting a positive picture of cognitive ageing, further research is needed to establish whether our findings reflect a deceleration of cognitive aging or a lower risk for the development of dementia-related disorders per se.


In this exploratory study, we used discovery proteomics to follow the release of proteins from bovine knee articular cartilage in response to mechanical injury and cytokine treatment. We also studied the effect of the glucocorticoid dexamethasone (Dex) on these responses.Bovine cartilage explants were treated with either cytokines alone , a single compressive mechanical injury, cytokines and injury, or no treatment, and cultured in serum-free DMEM supplemented with 1% ITS for 22 days. All samples were incubated with or without addition of 100 nM Dex. Mass spectrometry and Western blot analyses were performed on medium samples for the identification and quantification of released proteins.We identified 500 unique proteins present in all three biological replicates. Many proteins involved in the catabolic response of cartilage degradation had increased release after inflammatory stress. Dex rescued many of these catabolic effects. The release of some proteins involved in anabolic and chondroprotective processes was inconsistent, indicating differential effects on processes that may protect cartilage from injury. Dex restored only a small fraction of these to the control state, while others had their effects exacerbated by Dex exposure.We identified proteins that were released upon cytokine treatment which could be potential biomarkers of the inflammatory contribution to cartilage degradation. We also demonstrated the imperfect rescue of Dex on the effects of cartilage degradation, with many catabolic factors being reduced, while other anabolic or chondroprotective processes were not. One glucocorticoid, dexamethasone (Dex) has been demonstrated to rescue the loss of aggrecan and collagen constituents as well as chondrocyte viability in human and bovine cartilage explant models of inflammatory tissue injury and post-traumatic OA (PTOA) ,2, suggeLi et al.  demonstre.g., isolated cells versus intact cartilage). Thus, study conclusions often differ greatly, even when studying intact cartilage explants, complicating the discussion of safety and efficacy of Dex [At the same time, deleterious effects of Dex on cartilage tissue have been reported. Several studies using isolated human chondrocytes showed that even low doses of Dex can cause cell death and reduce cell proliferation, suggesting potential cytotoxic and catabolic side effects. However, the observed effects of Dex depends greatly on dose, model, duration of treatment and context  use a global discovery proteomics approach to quantify the effects of inflammation and mechanical impact injury on cartilage explants, and (2) study the effects of Dex in the presence and absence of inflammatory and injurious mechanical challenges. We believe this approach can lead to an increased understanding of the potential benefits of a glucocorticoid such as Dex, as well as the identification of biomarkers of cartilage degradation in the presence and absence of such treatments.22.1l-Glutamine (Gibco), 0.1 mM nonessential amino acids (Sigma), 0.4 mM proline (Sigma), 20 μg/ml ascorbic acid (Sigma), 100 units/ml penicillin G, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Sigma), and 1% insulin-transferrin-selenium .Cartilage disks  were harvested from the femoropatellar grooves of 1-2-week-old bovines  as described [2.2After pre-equilibration, samples were treated for 22 days as in 2.3Supplemental Methods) [Culture medium (50 μL) from days 4, 8, 12, 16, and 20  was prep2.42-transformed and principle component analysis (PCA) was performed on treatments N, I, C, and IC using the “prcomp” function in the R-package factoextra, with ‘center’ and ‘scale’ set to true to z-score values. Pairwise comparisons between treatments were performed on the summed peptide abundance over all five timepoints. Statistical analysis was performed using the R package limma, comparing log2-fold changes within each animal [P-values were obtained using empirical Bayes statistics. Adjusted p-values were calculated using the Benjamini-Hochberg method with a q-value of 0.05.Proteins identified in all three animals were filtered, keeping only proteins identified and quantified in at least 5% of samples. Assuming that missing values were due to low abundance, missing values were imputed using quantities equal to half the lowest identified abundance for each protein. Abundance data were logh animal . P-value2.5p < 0.05) and were present in at least three timepoints for one treatment condition across all three animals. Grouping criteria were based on patterns identified with hierarchical clustering of proteins with differential release from control, and validated against the raw, non-imputed data by two operators .Proteins were classified into three groups based on their response to treatments C and IC compared to control. The grouping criteria were: (I) decreased release in treatments C and/or IC, (II) increased release by treatment C alone, or (III) increased release in IC with or without increased release in C. Proteins were selected if they had a differential effect from the control  . The sam Methods ), with t Methods , separattibodies  or anti-cificity ) and immSupplemental Methods [The breakdown of COMP was analyzed as described . Twe Methods )). Sampl32-transformed abundance data  and 115 proteins  respectively, were observed to have a difference from control . 195 pro3.2p < 0.0001), respectively, versus 42% for the entire population of 188 proteins). Group II was enriched (p < 0.0001) for intracellular proteins (78% versus 47% in the entire population), while consisting of only 4% extracellular proteins.The 188 filtered proteins were classified into three response groups A–C. We f3.3STRING network analysis revealed two small network interaction clusters for proteins in Group I A: ECM-re3.4The effects of Dex on the release of the 188 selected proteins to the medium is shown in 3.5Cytokine and injury treatments had no effect on the release of aggrecan fragments by proteomic analysis, but were evaluated in parallel by the DMMB assay for sGAG release A. TreatmWhile our MS analyses of COMP release in the present study indicated no clear treatment effects E, Wester4ex vivo explant studies have shown that sustained low doses of Dex can rescue sGAG loss, reduce proteolytic enzyme synthesis, and maintain chondrocyte viability under inflammatory cytokine challenge [This study incorporates a global discovery approach to characterize the effects of inflammation and mechanical injury on cartilage explants, including the role of therapeutic intervention with Dex. This glucocorticoid treatment has been used with intra-articular injection for treatment of arthritic or post-surgery knee pain  with somhallenge .The present study is based on label-free protein quantification allowing a semi-quantitative estimation of relative protein abundances. Cytokine treatment, in particular, exhibits a large effect on proteins released into the explant culture media, while treating with mechanical injury alone has a small effect, as demonstrated by the overlap of the two treatments in PCA space B.4.1After an acute joint injury, the progression of PTOA is initiated by an early response characterized by the production of inflammatory factors and proteases, including MMPs, in reaction to inflammatory stress and chondrocyte death . The traIn our model, we confirmed the PTOA-like effects of IL-6 and TNFα on bovine cartilage explants by examining the release of sGAGs over the three-week course of the experiment A. We fou4.2Group III, proteins with increased release after exposure to inflammatory cytokines with or without applied injury, best represents the catabolic response of cartilage to PTOA stress. This group is the largest and represented by MMP-13 C, an actl-lactate dehydrogenase, PGK1, phosphoglycerate mutase 1), endoplasmic reticulum processing , or ribosomes . The biological reason for the increased release of these proteins may be due to transcriptional or translational changes in response to cytokine treatment, or a result of cell death causing these highly abundant intracellular proteins to be released from necrotic cells.Inflammatory stress also disrupts intracellular processes and leads directly to chondrocyte death . The dys4.3The increased release of one third of the proteins in Group III was partially or completely rescued by Dex, including all four MMPs in this group. This finding agrees with literature supporting the observations that Dex inhibits the production of MMPs under arthritic stresses and prevents ECM breakdown as measured by sGAG loss and histological staining . This ef4.4As catabolic processes continue in diseased cartilage, chondrocytes begin to produce chondroprotective and anabolic factors that initiate reparative processes to reverse proteolytic and inflammatory damage . Group IThe release of lysyl oxidase (LOX), an enzyme that regulates fibrillogenesis and collagen synthesis , was alsCertain proteins identified in Group III also reveal an increase in some chondroprotective processes. For example, the glycoprotein osteoprotegerin (TNFRSF11B) can suppress osteoclastogenesis and bone resorption . Thrombo4.5Dex does not reverse the effect of inflammatory cytokines for many proteins in Group I. For example, the release of LOX and testican-1  was rescued up to or above control levels, but the release of SPARC was not affected by Dex, suggesting that the actions of Dex on cartilage under arthritic stress do not result in a perfect rescue. This is also reflected in the behavior of collagens II and IX, as Dex did not rescue or further decreased their release (which was attributed to a decrease in synthesis). Previous studies on the effects of Dex on collagen synthesis have often reported conflicting results .4.6Highly relevant to the consideration of Dex as a PTOA disease-modifying drug are “side effects” of Dex: Dex causes a significant increase in the release of some proteins in all treatment conditions, such as CTGF D. CTGF i4.7Proteomics is a powerful tool to measure changes in global protein expression at a high level and identify changes to entire pathways, but has some limitations. This analysis utilized data from a small number (48) of single-peptide identifications, which are less reliable for quantitation than proteins with more peptide coverage, but kept for analysis because of their identification at a FDR of 0.01 and their presence in at least 5% of samples. An MS approach summing peptide abundances reflects total protein level and is less sensitive to small changes in cleavage events, such as with COMP and aggrecan. Our proteomics results do not reveal a clear effect on treatment with cytokines; in contrast, the western blots show clear proteolytic cleavage COMP: B. Some l5In this exploratory study, we used a discovery proteomics approach to follow the release of proteins in response to mechanical damage and cytokine treatment of bovine knee articular cartilage. We also investigated the effect of a low, continual dose of the glucocorticoid Dex. The majority of differentially expressed proteins were increased upon treatment but some proteins, including the fibrillar collagens II and IX, were reduced. A large number of the proteins with increased release after disease treatment had reduced release in the presence of Dex. These disease-induced proteins could be potential biomarkers of the inflammatory contribution to cartilage degradation and demonstrate the protective effect of Dex against matrix breakdown and protease release. Analyzing proteins released from the cartilage also allowed some insight into the dysregulation of anabolic processes after disease induction: the release of some anabolic factors was increased after cytokine exposure, suggesting attempts to protect against and repair catabolic effects, while many other anabolic factors had their release suppressed by cytokine exposure. Dex treatment had mixed effects on these changes, highlighting the need for further experiments to explore the effect of Dex on intracellular processes in tissue models of OA. The design of this study will allow for further exploration of regulation of protein release kinetics after disease or Dex treatment.All were involved in the conception and design of the study as well as interpretation of the data. YW, PL, VT, AS conducted the experimental work and data analysis. PÖ conducted the mass spectrometry work and data analysis. Bioinformatics analysis was mainly performed by MR and RMB. RMB and PÖ drafted the manuscript while all authors critically revised the manuscript and gave final approval of the article.The authors do not have any conflict of interest.