Plasma Levels Research Articles

B-038 A Novel Ultrasensitive Single-Molecule Counting Technology for Quantitation of Low Abundance Biomarkers

Abstract Background There is a need for sensitive, robust, and scalable analytical methods for accurate, early detection of disease using less invasive sampling methods. Often, smaller volumes and low marker concentrations present challenges to achieving sufficient sensitivity and accuracy with traditional immunoassay methods. This is particularly the case for measuring markers for Alzheimer's and other neurological diseases in blood, where levels are typically at much lower concentrations than in CSF. Here we introduce a novel ultrasensitive technology for flow-based single molecule detection in an optofluidic chip. We have developed an Ultrasensitive Detection Module (UDM) device and optofluidic cartridge that enable easy integration of this technology into existing immunoassay systems. UDM sensitivity data is shown for three prototype biomarker assays in comparison to the Lumipulse G1200 (Fujirebio Inc., Japan). We also present analytical validation data for three Alzheimer’s Disease plasma biomarker assays developed on a fully automated benchtop analyzer with integrated UDM detector. Methods Ultrasensitive detection is achieved by integrated low-loss waveguide in an optofluidic chip that enables maximal excitation of fluorescent molecules traveling through a narrow fluidic channel. The resulting high-intensity emission is detected as a single digital event, enabling individual molecules to be counted without need for amplification. Fluorescence-based immunoassays were developed for detection with the UDM using high performance monoclonal antibodies and reagents (Fujirebio Inc., Japan). Capture antibodies were immobilized on magnetic particles and detection antibodies conjugated to a fluorescent reporter construct (“reporter-Ab”). Multiple incubation and wash steps were followed by dissociation of immune complexes and separation of reporter-Ab from magnetic particles. Released reporter-Ab molecules were injected into the UDM device for digital detection. Signal to noise performance was evaluated in the UDM using reporter construct alone (no assay), as well as for prototype PSA, IL-6, and TSH assays, to establish baseline performance for analytical sensitivity. Plasma assays for pTau181, Aβ1-40, and Aβ1-42 were developed and optimized for the automated benchtop analyzer and validated for analytical sensitivity (LOD/LOQ), linearity, parallelism, precision and repeatability using clinical samples. Results Detection signal-to-noise testing of the reporter construct alone (no immunoassay) demonstrated up to 1000-fold higher sensitivity than the Lumipulse G1200. Prototype assays for PSA, TSH, and IL-6 showed up to 118-fold, 36-fold, and 80-fold higher signal to noise ratio, respectively, when compared to the Lumipulse G1200. Plasma assays for pTau181, Aβ1-40, and Aβ1-42 showed preliminary LLOQs calculated at 0.03 pg/mL, 0.17 pg/mL and 0.37 pg/mL, respectively. Conclusions We have developed a new single molecule counting platform able to detect neurological biomarkers at sub-pg/ml levels in plasma, with the potential to provide sensitive and accurate diagnostic testing of low abundance biomarkers in blood. Such technologies can create new clinical value through higher detection sensitivity and accuracy in areas like Alzheimer’s and other neurological diseases, supporting clinical research and translation to clinical practice.

B-302 Therapeutic Drug Monitoring (TDM) of Hydroxychloroquine in Whole Blood: Analysis of Over 10,000 Patient Results

Abstract Background Hydroxychloroquine (HCQ), a mainstay of systemic lupus erythematosus (SLE) therapy, improves survival and reduces flares. Therapeutic drug monitoring (TDM) may be useful in 1) identifying and improving adherence issues, 2) titrating daily dose to achieve therapeutic benefits while 3) minimizing retinopathy due to longterm cumulative exposure. Whole blood (WB) is the correct specimen for HCQ TDM because WB HCQ levels are higher than serum or plasma levels, more stable and reflective of the last month of HCQ ingestion, and are better correlated to efficacy and retinopathy risk. Here, we report WB HCQ concentrations in 10,463 clinical patient samples and their distribution. Methods HCQ concentrations in whole blood are measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) and reported in nanograms per milliliters whole blood. Recommended sample collection is ∼ 3 - 6 months into regular stable dosing to reach pharmacokinetic steady state and correlate with clinical efficacy. Results Here, we report the analysis of 10,463 patient samples for whole blood hydroxychloroquine (WB HCQ) concentrations. More than one-third (3752, 35.9%) were within the target range of > 1000 and < 2000 ng/mL. About one-quarter (2568, 24.5%) were within a tighter therapeutic target window, 1000 to 1500 ng/mL. The vast majority (9852, 94.2%,) of all WB HCQ were < 2000 ng/mL with a small percentage >2000 ng/mL (611, 5.8% between 2000 - 9502 ng/mL). About one-third (31.3%) were higher than 1177 ng/mL which corresponds to higher retinopathy risk. Adherence issues were evident in 10.5% (1102) with undetectable (483, 4.6%) and very low (<200 ng/mL) drug (619, 5.9%) (yellow). Depending upon the target threshold, levels < 500 ng/mL or <1000 ng/mL may be considered subtherapeutic as seen in 2498 (23.9%) and 4263 (40.7%) samples, respectively. Conclusions Due to wide pharmacokinetic variability and adherence issues, a given prescribed dose of HCQ does not correspond to WB HCQ, e.g. following 4, 5 or 6 mg/kg/day, levels are known to vary widely from subtherapeutic <500 to >2000 ng/mL. Here, our clinical database reiterates this wide range in WB HCQ levels from undetectable (<25) to 9502 ng/mL in 10,463 patient samples. One-quarter or one-third were within the therapeutic target range of 1000-1500 or 1000-200 ng/mL where >1000 has been associated with better disease control and survival. At the same time, levels less than 1177 avoid higher retinopathy risk. A small percentage of samples, 5.8%, were higher than 2000 ng/mL, suggesting the usefulness in TDM to fine-tune daily dose. In 10%, there was evidence of poor adherence, with very low or no detectable drug, and one quarter to 40% were subtherapeutic (<500 or <1000). The potential benefits of HCQ are many and include reduced flares and improved lupus nephritis, thrombosis risk, and long-term survival. At the same time, prescribed dose does not predict therapeutic levels. TDM-based counseling and dose adjustment may be instrumental in optimizing HCQ benefits while minimizing retinopathy risk. In conclusion, our analysis demonstrates the growing use of whole blood HCQ TDM to facilitate dose titration to safe and most effective target levels.

Mechanism of action and impact of thiol homeostasis on efficacy of an enzyme replacement therapy for classical homocystinuria

Homocystinuria (HCU) due to cystathionine beta-synthase (CBS) deficiency is characterized by elevated plasma and tissue homocysteine levels. There is no cure, but HCU is typically managed by methionine/protein restriction and vitamin B6 supplementation. Enzyme replacement therapy (ERT) based on human CBS has been developed and has shown significant efficacy correcting HCU phenotype in several mouse models by bringing plasma total homocysteine below the clinically relevant 100 μM threshold. As the reactive nature of homocysteine promotes disulfide formation and protein binding, and ERT is unable to normalize plasma total homocysteine levels, the mechanism of action of ERT in HCU remains to be further characterized. Here we showed that only a reduced homocysteine serves as a substrate for CBS and its availability restricts the homocysteine-degrading capacity of CBS. We also demonstrated that cells export homocysteine in its reduced form, which is efficiently metabolized by CBS in the culture medium. Availability of serine, a CBS co-substrate, was not a limiting factor in our cell-based model. Biological reductants, such as N-acetylcysteine, MESNA or cysteamine, increased the availability of the reduced homocysteine and thus promoted its subsequent CBS-based elimination. In a transgenic I278T mouse model of HCU, administration of biological reductants significantly increased the proportion of protein-unbound homocysteine in plasma, which improved the efficacy of the co-administered CBS-based ERT, as evidenced by significantly lower plasma total homocysteine levels. These results clarify the mechanism of action of CBS-based ERT and unveil novel pharmacological approaches to further increase its efficacy.

B-283 Biological Consequences of Nitrous Oxide Abuse: Insights from Targeted Metabolomics Investigation

Abstract Background The recreational use of nitrous oxide (N2O), commonly known as laughing gas, has experienced a notable surge in popularity in recent times. According to the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA), N2O is recognized as one of the most widely used psychoactive substances in Europe as of 2022. Its use also extends to Asia and America, according to literature. However, prolonged consumption of N2O has been associated with neurological complications linked to the functional inactivation of vitamin B12, disrupting One Carbon Metabolism. This study aims to explore the metabolic effects of N2O intoxication through a targeted metabolomics approach. Methods We conducted a prospective collection of clinical and biological information from 37 N2O users and 37 non-users (control group). Utilizing a targeted metabolomics method, we analyzed plasma amino acids using liquid chromatography coupled with mass spectrometry. Our statistical analysis encompassed principal component analyses, random forests, Mann-Whitney tests, Student's t-tests, and Spearman rank tests. Results Metabolomic analyses have demonstrated effective differentiation between patient groups. In comparison to controls, N2O consumers displayed reduced plasma levels of taurine, cystine, and methionine, alongside elevated plasma levels of serine and sarcosine. These findings suggest a potential influence of N2O on the cystine-methionine metabolic pathway. The decrease in methionine levels may be attributed to the decreased activity of methionine synthase (MS) due to vitamin B12 inactivation, which could partially explain the observed increase in serine levels. The rise in sarcosine levels could be explained by an increase in betaine-homocysteine S-methyltransferase (BHMT) activity. Moreover, dysfunction in cystathionine-β-synthase (CBS) or cystathionine γ-lyase (γ-CYS) may lead to the accumulation of upstream metabolites (such as serine) and a reduction in downstream metabolites (such as cysteine and taurine). Conclusions This study sheds light on potential new impacts of N2O, particularly on the plasma levels of serine, cystine, and taurine, indicating a potential modulation of enzymes involved in the conversion of homocysteine to taurine (CBS and/or γ-CYS). Additional investigations are required in a larger group of patients, which corresponds to the aims of the EFLM Task & Finish Group: Biomarkers of Diagnosis and Follow-up of Nitrous Oxide Abuse: https://www.eflm.eu/site/who-we-are/committee/science-committee/fu/tfg-biomarkers-of-diagnosis

Pharmacokinetic interaction of quetiapine and lamotrigine – victim and perpetrator?

ABSTRACT Objective We aimed to investigate the ambiguous findings of earlier research regarding the reduction of quetiapine plasma levels when combined with lamotrigine, most likely via UDP-glucuronosyltransferase induction by lamotrigine. Methods One thousand one hundred and fifty samples, divided into four groups of patients receiving either quetiapine immediate- (IR) or extended-release (XR) without or in combination with lamotrigine were compared regarding absolute and dose-adjusted plasma concentrations. Furthermore, samples of intra-individual controls were analyzed. Results Patients receiving quetiapine IR in combination with lamotrigine showed 31% lower plasma (p = 0.002) and 23% lower dose-adjusted plasma concentrations (p = 0.004) compared to those receiving IR monotherapy. The proportion of patients with quetiapine plasma concentrations below the lower limit of the therapeutic reference range was 50% and 30% in the combination group and in patients receiving monotherapy, respectively (p = 0.03). However, no significant differences regarding plasma concentration (p = 0.13) and dose-adjusted plasma concentration (p = 0.42) were observed in patients with combination vs. monotherapy with the XR formulation of quetiapine. In the intra-individual controls, no trends could be identified, possibly due to insufficient number of samples (p > 0.05). Conclusions The combination of quetiapine IR with lamotrigine is associated with significantly lower drug concentrations of quetiapine, potentially impacting quetiapine effectiveness. For quetiapine ER, a significant interaction is less likely.

Transpulmonary Plasma Endothelin-1 Arterial:Venous Ratio Differentiates Survivors from Non-Survivors in Critically Ill Patients with COVID-19-Induced Acute Respiratory Distress Syndrome.

Endothelin-1 (ET-1) is a potent vasoconstrictor produced by endothelial cells and cleared from circulating blood mainly in the pulmonary vasculature. In a healthy pulmonary circulation, the rate of local production of ET-1 is less than its rate of clearance. In the present study, we aimed to investigate whether the abnormal pulmonary circulatory handling of ET-1 relates to poor clinical outcomes in patients with coronavirus disease 2019 (COVID-19)-induced acute respiratory distress syndrome (ARDS). To this end, central venous and systemic arterial ET-1 plasma levels were simultaneously measured on Days 1 and 3 following ICU admission in mechanically ventilated COVID-19 patients with ARDS (COVID-19 ARDS, N = 18). Central venous and systemic arterial ET-1 plasma levels were also measured in two distinct SARS-CoV-2-negative mechanically ventilated critically ill patient groups, matched for age, sex, and critical illness severity, with ARDS (non-COVID-19 ARDS, N = 14) or without ARDS (non-COVID-19 non-ARDS, N = 20). Upon ICU admission, COVID-19-induced ARDS patients had higher systemic arterial and central venous ET-1 levels compared to the non-COVID-19 ARDS and non-COVID-19 non-ARDS patients (p < 0.05), yet a normal systemic arterial:central venous (A:V) ET-1 ratio [0.63 (0.49-1.02)], suggesting that pulmonary ET-1 clearance is intact in these patients. On the other hand, the non-COVID-19 ARDS patients demonstrated abnormal ET-1 handling [A:V ET-1 ratio 1.06 (0.93-1.20)], while the non-COVID-19 non-ARDS group showed normal ET-1 handling [0.79 (0.52-1.11)]. On Day 3, the A:V ratio in all three groups was <1. When the COVID-19 ARDS patients were divided based on 28-day ICU mortality, while their systemic arterial and central venous levels did not differ, the A:V ET-1 ratio was statistically significantly higher upon ICU admission in the non-survivors [0.95 (0.78-1.34)] compared to the survivors [0.57 (0.48-0.92), p = 0.027]. Our results highlight the potential importance of ET-1 as both a biomarker and a therapeutic target in critically ill COVID-19 patients. The elevated A:V ET-1 ratio in non-survivors suggests that the early disruption of pulmonary ET-1 handling may be a key marker of poor prognosis.

Long-Term Casein-Bound Lactulosyllysine Consumption Induced Nonobese Nonalcoholic Fatty Liver Disease by Promoting Carbonyl Glycation in the Liver of C57BL/6 Mice.

Lactulosyllysine (LL) is abundant in thermally processed dairy products, with its concentration increasing in response to more intense heat treatment. However, there are limited studies on the potential harmful effects of LL on human health. This study investigated the negative impact of casein-bound LL on liver health by feeding healthy C57BL/6 mice diets containing varying levels of casein-bound LL. After 16 weeks of LL diet administration, mice exhibited a nonobese nonalcoholic fatty liver disease (NONAFLD) phenotype, characterized by reduced body weight gain, hypolipidemia, and intrahepatic lipid accumulation. Nontarget metabolomic analysis showed that casein-bound LL induced alterations in plasma levels of compounds associated with lipid degradation. Mechanistically, casein-bound LL may impair the function of 5'-adenosine monophosphate-activated protein kinase and apolipoprotein B100 by inducing dicarbonyl stress, thereby promoting carbonyl glycation in the liver. Consequently, the long-term consumption of LL-rich dairy products may be a contributing factor to the risk of developing NONAFLD.

A-001 The Postprandial Levels of the Metabolites in the Metaorganismal Trimethylamine N-Oxide (TMAO) Pathway are Altered in A Diet-, Microbe-, and Sex-Specific Manner

Abstract Background Cardiovascular disease (CVD) remains the leading cause of death in developed countries. Elevated levels of the gut-microbe-associated metabolite trimethylamine-N-oxide (TMAO) have been associated with increased risk for CVD mortality in many large independent studies. In fact, large laboratory corporations such as Labcorp and Quest Diagnostic now offer TMAO diagnostic tests for the assessment of CVD risk and as a marker for disease-associated dysbiosis, using samples obtained from fasting patients. Although the strong association between TMAO levels and CVD risk has been established in fasting blood samples, here we are investigating the potential for a single meal to dynamically alter TMAO-related metabolites based on sex, diverse dietary substrates, and microbial influences. Methods 35 healthy participants were randomized to one of four study groups with approximately 8-9 subjects in each arm. Half of the participants were randomly assigned to receive a three-day broad spectrum antibiotic regimen (ciprofloxacin, metronidazole, and vancomycin) while the others received no antibiotics. These two groups were further subdivided into a group consuming a highly processed meal (originating from local fast food restaurants) or alternatively a whole food meal (containing a variety of fruits, vegetables, and healthy fiber). Blood samples were taken at baseline (after an overnight fast), and then postprandially at 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, and 6 hours after meal ingestion. Targeted plasma metabolites were quantified using a stable isotope dilution, liquid chromatography - tandem mass spectrometry (LC-MS/MS) method Results While plasma TMAO levels between food groups did not change significantly within the total cohort, there were clear individualized responses to either highly processed or whole food meals. Separation based on sex showed that TMAO levels were significantly reduced in females at 6 hours but remained steady in males for both food groups. Particularly, in the processed food group, TMAO levels were significantly lower in females than males at the 6-hour time point. Neither of these observations held true for TMAO precursors choline, carnitine, or betaine. However, plasma levels of a dietary precursor for TMAO, γ-butyrobetaine, showed clear diet-microbe-host interactions. For males in the processed food group, plasma γ-butyrobetaine levels were significantly increased in subjects on broad spectrum antibiotics. Conclusions Our results show the postprandial levels of TMAO, and its nutrient precursors dynamically change in a diet-, microbe-, and sex-dependent manner. These findings provide new insights into the postprandial levels of TMAO-related metabolites and may inform precision nutritional approaches in those who could benefit from TMAO-lowering strategies.

Altered B-cell, plasma cell and antibody immune profiles in blood of systemic mastocytosis

Systemic mastocytosis (SM) is a heterogeneous disease characterised by an expansion of KIT-mutated constitutively activated mast cells (MC) which release MC mediators that might act on the tumour microenvironment including other immune cells. Here we investigated the blood distribution of B-cell, plasma cell (PC) and antibody-isotype compartments in SM. We used spectral flow cytometry and the EuroFlow Immunomonitoring panel and Lymphocyte Screening Tube to quantify B-cells, PC and their subsets in blood of 108 SM patients - 35 bone marrow mastocytosis (BMM), 64 indolent SM (ISM), 9 aggressive SM (ASM)- vs 117 age-matched healthy donors (HD) and paired bone marrow (BM) samples of 31 SM vs 17 controls, respectively. In parallel, immunoglobulin (Ig) M, IgD, IgG, IgA and IgE plasma levels of were measured. Compared to HD, SM patients showed an increased immature B-cell production in BM (P=0.003) associated with a greater release of pre-germinal center immature (P<0.001) and naive CD5+ B-lymphocytes (P<0.001) to blood, but a pronounced decrease in PC counts of all different IgH-isotypes and subclasses (P≤0.001) together with overall increased IgM (P=0.001) and IgD (P<0.001) plasma levels. Of note, different immune profiles were found per diagnostic subtype of the disease with progressively greater counts in blood of immature B-lymphocytes together with decreased IgMD+, IgG2+, IgA1+ and IgA2+ MBC (P≤0.032) and elevated IgM (P=0.017) plasma levels in ASM cases, increased IgM (P=0.001) and IgD (P=0.001) plasma levels in ISM patients and exacerbated IgE (P<0.001) with decreased IgG (P=0.008) plasma levels in BMM cases. Our results reveal a significant dysregulation of the B-cell and PC compartments in blood of SM patients, consistent with distinctly altered antibody-isotype profiles in plasma of BMM vs ISM vs ASM patients.

Plasma Interleukin-13 Levels Correlate With the Severity of Symptoms Induced by Functional Dyspepsia.

Functional dyspepsia (FD) is a gastrointestinal functional disorder of the upper gastrointestinal tract that affects the quality of life of patients and poses a significant economic burden. It has been proposed that the local inflammatory immune response at the duodenum is associated with an increase in intestinal permeability, favoring the recruitment of Th2 cells and granulocyte degranulation. Moreover, systemic immune response could also be related to the symptoms of FD. The objective of this study was to evaluate the systemic immune response in Uruguayan patients with FD by analyzing the cytokine levels in plasma and the frequency of circulating T cells associated with duodenal recruitment. An analytic and cross-sectional study in 30 patients with FD and 15 healthy controls (HCs) was carried out. Patients were diagnosed with FD according to the Roma IV Committee definition. Cytokine levels were measured in plasma by a specific assay. Expression of α4β7 and CC chemokine receptor9 in circulating T cells was evaluated by flow cytometry. Higher levels of interleukin (IL)-5, IL-13, and IL-8 and lower levels of IL-10 and IL-12p70 were detected in patients with FD than in HC. Furthermore, a positive linear correlation between IL-13 and the severity of FD symptoms was found. CD4 + T cells from patients with FD expressed higher levels of α4β7 and CC chemokine receptor9 than those from HC. An increase of Th2-like cytokines and a positive correlation between the levels of plasma IL-13 and the severity of symptoms in patients with FD from Uruguay were detected.

Simultaneous Determination of Methotrexate Concentrations in Human Plasma and Cerebrospinal Fluid Using Two-Dimensional Liquid Chromatography: Applications in Primary Central Nervous System Lymphoma.

In this study, we aimed to develop a method for the simultaneous quantification of methotrexate (MTX) samples extracted from human plasma and cerebrospinal fluid (CSF), using two-dimensional liquid chromatography (2D-LC). Furthermore, we intended to verify whether intravenous mannitol could increase MTX concentration in the CSF of patients. The mobile phase of PUMP1 consisted of 10.0 mmol/L ammonium acetate and acetonitrile. PUMP2 solution consisted of an aqueous solution of 10.0 mmol/L ammonium acetate. The mobile phase of PUMP3 comprised 50.0 mmol/L ammonium acetate and acetonitrile, with a flow rate of 1.0 mL/min. The developed method was successfully employed to simultaneously determine drug levels in plasma and CSF from the patients treated with MTX. CSF samples were obtained by lumbar puncture 0.5 - 2 h after starting the high-dose methotrexate (HD-MTX) infusion (over 4 h) and immediately before the intrathecal (IT) administration of MTX. Venous blood samples were drawn 4 h after the start of infusion. The calibration curve was linear, with a range of 0.07 - 2.38 µmol/L for CSF samples and a range of 0.11 - 5.51 µmol/L for plasma samples. Precision (> 95%) and accuracy (> 97%) were within the acceptance criteria for each quality control (QC) level. Inter- and intra-day accuracy and precision values met the acceptance criteria for each QC level. The correlation between MTX concentrations in the plasma and CSF was moderate (r = 0.502). No significant difference was observed in MTX concentration in CSF between patients using intravenous mannitol and those not using intravenous mannitol (P = 0.682). The developed method was useful for therapeutic drug monitoring of MTX and suitable for assessing the risks and benefits of chemotherapy in patients with primary central nervous system lymphoma. Intravenous mannitol did not increase MTX concentration in the CSF of patients.

In vitro fertilization-induced extreme hypertriglyceridemia with secondary acute pancreatitis in emergency department: A case report and literature review

Abstract Acute pancreatitis is one of the severe complications of hypertriglyceridemia, which needs to be recognized early to provide appropriate treatment. Hypertriglyceridemia-induced pancreatitis has several causes, in which in vitro fertilization (IVF) is a rare etiology that is becoming increasingly popular. We report a 33-year-old female patient with a history of hypertension who has failed an IVF cycle and started a new IVF procedure 1 month before admission. She was diagnosed with severe triglyceridemia-induced acute pancreatitis with extremely high serum triglycerides (TGs) levels (18,547 mg/dL). We combined plasmapheresis and intravenous (IV) insulin and significantly reduced blood TG over a short time. She was discharged with a TG level of 366.7 mg/dL on the 10th day. It is essential to monitor serum TG levels in plasma before, during, and after this therapy, especially in the 1st month after initiating IVF. Although plasmapheresis combined with IV insulin is not officially recommended for acute triglyceridemia-induced pancreatitis, the therapy can be considered in cases with extremely high serum TG levels.

The rheumatoid arthritis gut microbial biobank reveals core microbial species that associate and effect on host inflammation and autoimmune responses.

Gut microbiota dysbiosis has been implicated in rheumatoid arthritis (RA) and influences disease progression. Although molecular and culture-independent studies revealed RA patients harbored a core microbiome and had characteristic bacterial species, the lack of cultured bacterial strains had limited investigations on their functions. This study aimed to establish an RA-originated gut microbial biobank (RAGMB) that covers and further to correlates and validates core microbial species on clinically used and diagnostic inflammation and immune indices. We obtained 3200 bacterial isolates from fecal samples of 20 RA patients with seven improved and 11 traditional bacterial cultivation methods. These isolates were phylogenetically identified and selected for RAGMB. The RAGMB harbored 601 bacterial strains that represented 280 species (including 43 novel species) of seven bacterial phyla. The RAGMB covered 93.2% at species level of medium- and high-abundant (relative abundances ≥0.2%) RA gut microbes, and included four rare species of the phylum Synergistota. The RA core gut microbiome was defined and composed of 20 bacterial species. Among these, Mediterraneibacter tenuis and Eubacterium rectale were two species that statistically and significantly correlated with clinically used diagnostic indices such as erythrocyte sedimentation rate (ESR) and IL-10. Thus, M. tenuis and E. rectale were selected for experimental validation using DSS-treated and not DSS-treated mice model. Results demonstrated both M. tenuis and E. rectale exacerbated host inflammatory responses, including shortened colon length and increased spleen weight, decreased IL-10 and increased IL-17A levels in plasma. Overall, we established the RAGMB, defined the RA core microbiome, correlated and demonstrated core microbial species effected on host inflammatory and immune responses. This work provides diverse gut microbial resources for future studies on RA etiology and potential new targets for new biomedical practices.

Gal-3 blocks the binding between PD-1 and pembrolizumab

IntroductionImmune checkpoint inhibitors (ICI) have revolutionized the treatment of metastatic malignant melanoma (MM) and improved long-term survival. Despite the impressive results, some patients still have progressive disease, and the search...

Impact of Feeding Artemia franciscana Enriched with Various Oil Resources on Growth, Blood Biochemical and Behavioral Indices, and Survival of Oreochromis Niloticus

Abstract The main objective of the present trial was to examine the efficacy of feeding tilapia fry fish on Artemia franciscana diets supplemented with various oil emulsion resources in terms of performance, behavior indices, survival rate, blood biochemical parameters, and immunological response. Four hundred Nile tilapia fry (weighing 0.15±0.05 g and measuring 2.17±0.08 cm) were randomly allocated into four equal groups (each with five repetitions) and acclimatized for fifteen days. The first group served as the control and received unenriched Artemia franciscana (G0), while the remaining three groups were fed Artemia franciscana diets enriched with different oil resources (0.5 mL oil per liter for 6 hours): soybean oil (G1), sesame oil (G2), and rice bran oil (G3). Behavioral observations were recorded during the 45-day experimental period. At the end of the feeding trial, the chemical composition and fatty acid content of both Artemia and fingerlings were analyzed. Furthermore, the growth performance, survival, and immune response of the fingerlings were evaluated. The results indicated noticeable improvements in behavioral measurements (feeding, foraging and schooling), performance (final length, final weight, net weight gain, feed conversion ratio and specific growth rate), survival, and immune response among fry fish supplemented with enriched Artemia, particularly those enriched with soybean oil. Additionally, the chemical composition and fatty acid content of both Artemia and fish fry were significantly enhanced when oil emulsions are applied, with soybean oil demonstrating the most prominent improvements. Whereas, supplementing fry fish Artemia diets with oil resulted in lower liver enzyme activity and higher protein component levels in plasma in comparison to the control group. In brief, feeding Nile tilapia fry fish Artemia diets enriched with a soybean oil emulsion (0.5 mL/L) is recommended for promoting high performance, immunological activity, and survival throughout the early stage till fingerlings phase.

Kinetics of 15 per- and polyfluoroalkyl substances (PFAS) after single oral application as a mixture – A pilot investigation in a male volunteer

Per- and polyfluoroalkyl substances (PFAS) are ubiquitous environmental contaminants with half-lives in humans in the range of years in case of the long-chain compounds, leading to accumulation and measurable levels in plasma. In contrast, short-chain and “alternative” PFAS have lower levels or are not detectable in humans with background exposure. This may be due to lower exposure, but also due to much shorter half-lives compared to long-chain compounds. To get better data on kinetics, a healthy volunteer orally ingested a mixture of fifteen predominantly 13C-labeled PFAS (“MPFAS”) in a pilot investigation (MPFBA, MPFPeA, MPFHxA, MPFHpA, MPFOA, MPFNA, MPFDA, MPFUdA, MPFDoA, PFBS, MPFHxS, MPFOS, DONA, HFPO-DA, 6:2FTS). After application, concentrations were measured over 450 days in plasma, urine and feces, using UHPLC-MS/MS analysis after extraction. The compounds were absorbed quickly and almost completely. Data analysis revealed volumes of distribution between 110 and 177 mL/kg bw for most compounds, but higher values for MPFDA, MPFUdA and MPFDoA (maximum of 354 mL/kg bw). Half-lives were found to vary extremely, from 0.5 days (MPFPeA) and 1.5 days (MPFHxA) to 51 days (PFBS) and 152 days (MPFHpA) in case of the short-chain and “alternative” compounds. For the long-chain compounds, half-lives in the range of several years were confirmed for MPFOA, MPFNA, MPFHxS and MPFOS, but with even higher chain-lengths of the carboxylic acids, the half-lives were found to decrease, with the shortest half-life for MPFDoA (295 days). Elimination from the body was completely explained by the urinary losses in case of the short-chain and “alternative” PFAS, and in part by the fecal losses in case of the long-chain PFCA. Overall, elimination kinetics seem to be determined by several different renal and gastrointestinal factors (fraction unbound in plasma, binding affinity to organic anion transporters causing netto secretion or reabsorption, fecal loss with mechanisms to be clarified).

Anti-factor H autoantibodies in patients with lupus nephritis

IntroductionLupus nephritis (LN) is a disease marked by autoantibodies against complement components. Autoantibodies against negative complement regulator factor H (anti-FH) are prevalent in aHUS, are associated with deletion of factor H-related protein 1 (FHR1) gene, and have overt functional consequences. They are also observed in C3 glomerulopathies. The frequency and relevance of anti-FH in LN are poorly studied. AimThe aim of our investigation was to screen for the presence of anti-FH and FHR1 gene deletion in a cohort of LN patients and to evaluate their association with LN activity. MethodELISA test and Western blot for detection of anti-FH and FHR1 deletion were used, respectively. Patients’ clinical and laboratory parameters regarding anti-FH role were processed by statistical analysis. ResultsAnti-FH were found at low level in a small number of LN patients – 11.7% (7/60) and were not associated with deletion of FHR1. Anti-FH did not correlate with ANA titers, anti-dsDNA, C3/C4 hypocomplementemia, eGFR, proteinuria, or active urinary sediment in LN patients. A weak correlation was found between anti-FH and anti-C3 levels. Anti-FH were linked with endocapillary proliferation and histological activity index. Four anti-FH positive patients had severe to moderate LN as per the BILAG renal score. ConclusionsAnti-FH autoantibodies are an accessory finding in LN and are more likely to manifest during the active phase of the disease. Due to their low frequency and plasma levels, they do not seem suitable for routine laboratory investigation in patients with LN.

Antipsychotic Drugs: A Concise Review of History, Classification, Indications, Mechanism, Efficacy, Side Effects, Dosing, and Clinical Application.

The introduction of the first antipsychotic drug, chlorpromazine, was a milestone for psychiatry. The authors review the history, classification, indications, mechanism, efficacy, side effects, dosing, drug initiation, switching, and other practical issues and questions related to antipsychotics. Classifications such as first-generation/typical versus second-generation/atypical antipsychotics are neither valid nor useful; these agents should be described according to the Neuroscience-based Nomenclature (NbN). Antipsychotic drugs are not specific for treating schizophrenia. They reduce psychosis regardless of the underlying diagnosis, and they go beyond nonspecific sedation. All currently available antipsychotic drugs are dopamine blockers or dopamine partial agonists. In schizophrenia, effect sizes for relapse prevention are larger than for acute treatment. A major unresolved problem is the implausible increase in placebo response in antipsychotic drug trials over the decades. Differences in side effects, which can be objectively measured, such as weight gain, are less equivocal than differences in rating-scale-measured (subjective) efficacy. The criteria for choosing among antipsychotics are mainly pragmatic and include factors such as available formulations, metabolism, half-life, efficacy, and side effects in previous illness episodes. Plasma levels help to detect nonadherence, and once-daily dosing at night (which is possible with many antipsychotics) and long-acting injectable formulations are useful when adherence is a problem. Dose-response curves for both acute treatment and relapse prevention follow a hyperbolic pattern, with maximally efficacious average dosages for schizophrenia of around 5 mg/day risperidone equivalents. Computer apps facilitating the choice between drugs are available. Future drug development should include pharmacogenetics and focus on drugs for specific aspects of psychosis.

Effects of oxygen levels and temperature on growth and physiology of pikeperch juveniles cultured in a recirculating aquaculture system

This study aimed to understand how environmental factors, specifically water temperature and oxygen saturation, affect the growth performance and physiology of pikeperch (Sander lucioperca) juveniles in recirculating aquaculture systems (RASs). Given the importance of optimising growth conditions in aquaculture to maximise efficiency, it aims to assess whether different combinations of oxygen levels and temperatures can enhance growth while maintaining the physiological health and welfare of the fish. The experimental design included the culturing pikeperch juveniles (22.7 ± 7.1 g) were exposed to hypoxia (78 ± 14%), normoxia (105 ± 12%), and hyperoxia (140 ± 18%) conditions for 72 days. This was conducted at two temperatures, 20 °C and 23 °C, each in a separate but identical RAS. The level of oxygen supply was controlled with micro bubble diffusers on the bottom of each tank. The hyperoxia at 23 °C positively affected total length, BW, specific growth rate, feed intake and feed conservation rate (FCR). The slowest growth and feed intake, along with the highest FCR, were observed in hypoxia at 20 °C. Fish reared under 23 °C exhibited significantly higher visceral-somatic index (3.54 ± 0.83 at 23 °C and 2.76 ± 0.73 at 20 °C) regardless of oxygen levels. It was primarily responsible for the observed growth difference (Final BW: 58.3 ± 18.8 g at 23 °C and 53.0 ± 18.3 g at 20 °C). The water temperature also affected haematocrit, haemoglobin, leucocyte count, mean corpuscular haemoglobin, mean corpuscular volume (MCV) of the blood cells; the concentration of lymphocytes, neutrophile granulocyte bands and segments. Among biochemical markers, temperature affected cytoplasmic and mitochondrial enzymes, ammonia and triglyceride levels in blood plasma. Elevated antioxidant activity was observed in muscle, intestine and liver tissues. Oxygen levels demonstrated significant effects on growth, feed intake and conversion, the MCV of the blood cells, the concentration of the glucose, lactate and ammonia in blood plasma, and antioxidant biomarkers in the liver tissue. The analysis indicated a significant effect of oxygen on energy metabolism. The results showed hyperoxia under 23 °C create conditions for the highest growth and feed intake, high feed utilisation. There are, however, concerns about the physiological conditions and welfare of intensively cultured pikeperch juveniles, as higher feed intake led to increased visceral fat content in the body, elevated antioxidant activity in the liver, muscle and intestine tissues, morphology of blood cell, and energy metabolism.

Fam3a-mediated prohormone convertase switch in α-cells regulates pancreatic GLP-1 production in an Nr4a2-Foxa2-dependent manner

BackgroundFam3a has been demonstrated to regulate pancreatic β-cell function and glucose homeostasis. However, the role and mechanism of Fam3a in regulating α-cell function remain unexplored. MethodsGlucagon and glucagon-like peptide-1 (GLP-1) levels in pancreas and plasma were measured in global Fam3a knockout (Fam3a−/−) mice. Human islet single-cell RNA sequencing (scRNA-seq) datasets were utilized to analyze gene expression correlations between FAM3A and PCSK1 (encoding PC1/3, which processes proglucagon into GLP-1). Mouse pancreatic α-cell line αTC1.9 cells were transfected with Fam3a siRNA or plasmid for Fam3a knockdown or overexpression to explore the effects of Fam3a on PC1/3 expression and GLP-1 production. The downstream mediator (including Nr4a2) was identified by transcriptomic analysis, and its role was confirmed by Fam3a knockdown or overexpression in αTC1.9 cells. Based on the interacted protein of Nr4a2 and the direct binding to Pcsk1 promoter, the transcription factor Foxa2 was selected for further verification. Nuclear translocation assay and dual-luciferase reporter assay were used to clarify the involvement of Fam3a-Nr4a2-Foxa2 pathway in PC1/3 expression and GLP-1 production. Moreover, α-cell-specific Fam3a knockout (Fam3aα−/−) mice were constructed to evaluate the metabolic variables and hormone levels under normoglycemic, high-fat diet (HFD)-fed and streptozotocin (STZ)-induced diabetic conditions. Exendin 9–39 (Ex9), a GLP-1 receptor antagonist, was used to investigate GLP-1 paracrine effects in Fam3aα−/− mice and in their primary islets. ResultsCompared with wild-type mice, pancreatic and plasma active GLP-1 levels were increased in Fam3a−/− mice. Analysis of human islet scRNA-seq datasets showed a significant negative correction between FAM3A and PCSK1 in α-cells. Fam3a knockdown upregulated PC1/3 expression and GLP-1 production in αTC1.9 cells, while Fam3a overexpression displayed inverse effects. Transcriptomic analysis identified Nr4a2 as a key downstream mediator of Fam3a, and Nr4a2 expression in αTC1.9 cells was downregulated and upregulated by Fam3a knockdown and overexpression, respectively. Nr4a2 silencing increased PC1/3 expression, albeit Nr4a2 did not directly bind to Pcsk1 promoter. Instead, Nr4a2 formed a complex with Foxa2 to facilitate Fam3a-mediated Foxa2 nuclear translocation. Foxa2 negatively regulated PC1/3 expression and GLP-1 production. Besides, Foxa2 inhibited the transcriptional activity of Pcsk1 promoter at specific binding sites 10 and 6, and this inhibition was intensified by Nr4a2 in αTC1.9 cells. Compared with Flox/cre littermates, improved glucose tolerance, increased active GLP-1 level in pancreas and plasma, upregulated plasma insulin level in response to glucose, and decreased plasma glucagon level were observed in Fam3aα−/− mice. Primary islets isolated from Fam3aα−/− mice also showed an increase in active GLP-1 and insulin release. In addition, the insulinotropic effect of intra-islet GLP-1 was blocked by Ex9 in Fam3aα−/− mice and in their primary islets. Similarly, HFD-fed Fam3aα−/− mice also exhibited an improved glucose tolerance. Both HFD-fed and STZ-induced diabetic Fam3aα−/− mice showed an increased pancreatic active GLP-1 level, an elevated plasma insulin level and a reduced plasma glucagon level. ConclusionsFam3a deficiency in α-cells enhances pancreatic GLP-1 production to improve β-cell function via paracrine signaling in an Nr4a2-Foxa2-PC1/3-dependent manner. Our study unveils a novel strategy for reprogramming α-cell proglucagon processing output from glucagon to GLP-1 and deepen the understanding of crosstalk between α-cells and β-cells.