CDKL5 deficiency disorder (CDD) is an early-onset developmental and epileptic encephalopathy characterized by frequent drug-resistant seizures, cerebral visual impairment, motor dysfunction, and sleep and gastrointestinal disturbances. Preliminary evidence suggests that highly purified cannabidiol (CBD) may reduce seizure frequency, but data on its effects on comorbidities are lacking. This study aimed to evaluate the efficacy and safety of CBD in individuals with CDD. We conducted a prospective, open-label, single-center study including patients with CDD aged >1 year. Outcomes included motor seizure frequency, caregiver- and clinician-rated Clinical Global Impression (CGI), and changes in sleep, motor abilities, and EEG at 3, 6, and 12 months. CBD plasma levels were measured with High-Performance Liquid chromatography-Mass Spectrometry (HPLC-MS). Eight of nine patients (all females; median age 10 years, range 1-24) completed the study, with a retention rate at 12 months of 8/9 (89%). One discontinued at 6 months due to a skin rash. A > 50% seizure reduction was observed in 8/9 patients at 3 months, 6/9 at 6 months, and 1/8 at 12 months. Seven patients showed some degree of vigilance improvements, three in motor performance, and two in sleep and constipation. All caregivers reported at least minimal overall improvement (CGI score 3) at 3 months, and three reported marked improvement (CGI score 2), with a peak at 3 months. Five patients showed adverse events during the trial, but none were considered serious. The median CBD dose at all time-points was 15.6 mg/kg/day (IQR 10.0-18.9) corresponding to a plasma dose of 69.9 ng/mL (IQR 29.8-114.6) and the median concentration/dose ratio was 4.7 (IQR 2.7-6.8). The safety and efficacy of highly purified CBD in CDD were consistent with previous reports in the literature, with possible benefits beyond seizure control. Further studies are warranted to assess non-seizure outcomes and compare long-term efficacy across treatment options. We studied nine girls with CDKL5 deficiency disorder who had frequent, hard-to-treat seizures. They received cannabidiol for up to 1 year, added to their usual medicines. Most children had fewer seizures in the first months of treatment. Some families also noticed better alertness, eye contact, movement, sleep, or constipation. Side effects were usually mild and manageable. Although seizure frequency often returned to baseline by the end of the study, most families chose to continue cannabidiol. Because this was a small study without a placebo group, these results are preliminary, and larger controlled trials are needed.

ABSTRACT Cardiomyopathy and stroke are major complications of Chagas disease (CD). We investigated if hypercoagulability, endothelial cell (EC) dysfunction, and blood stasis cause cardiac-cerebral fibrinogenesis in CD. C57BL/6 mice infected with Trypanosoma cruzi (Tc) were evaluated at acute (AT), indeterminate (IT) and chronic (CT) stages of CD. Mice were given anti-parasite, bicistronic immunogens (BCV or BCVR) to determine the role of Tc in fibrinogenesis. We monitored (1) platelet and coagulation cascade activation; (2) cardiac-cerebral blood vessels injury; (3) proinflammatory/prothrombotic phenotypes of microglia, macrophages, and vascular cells; and (4) fibrinogenesis. After quiescent acute phase, plasma levels of coagulation and other factors of platelet activation/aggregation, hypercoagulability, and clotting capacity were increased in IT-CT mice. Proinflammatory and prothrombotic cytokines/chemokines in cardiac-cerebral blood vessels were also significantly increased in IT-CT mice. Loss of cardiac EC and a marked increase in their proinflammatory/cell adhesion response was not compensated by pro-angiogenic/wound-healing response in cardiac-cerebral tissues of infected mice. Microglial proinflammatory activation preceded the vascular inflammation suggesting a feedback cycle of EC activation in brain of infected mice. Treatment with BCV/BCVR controlled the parasite persistence and hypercoagulability/vascular inflammation in infected mice. Importantly, BCV/BCVR treatment abolished the fibrin clots that otherwise were pronounced in heart and brain of IT-CD mice. We conclude that acute Tc infection triggered a subdued hemodynamic disorder, and persistence of low-grade parasites contributed to cardiovascular/cerebrovascular proinflammatory and prothrombotic response and fibrin deposition in mice. BCV/BCVR offer potential immunotherapies for reducing the recurrent clot formation and risk of stroke in CD. Commonly used abbreviations AT, IT, and CT: Acute, intermediate, and chronic stages of T. cruzi infection, respectively; APTT: Activated partial thromboplastin time; BCV: Bicistronic immunogen; BCVR: BCV adjuvanted with RIG-I agonist; CD: Chagas disease; EC: Endothelial cells; INR: International normalized ratio; LV: Left ventricle; PT: Prothrombin time; SMC: Smooth muscle cell; TF: Tissue factor; Tc : Trypanosoma cruzi; uPA: Urokinase plasminogen activator; VCAM1: Vascular Cell adhesion molecule 1; VEGF: Vascular endothelial growth factor.

Current pretransplant clinical scoring systems fail to accurately estimate post-liver transplant (LT) survival, making recipient selection challenging. Persistent immune dysfunction contributes to early LT recipient mortality but is not captured by existing models. To identify plasma biomarkers of pretransplant immune dysfunction and to develop a biomarker-based pre-LT risk stratification tool to predict post-LT mortality. Prospective biomarker analysis of consecutively enrolled adult LT recipients was conducted. Healthy controls were included for baseline comparison. Patients undergoing LT at Houston Methodist Hospital (October 1, 2013, to December 31, 2017) and Rutgers/University Hospital (January 1, 2019, to March 31, 2021) were enrolled under an institutional review board-approved protocol, and data were censored on March 31, 2023, and analyzed. Patients with cirrhosis older than 18 years undergoing deceased donor LT were included. Exclusion criteria were age greater than 70 years, cancer other than hepatocellular carcinoma, retransplant, status 1A, intraoperative mortality, multivisceral transplant (except liver-kidney), and sample availability. Plasma cytokine, chemokine, and immune exhaustion biomarkers were quantified using multiplex Luminex assays at the time of transplant. Clinical, demographic, and laboratory data were extracted from institutional databases. The primary outcome was all-cause mortality within 1 year of LT. Secondary outcomes included graft survival, infections, rejection, readmissions, and 24-month survival. A total of 779 adult LTs were performed between 2007 and 2017, with prospective biomarker analysis of 279 consecutively enrolled LT recipients between 2018 and 2022. Median (IQR) participant age was 56.7 (48.2-62.5) years, and 110 of 279 patients (39.4%) were female. Pre-LT plasma levels of B-cell activating factor, C-C motif chemokine ligand 1, eotaxin, fractalkine, interleukin 1β (IL-1β), sIL-6Rβ, metalloproteinase (MMP) 2, and MMP3 were significantly associated with 1-year post-LT mortality. Multivariable Cox proportional hazards modeling identified fractalkine and MMP3 as independent predictors of early post-LT mortality. These were used to develop the Liver Immune Frailty Index (LIFI), stratifying patients into low, moderate, and high risk. One-year mortality was 1.9%, 10.3%, and 63.6% for LIFI-low, -moderate, and -high, respectively. Relative risk of death within 1 year was 5.43 (95% CI, 1.59-18.60; P < .001) for LIFI-moderate and 33.41 (95% CI, 11.48-97.25; P < .001) for LIFI-high compared to LIFI-low. LIFI demonstrated strong discrimination (C statistic, 0.83) and was associated with post-LT infections, longer hospital stays, and reduced patient survival. Per the results of this diagnostic/prognostic study, LIFI identifies LT candidates with severe immune dysfunction at high risk for early post-LT mortality, offering a preoperative, objective tool to refine transplant candidacy, guide perioperative management, and improve LT outcomes.

Introduction The complement system plays a vital role in the immune response against tuberculosis (TB), aiding in the recognition and clearance of Mycobacterium tuberculosis. However, its imbalance can result in either insufficient immune activation or excessive inflammation, both of which may contribute to poor treatment outcomes. Methods This study investigates whether baseline complement profiles are associated with unfavorable treatment responses in pulmonary TB patients. Using the Magpix multiplex cytokine assay, plasma levels of complement components (C2, C3, C3b/iC3b, C4, C4b, C5, C5a, C1q, MBL) and regulatory proteins (Factor B, Factor D, Factor H, Factor I) were measured in TB patients with poor treatment outcomes (n=68) and disease-free controls (n=108). Results At both baseline (pre-treatment) and month two of anti-TB therapy, cases had significantly elevated levels of C3, C3b, C4b, C5, C5a, and C1q, and reduced levels of Factor B and Factor H compared to controls. Regression modelling revealed that C3, C3b, C4b, C5, C5a and C1q were associated with increased risk of unfavorable outcomes in unadjusted and adjusted analyses in the study cohort. Discussion These findings suggest that early and sustained complement activation, particularly through the classical pathway, is associated with adverse outcomes in TB. Complement dysregulation may thus serve as a potential prognostic marker for identifying individuals at risk of poor treatment response.

The renin-angiotensin system (RAS) plays a critical role in vascular homeostasis and inflammation, and its dysregulation has been implicated in the pathophysiology of COVID-19. We investigated the dynamics of RAS peptides and markers of endothelial dysfunction in relation to respiratory disease progression in hospitalized COVID-19 patients. In this single-centre prospective observational study, 155 adult patients with confirmed SARS-CoV-2 infection were enrolled at hospital admission. Plasma levels of renin, angiotensin I (Ang I), angiotensin II (Ang II), angiotensin 1-7 (Ang 1-7), the Ang II/Ang I ratio (as a surrogate of ACE activity), and asymmetric dimethylarginine (ADMA)-a marker of endothelial dysfunction -were measured at baseline (D0) and day 3 (D3). Endothelial injury was further assessed using the Endothelial Activation and Stress Index (EASIX). Patients were stratified by respiratory trajectory using the WHO ordinal scale. Biomarker kinetics were analysed with baseline-adjusted regression models, and 28-day clinical status was evaluated using partial proportional-odds regression. Of 155 patients, 89 (57%) experienced worsening respiratory status. These patients exhibited progressive RAS activation with higher renin and Ang I at D3 (p < 0.001), a decline in the Ang II/Ang I ratio (p < 0.001), and a rise in Ang 1-7 (p < 0.001). ADMA and EASIX levels increased in parallel, with significantly higher ADMA in worsening vs. non-worsening patients at D3 (0.72 [0.62-0.87] vs. 0.61 [0.50-0.70] µM/L; p < 0.001). Biomarker trajectories differed according to disease course, with significant interaction terms between baseline values and respiratory deterioration. At 28days, outcomes were associated with renin, Ang I, Ang 1-7, and the Ang II/Ang I ratio, but not with Ang II. Elevated baseline ADMA also independently predicted worse prognosis. Worsening respiratory status in COVID-19 is associated with delayed activation of the RAS, a shift toward the alternative Ang 1-7 pathway, and parallel increases in endothelial dysfunction markers. These findings suggest that serial measurements of RAS peptides and ADMA may aid in identifying high-risk phenotypes and inform personalized therapeutic strategies in COVID-19.

Background The advantages of lithium in treating bipolar disorder (BD) are inconsistent with a declining trend in lithium prescriptions worldwide. Understanding lithium prescription patterns and serum concentrations could improve lithium clinical practices. Methods This multicentre study used latent variable analysis to explore lithium prescription patterns and changing trends in lithium serum concentrations. A regression model was used to identify their underlying associated factors. Results High- and low-dose prescription patterns were discovered in patients with mania and bipolar depression (BD-D), respectively. Patients with BD-D tended to receive lower lithium dosages. Lithium combination therapy was mainstream for BD. Factors associated with prescription patterns differed between patients with mania and BD-D. The lithium concentration-to-dose (C/D) ratio initially decreased but then increased. The final lithium serum concentration was mainly associated with dose titration. Conclusions In this study, lower lithium dosage combined with second-generation antipsychotics is commonly used in the treatment of BD, and lithium prescription patterns fail to follow the guideline recommendations for BD. There are episode-specific factors associated with lithium prescriptions. A non-linear trend in the C/D ratio appears to be one of the factors contributing to lithium delay effects. Adjusting the lithium dosage is a direct way to change its serum concentration. Key Points Lower lithium dosage combined with SGAs is commonly used in the treatment of BD, and lithium prescription patterns fail to follow the guideline recommendations for BD. Factors associated with lithium prescription patterns differed between patients with mania and BD-D. In the context of a standardised lithium dosage (1 g), lithium plasma levels initially decrease before gradually increasing, which appears to be one of the factors contributing to lithium delay effects.

Altered lipid metabolism has been implicated in neurodegenerative disorders; however, its relevance to dementia with Lewy bodies (DLB) remains incompletely characterized. This study examined associations between plasma lipid profiles, lipid-related inflammatory indices, and clinical and neurodegenerative features in drug-naive patients with DLB, with comparisons to age-matched healthy controls. Plasma levels of high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol did not differ significantly between patients with DLB and controls, whereas triglyceride (TG) levels were significantly lower in DLB (p = 0.002). Within the DLB cohort, higher HDL-C levels were associated with less severe neurodegenerative changes, including milder nigrostriatal degeneration (β = 0.448, p = 0.005), lower motor symptom severity (β = -0.324, p = 0.047), and a reduced prevalence of visual hallucinations (OR = 0.924, p = 0.006). In contrast, higher TG levels and an increased TG-to-HDL-C ratio were associated with more pronounced neurodegenerative involvement (TG: β = -0.358, p = 0.021; TG-to-HDL-C ratio: β = -0.426, p = 0.006). Greater neurodegenerative severity was also associated with lipid-related inflammatory markers, including the neutrophil-to-HDL-C ratio (β = -0.491, p = 0.001) and the platelet-to-HDL-C ratio (β = -0.301, p = 0.040), suggesting that inflammation may act as a contributory or mediating process. Collectively, these findings indicate that plasma lipid profiles-particularly HDL-C-are associated with neurodegeneration and clinical manifestations in DLB, extending beyond isolated dopaminergic involvement. An HDL-C-related neurodegenerative framework, with inflammation as a modifying process, may better reflect the underlying pathophysiology of DLB and warrants further longitudinal investigation.

During the perioperative period, increased vascular permeability is associated with elevated morbidity and mortality. Obstructive jaundice (OJ) has been demonstrated to increase vascular permeability. The endothelial glycocalyx (EG), comprising hyaluronic acid (HA), chondroitin sulfate (CS), and syndecan-1 (SDC1), is essential for vascular integrity. However, its role in OJ-related vascular dysfunction remains unclear. This study aimed to investigate whether EG degradation contributes to vascular hyperpermeability in OJ patients. Plasma was preoperatively collected from 35 OJ patients and 33 non-jaundiced controls. EG components (HA, CS, SDC1) and vascular permeability-related mediators, including vascular endothelial growth factor (VEGF), thrombin, sphingosine-1-phosphate, atrial natriuretic peptide, cyclic adenosine monophosphate, and nitric oxide (NO) were quantified. Transendothelial electrical resistance was used to assess endothelial permeability. OJ patients showed significantly increased vascular permeability, which correlated with elevated levels of HA, CS, and SDC1. Concurrently, plasma levels of VEGF, thrombin, atrial natriuretic peptide, and NO were elevated, while the endothelial-stabilizing mediator cyclic adenosine monophosphate was reduced, indicating synergistic EG breakdown and dysregulation of permeability pathways. This study confirms that EG degradation is associated with OJ-induced vascular hyperpermeability. Targeting EG integrity may offer a therapeutic strategy to improve perioperative outcomes, though further studies are needed to establish causality.

Background. The purpose was to determine prognostic factors of improving left ventricular diastolic function (LV DF) in resistant hypertension patients who received multicomponent antihypertensive therapy for three years. Materials and methods. One hundred and two patients with true resistant hypertension were included. They received triple fixed combination (renin-angiotensin-aldosterone system blocker/calcium antagonist/diuretic) to which the fourth drug (spironolactone, eplerenone, moxonidine, torasemide or nebivolol) has been added. The state of LV DF was evaluated at baseline and by the end of the study. Office and 24-h ambulatory blood pressure (BP) measurements, echocardiography, clinical characteristics, neurohumoral and proinflammatory status were assessed. Results. Impaired LV DF was detected in 75.5 % of patients. The first degree of LV diastolic dysfunction was observed more often, in 63.7 % of cases. The patients were divided into 2 groups: the first one included people without initial impairment of LV DF (n = 25), the second one consisted of those with LV diastolic dysfunction (n = 77). The latter were older, had a longer duration of hypertension, higher body mass index, 24-h urinary albumin excretion, office and 24-h ambulatory BP, they also more often (by 2 times) had disorders of circadian BP rhythm and concomitant diabetes. Left ventricular diastolic dysfunction in 100 % of cases was associated with severe LV hypertrophy, increased plasma concentration of inflammatory proteins (C-reactive protein, fibrinogen), cytokines (interleukin-6, tumor necrosis factor ), increased activity of leukocyte elastase, macrophage matrix metalloproteinase-12. The concentration in the blood of aldosterone, active renin, 24-h urinary excretion of metanephrines did not differ between the groups. Conclusions. Improvement and stabilization of LV DF occurred in parallel with regression of LV hypertrophy (normalization of LV mass index in 35.1 % of patients and its significant decrease in 64.9 %) against the background of a decrease in BP and in the proportion of patients with impaired circadian BP rhythm. The independent factors of the E/E' ratio were baseline plasma levels of aldosterone ( = 0.556, P = 0.0001), glucose ( = 0.366, P = 0.0001), active renin ( = –0.223, P = 0.004), 24-h urinary albumin excretion ( = 0.188, P = 0.016), age of patients ( = 0.192, P = 0.023). The odds of an improvement in LV DF increased by 3.7 times, if patients with resistant hypertension had no diabetes; LV hypertrophy regression occurred.

Device-based therapies may augment decongestion in acute decompensated heart failure. We investigated the efficacy and safety of prolonged mechanical superior vena cava occlusion using the preCARDIA system in a swine model of heart failure. Over 6h of preCARDIA activation, right- and left-sided filling pressures were significantly reduced. Plasma levels of the brain injury marker ubiquitin C-terminal hydrolase L1 and intracerebral arterial pressure were not significantly changed. Post-mortem evaluation did not identify gross cerebral or histologic injury. Future studies are needed to compare continuous versus cyclic preCARDIA activation and to further confirm the neurologic safety of sustained superior vena cava occlusion.

Patients with aneurysmal subarachnoid hemorrhage (aSAH) are at high risk of secondary ischemia, and timely insight into cerebral metabolism may improve clinical management. Cerebral microdialysis offers continuous metabolic monitoring but is invasive, focal, and confined to specialized centers. Peripheral blood and cerebrospinal fluid (CSF) could provide less invasive, more accessible surrogates for assessing cerebral metabolic status. This study aimed to evaluate glucose and lactate dynamics across blood, CSF, and brain parenchyma in patients with severe aSAH. A total of 39 patients with aSAH undergoing multimodal neuromonitoring were retrospectively analyzed. Glucose and lactate levels from plasma, CSF, and cerebral microdialysis were matched within 90-min intervals relative to each microdialysis measurement. Associations were analyzed using linear mixed-effects models. Plasma (p < 0.001) and CSF (p < 0.001) glucose levels were significantly associated with cerebral glucose concentrations. Moderate correlations were observed between plasma-brain (r = 0.54) and plasma-CSF (r = 0.55), while CSF-brain correlation was weaker (r = 0.36). For lactate, significant associations were found between CSF and brain (p = 0.04; r = 0.24) and between plasma and CSF (p < 0.001; r = 0.33), but plasma-brain lactate showed no significant relationship. The plasma-brain glucose association weakened slightly over time (p = 0.008) and strengthened during episodes of low brain tissue oxygenation (pbtO2 < 15mm Hg; p = 0.01). Insulin had no effect on glucose relationships but significantly attenuated the plasma-brain lactate association (p < 0.001). The presence of metabolic crisis (lactate/pyruvate ratio > 40) strengthened the CSF-brain lactate association (p = 0.04). CSF cell count had no significant effect. In severe aSAH, glucose and lactate levels in blood and CSF reflect cerebral values in a compartment- and metabolite-specific manner, but with high variability influenced by clinical conditions. These findings suggest that blood and CSF provide only limited information about cerebral metabolism, highlighting the complementary value of cerebral microdialysis for individualized, brain-targeted monitoring.

Multiple sclerosis (MS) shows a highly heterogeneous course, with some patients accumulating severe disability early while others remain relatively preserved even after decades. A key driver of disability progression is smoldering inflammation, a chronic, compartmentalized immune process at the edge of chronic active lesions. However, the factors driving smoldering inflammation in MS remain incompletely understood. We investigated the role of genetic variation in smoldering inflammation-related genes across two independent MS cohorts, using a discovery-replication design in a total of 2,817 patients. We identified a locus in the HIF1A (Hypoxia-Inducible Factor 1-alpha) gene that is associated with a more favorable disease course at over 20years from disease onset. Using additional independent cohorts, we found that carriers of the HIF1A protective allele exhibited lower paramagnetic rim lesion volume on MRI, lower plasma and cerebrospinal fluid neurofilament levels, and reduced microglial/macrophage inflammation with less axonal injury in post-mortem progressive MS tissue. By integrating single-nucleus RNA sequencing and spatial transcriptomics, we showed that the HIF1A variant dynamically modulates gene expression in a cell-type specific and context-dependent manner in the MS brain. Collectively, these findings highlight a protective HIF1A variant associated with a more favourable long-term disease course and reduced smoldering inflammation, opening new avenues to translate this genetic discovery into new potential strategies to tackle disease progression.

Background/Objectives: After fermentation, yogurt is often supplemented with probiotics, yet sweetened with added sugars that can negatively impact cardiometabolic health. Honey provides rare sugars, oligosaccharides and phenolics that may promote gut and cardiometabolic health. We aimed to determine the impact of yogurt sweetened with commercial clover blossom honey on pro-inflammatory Th17 cytokines and microbial-derived metabolites in healthy postmenopausal women. Methods: In a randomized controlled crossover dietary intervention trial, postmenopausal women (45–65 years of age) consumed two 150 g servings of yogurt for breakfast for 4 weeks, with each serving sweetened with a tablespoon of clover blossom honey or an isocaloric amount of sugar. Blood samples were collected for the measurement of plasma lipids, bile acids (BA) and Th17 cytokines, along with fecal short-chain fatty acids (SCFA). The primary outcome was plasma interleukin (IL)-23. Results: Neither dietary intervention significantly changed IL-23, plasma lipids, fecal SCFA or plasma BA. Compared to sugar-sweetened yogurt, IL-33 was significantly lower after 4 weeks of honey-sweetened yogurt intake. Conclusions: In a healthy population of postmenopausal women, the daily intake for 4 weeks of honey-sweetened yogurt did not significantly impact our primary outcome of IL-23. Instead, lower plasma levels of IL-33 were observed with honey compared to sugar-sweetened yogurt intake. The impact of the intervention on this cytokine was independent of changes in fecal SCFA and plasma BA. Confirmatory studies, in a larger population with levels of honey intake within dietary recommendations for added sugar, are warranted.

Trastuzumab used for the treatment of patients with HER2-positive breast cancer induces cardiotoxicity. The NRG1/HER pathway plays a central role in human cardiovascular physiology; however, the link between exercise, NRG1, and cardiotoxicity is unclear. Patients were randomized to receive adjuvant trastuzumab in combination with a training program (TG) or trastuzumab alone (CG). The aim of this study was to assess the effect of a 12-week supervised exercise training on the circulating level of NRG1. Secondary objectives were to assess the correlation between NRG1 level and cardiotoxicity. 89 patients were randomized (TG; n = 46; CG; n = 43), 76 have a NRG1 concentration available at baseline. After the exercise program, plasma levels of NRG1 decreased significantly in the TG (mean difference -0.20ng/ml; 95% CI, -0.32, - 0.07) whereas they remained stable in the CG (mean difference - 0.05ng/ml; 95% CI, - 0.20, 0.10). Notably, baseline NRG1 concentrations were higher in the TG group. However, no correlation between NRG1 changes and either cardiorespiratory fitness (V̇O2 max) and left ventricular ejection fraction (LVEF) was observed (R = 0.087, p = 0.53; R = -0.157, p = 0.26; and R = -0.131, p = 0.33, respectively). A 12-week interval training program significantly decreased NRG1 concentration in HER2-positive patients with breast cancer treated with adjuvant trastuzumab therapy, despite the trained group presenting higher baseline NRG1 values compared to the control group. In addition, this change was neither associated with V̇O2 max nor with LVEF. This trial was registered with ClinicalTrials.gov under the number NCT02433067.

The roles of nesfatin-1 in the lateral parabrachial nucleus on feeding and glucose metabolism in type 1 diabetic rats.

Two simple, rapid, cost-effective, and environmentally friendly chromatographic methods were developed and validated for the simultaneous determination of metformin (MEF), linagliptin (LIN), and empagliflozin (EMP) in human plasma, with successful application to pharmacokinetic study. Plasma sample preparation was performed using a straightforward protein precipitation technique employing acetonitrile: methanol: trichloroacetic acid (50:49:1, by volume), which provided high extraction recovery and minimal matrix interference. The first method was based on high-performance liquid chromatography with diode array detection (HPLC-DAD) using an ODS Hypersil C18 column and isocratic elution with a mobile phase consisting of acetonitrile, methanol, and phosphate buffer (pH 3) in a ratio of (40:40:20, by volume), at a flow rate of 1.3 mL/min, with detection at 230nm. The second method employed high-performance thin-layer chromatography (HPTLC) with densitometric detection at 225nm, using silica gel 60 F254 plates and n-hexane: methanol: glacial acetic acid (6:3:1, by volume) as the developing system. Excellent linearity was achieved over concentration ranges of 85-1650 ng/mL for MEF, 50-1100 ng/mL for EMP, and 45-950 ng/mL for LIN using the HPLC method, and 500-2800, 100-800, and 50-550 ng/band, respectively, using the HPTLC method, with correlation coefficients exceeding 0.998. The lower limits of quantitation for the HPLC method were 85, 50, and 45 ng/mL for MEF, EMP, and LIN, respectively. Both methods demonstrated satisfactory accuracy, precision, recovery (> 92%), stability, and negligible matrix effects in accordance with European Medicines Agency guidelines. The validated methods were successfully applied to a pharmacokinetic study in healthy volunteers, yielding mean Cmax values of 877.5 ± 162.2 ng/mL (MEF), 576 ± 87.5 ng/mL (EMP), and 680.8 ± 7.9 ng/mL (LIN), with Tmax values of 2.42 ± 0.38, 1.5 ± 0.61, and 5.3 ± 0.52h, respectively. The obtained pharmacokinetic parameters were consistent with reported literature, confirming the reliability and clinical applicability of the proposed green bioanalytical methods.

Alzheimer's disease (AD) develops gradually, with significant neurodegeneration already present by the time clinical symptoms emerge. Since synapses are affected early in the disease, synaptic proteins are being investigated as potential markers of the prodromal stage. Using data and plasma samples provided by the Consortium for the early identification of Alzheimer's disease-Quebec (CIMA-Q), we analyzed plasma levels of neuroligin (NLGN)-derived peptides in cognitively normal (CN) individuals and cognitively impaired (CI) individuals, including those with amnestic mild cognitive impairment (aMCI) and early-stage Alzheimer's disease (AD). Plasma levels of NLGN-derived peptides were assessed by quantifying tryptic peptides using liquid chromatography coupled with tandem mass spectrometry. Our findings show that levels of specific NLGN peptides were significantly elevated in CI compared to CN individuals. Receiver operating characteristic (ROC) curve analysis revealed that some NLGN peptides could distinguish CI individuals. Furthermore, analysis based on Mini-Mental State Examination (MMSE) scores revealed that specific plasma phosphorylated tau peptides were significantly and positively correlated with selected NLGN-derived peptides in more advanced stages of cognitive decline. These results support further investigation into synaptic NLGN-derived peptides in the blood as promising tools for monitoring the earliest stages of AD.

Forkhead box protein A3 (FOXA3), also known as hepatocyte nuclear factor 3g (HNF3g), is a member of the FOX family of transcription factors and regulates lipid and glucose metabolism and liver regeneration. Hepatic FOXA3 is reduced in obesity and patients with metabolic dysfunction-associated steatohepatitis (MASH). So far, it remains unknown whether hepatic FOXA3 is essential for regulating lipid metabolism or metabolic dysfunction-associated liver disease (MASLD). In this study, we first investigated whether genetic inactivation of hepatocyte Foxa3 affected the development of MASLD/MASH in C57BL/6 mice and then explored whether loss of hepatocyte Foxa3 regulated atherosclerosis development in Ldlr-deficient mice. Inactivation of Foxa3 in hepatocytes did not affect the development of Western diet-induced MASLD/MASH in C57BL/6 mice but attenuated MASH development in Western diet-fed Ldlr-deficient mice. Moreover, genetic loss of hepatocyte Foxa3 ameliorated hyperlipidemia and atherosclerosis in Ldlr-deficient mice. In Ldlr-deficient mice, loss of hepatocyte Foxa3 resulted in reduced expression of lipogenic, pro-inflammatory, or fibrogenic genes in the liver and reduced cholic acid levels in plasma and bile. Thus, hepatocyte FOXA3 loss confers protection against the development of MASH and atherosclerosis in hyperlipidemic Ldlr-deficient mice.

Objective Autoimmune thyroiditis (AIT), a prevalent autoimmune disorder that frequently leads to hypothyroidism. A critical unmet need exists for disease-modifying therapies that target its underlying pathogenesis. This study aimed to identify and validate novel therapeutic targets for AIT. Methods We employed an integrative genomics approach, combining genome-wide association studies (GWAS) with molecular quantitative trait loci (QTL) analyses, including expression (eQTL), protein (pQTL), and DNA methylation QTL (mQTL), across two independent AIT cohorts for discovery and replication. We performed two-sample bidirectional Mendelian randomization (MR) with sensitivity analyses, followed by summary-data-based MR (SMR) and heterogeneity in dependent instruments (HEIDI) tests. Top candidates were further evaluated via phenome-wide association study (PheWAS) and computational drug screening. Guided by these findings, we quantified plasma levels of the top-priority candidate, Ribonuclease T2 (RNASET2), via ELISA in AIT patients and non-AIT controls. To functionally validate its therapeutic potential, we developed a novel three-dimension (3D) inflammatory thyrocyte spheroid model and evaluated potential therapeutic effects of recombinant RNASET2. Loss-of-function (small interfering RNA-mediated knockdown) and gain-of-function (recombinant protein RNASET2 rescue) experiments further supported RNASET2 as a therapeutic target. Results Multi-omics integration consistently nominated RNASET2 as a causal protective factor against AIT. Signals of pQTL and eQTL for RNASET2 were associated with decreased AIT risk, while three mQTLs were correlated with increased risk. PheWAS indicated minimal pleiotropic effects, supporting its therapeutic suitability. Computational drug screening nominated genistein, a soy isoflavone known to upregulate RNASET2 expression, as a repurposing candidate. Empirically, plasma RNASET2 levels were moderately elevated in AIT patients, potentially reflecting a compensatory anti-inflammatory response. Crucially, recombinant RNASET2 effectively mitigated inflammation and apoptosis in the thyrocyte spheroid model, confirming its functional protective role. Consistently, RNASET2 knockdown heightened susceptibility to inflammatory cell death and cytokine expression, a phenotype reversed by recombinant RNASET2 supplementation. Conclusions By integrating large-scale genomic analyses with functional validation, our study establishes RNASET2 as a promising therapeutic target for AIT. RNASET2 augmentation represents a potential disease-modifying strategy, providing a translational bridge from genetic discovery to clinical application.

To determine how certolizumab pegol (CZP) dose and dose adjustments influence CZP plasma trough levels to facilitate therapeutic drug monitoring of CZP. The effect of CZP dose and dose adjustments on CZP plasma trough levels was evaluated post hoc using longitudinal data from a 52-week randomized phase III trial (RAPID 1) and its open-label extension trial. Patients with active rheumatoid arthritis treated with methotrexate for ≥6 months were randomized to CZP 200 mg, 400 mg, or placebo every other week (EOW). Patients in the extension trial were initially treated with CZP 400 mg EOW, then reduced to 200 mg EOW after ≥6 months. Of 982 randomized patients, 846 patients entered the open-label extension trial. Median (interquartile range) plasma CZP concentrations after 12 weeks of treatment were 21.3 mg/L (14.7, 27.7) in the 200-mg group and 38.3 mg/L (29.2, 63.8) in the 400-mg group and increased from 18.3 (12.4, 26.5) to 43.4 (26.8, 63.3) mg/L after dose escalation from 200 to 400 mg EOW. Following CZP dose reduction from 400 mg to 200 mg, median CZP levels decreased from 36.1 (24.9, 49.0) to 17.2 (11.5, 23.1) mg/L. CZP plasma concentrations were influenced by both dose and dose adjustment in a predictable manner, with median plasma levels twice as high in the 400-mg group than in the 200-mg group, with a 2-fold increase after the dose increase from 200 to 400 mg. This facilitates the development of algorithms for therapeutic drug monitoring of CZP.