ELISA Immunoassay Research Articles

Bioavailability of the tumor necrosis factor alpha/regulated on activation, normal Tcell expressed and secreted (RANTES) biosystem inside the gestational sac during the pre-immune stages of embryo development.

In-vivo studies of the bioavailability of major components of the tumor necrosis factor alpha (TNFα) biosystem inside the gestational sac during embryogenesis have not been reported. We sought to determine the concentration of TNFα, soluble (s) TNFα receptors (sTNFR1, sTNFR2), and RANTES in the primate extraembryonic celomic fluid (ECF). A validated timed-pregnant baboon animal model (N: 10) for experimental research in pregnancy was used to collect paired maternal blood and ECF samples in ongoing pregnancies. The concentrations (pg/dL) of TNFα, sTNFR1, sTNFR2, and RANTES were then determined by ELISA immunoassays. All animals delivered at term healthy newborns. The differential concentration of TNFα, sTNFR1, sTNFR2, and RANTES between the maternal plasma and the ECF could be determined with ratios for TNFα (5.4), sTNFR2 (1.85) and RANTES (3.59) that contrasted with that of sTNFR1 (0.07), which favored the gestational sac compartment. No significant correlations were noted between maternal plasma and ECF TNFR1, sTNFR2 and RANTES. There was a trend for a correlation between TNFα in maternal plasma and ECF (R=0.74; p=0.07). We report the physiological concentrations of TNFα, sTNFR1, sTNFR2, and RANTES in extraembryonic celomic fluid during embryogenesis in primates.

Effect of Conbercept on Corneal Neovascularization in a Rabbit Model

ABSTRACT Objective To study the efficacy of Conbercept for the treatment of corneal neovascularization (NV) in a rabbit model. Methods NV was induced by placing sutures. Eight rabbits were used as a control. The other 136 rabbits were randomly divided into two equal groups, and 68 rabbits in each group were divided into four subgroups and given different treatments. Time-course photographs, histological examination, and enzyme-linked immunoassay ELISA analysis for vascular endothelial growth factor were performed at weeks 1, 2, and 3 after injection placement. Results At weeks 1, 2, and 3 after injection placement, there was less expression of corneal NV and VEGF in the conbercept-treated groups than in the saline-treated control groups and less corneal NV and VEGF were expressed in the early treatment group than in the late treatment group. At weeks 2 and 3 after injection, there were fewer corneal NV (length and area) in the early intrastromal injection group with conbercept than in the early subconjunctival injection group with conbercept and a smaller diameter of corneal NV than in the late intrastromal injection group treated with conbercept. Histological examination showed a smaller diameter of corneal NV in all eyes in conbercept-treated groups 1 w after injection than before injection. Treatment with subconjunctival injection with conbercept led to a larger diameter at weeks 2 and 3 than at week 1. Conclusions Subconjunctival and intrastromal administrations of conbercept effectively inhibit corneal NV in rabbits, and the latter has the better effect. The effect is the best in the group with cornea intrastromal injection of conbercept 1 w after suture. Early administration of conbercept may successfully inhibit corneal NV in an animal model.

Effect of Curcumin (Standard and Supplement) with Zinc on Reproductive Hormones in Polycystic Ovary Syndrome (PCOS) Rats

Polycystic ovary syndrome (PCOS) is a complex and heterogeneous disorder that affects a large percentage of women worldwide. The pathophysiology is not fully explained. In recent years, complementary and alternative medicine has emerged as a viable treatment option. This study examined how curcumin and zinc affected on reproductive hormones in rats with polycystic ovary syndrome (PCOS). It was induced by oral administration of letrozole (1mg/kg/day) for 21 days. PCOS groups were treated over 14 days with curcumin standard 200 mg/kg, curcumin supplement 200 mg/kg, and zinc 30 mg /kg (separately or in combination with two forms of curcumin), and metformin 50 mg/kg as standard treatment. The blood biochemistry of these groups was compared to those of the groups of healthy, PCOS, and those who received metformin. The immunoassay ELISA technique was employed for measuring the concentration of reproductive hormones. The results manifested that the administration of letrozole resulted in a significant elevation (p≤0.05) of luteinizing hormone, follicle-stimulating hormone, testosterone, and prolactin associated with a significant decrease p≤0.05) of estrogen and progesterone levels in the PCOS-designed model. The results of effect of curcumin supplement alone and with zinc showed a significant improvement in all parameters by reducing the luteinizing hormone, follicle-stimulating hormone, testosterone, and prolactin. Estrogen and progesterone elevated significantly for all groups, while the elevation was highly significant in both curcumin supplement alone and curcumin supplement with zinc. The treatment with curcumin supplement and their combination with zinc showed promising results in restoring reproductive hormones to the normal level.

N-Acetylcysteine Decreases Myocardial Content of Inflammatory Mediators Preventing the Development of Inflammation State and Oxidative Stress in Rats Subjected to a High-Fat Diet.

Arachidonic acid (AA) is a key precursor for proinflammatory and anti-inflammatory derivatives that regulate the inflammatory response. The modulation of AA metabolism is a target for searching a therapeutic agent with potent anti-inflammatory action in cardiovascular disorders. Therefore, our study aims to determine the potential preventive impact of N-acetylcysteine (NAC) supplementation on myocardial inflammation and the occurrence of oxidative stress in obesity induced by high-fat feeding. The experiment was conducted for eight weeks on male Wistar rats fed a standard chow or a high-fat diet (HFD) with intragastric NAC supplementation. The Gas-Liquid Chromatography (GLC) method was used to quantify the plasma and myocardial AA levels in the selected lipid fraction. The expression of proteins included in the inflammation pathway was measured by the Western blot technique. The concentrations of arachidonic acid derivatives, cytokines and chemokines, and oxidative stress parameters were determined by the ELISA, colorimetric, and multiplex immunoassay kits. We established that in the left ventricle tissue NAC reduced AA concentration, especially in the phospholipid fraction. NAC administration ameliorated the COX-2 and 5-LOX expression, leading to a decrease in the PGE2 and LTC4 contents, respectively, and augmented the 12/15-LOX expression, increasing the LXA4 content. In obese rats, NAC ameliorated NF-κB expression, inhibiting the secretion of proinflammatory cytokines. NAC also affected the antioxidant levels in HFD rats through an increase in GSH and CAT contents with a simultaneous decrease in the levels of 4-HNE and MDA. We concluded that NAC treatment weakens the NF-κB signaling pathway, limiting the development of myocardial low-grade inflammation, and increasing the antioxidant content that may protect against the development of oxidative stress in rats with obesity induced by an HFD.

Development of a Method for Detection of SARS-CoV-2 Nucleocapsid Antibodies on Dried Blood Spot by DELFIA Immunoassay.

Antibodies against the SARS-CoV-2 nucleocapsid protein are produced by the immune system in response to SARS-CoV-2 infection, but most available vaccines developed to fight the pandemic spread target the SARS-CoV-2 spike protein. The aim of this study was to improve the detection of antibodies against the SARS-CoV-2 nucleocapsid by providing a simple and robust method applicable to a large population. For this purpose, we developed a DELFIA immunoassay on dried blood spots (DBSs) by converting a commercially available IVD ELISA assay. A total of forty-seven paired plasma and dried blood spots were collected from vaccinated and/or previously SARS-CoV-2-infected subjects. The DBS-DELFIA resulted in a wider dynamic range and higher sensitivity for detecting antibodies against the SARS-CoV-2 nucleocapsid. Moreover, the DBS-DELFIA showed a good total intra-assay coefficient of variability of 14.6%. Finally, a strong correlation was found between SARS-CoV-2 nucleocapsid antibodies detected by the DBS-DELFIA and ELISA immunoassays (r = 0.9). Therefore, the association of dried blood sampling with DELFIA technology may provide an easier, minimally invasive, and accurate measurement of SARS-CoV-2 nucleocapsid antibodies in previously SARS-CoV-2-infected subjects. In conclusion, these results justify further research to develop a certified IVD DBS-DELFIA assay for detecting SARS-CoV-2 nucleocapsid antibodies useful for diagnostics as well as for serosurveillance studies.

Cotinine as a Sentinel of Canine Exposure to Tobacco Smoke.

The adverse health effects of both active and passive tobacco smoke have been well-known in humans for a long time. It is presumable that even pets, which intimately share the owner's lifestyle, may be exposed to the same risks. This study aimed to detect and quantify cotinine (a metabolite of nicotine) in the serum and hair of dogs using a specific commercial ELISA immunoassay kit. A total of 32 dogs, 16 exposed and 16 unexposed to the owner's smoke, were enrolled. The cotinine concentration was higher in the exposed than the unexposed group in both matrices (p < 0.001), with greater values in serum than in hair (p < 0.001). Exposed bitches had higher hair cotinine than male dogs (p < 0.001). Conversely, serum and fur cotinine concentrations were lower in female than male dogs of the unexposed group (p < 0.01). The exposure intensity, age, and weight of the dogs did not affect cotinine concentrations. A cut-off value of 2.78 ng/mL and 1.13 ng/mL cotinine concentration in serum and fur, respectively, was estimated to distinguish between the exposed and unexposed dogs. Cotinine was confirmed as a valuable marker of passive smoking also in dogs. Although owners do not perceive secondhand smoke as a risk for their dogs, greater awareness should be advisable, especially in pregnant animals.

Fluorescence ratio immunoassay for fumonisin B1 based on the oxidase characteristics of the growth of monodispersed 2-D MnO2 nanosheet on an individual gold nanoparticle (AuNP@MnO2).

Fumonisin B1 (FB1) is one of the important mycotoxins posing health risks in the area of food safety. Asensitive fluorescence ratio immunoassay has been establishedfor FB1 based on the growth of monodispersed 2-D MnO2 nanosheet on an individual gold nanoparticle (AuNP@MnO2). FB1 competed with the coated FB1-BSA to bind the FB1 monoclonal antibody. After awashing step, alkaline phosphatase-labeled goat anti-mouse IgG (ALP-IgG) with high catalytic activity was combined with FB1 monoclonal antibody. ALP reacts with ascorbic acid 2-phosphate (AAP) to produce ascorbic acid (AA), which decomposes AuNP@MnO2 to dehydroascorbic acid (DHAA). O-Phenylenediamine dihydrochloride (OPD) is oxidized to yellow-fluorescent substrate of 2,3-diaminophenazine (DAP) (excitation, 423 nm; emission, 570 nm) by AuNP@MnO2. Meanwhile, OPD can also be reduced to blue fluorescent substrate of OPDred (excitation, 350 nm; emission, 430 nm) by DHAA. The content of FB1 can be determined by fluorescence ratio of blue/yellow. The limit of detection (LOD) of the fluorescence ratio immunoassay for FB1 was 0.06 ng mL-1, and the linear range was from 0.25 to 60.00 ng mL-1. The effectiveness of the assay was verified in real maize samples, and satisfactory recoveries were attained. The correlation coefficient of these results between the fluorescence ratio immunoassay and commercial ELISA kit was 0.9999. This method provides a sensitive and selective tool for the detection of FB1 in maize samples.

Clinical and diagnostic significance of plasminogen activator inhibitor type 1 in the early development of coronary heart disease

Aim to determine the quantitative content of the tissue plasminogen activator inhibitor type 1 (PAI-1) and to determine its relationship with risk factors for coronary artery disease.&#x0D; Material and methods. The study included 67 male and female patients (36 men and 31 women) with coronary artery disease (CAD) and stable angina classes II-IV with the presence of hypertension. The mean age of the patients was 60.20.76 years. In all patients, clinical and anamnestic data were collected during the examination. For pain assessment, we used a verbal scale. We registered the anthropometric data and body mass index. Biochemical and instrumental methods of research were used according to the standards for diagnosing CAD. We assessed the occurrence of risk factors in patients included in the study. A complex of general clinical laboratory and instrumental tests was used, as well as the method of enzyme immunoassay ELISA, which determined the level of PAI-1, platelet aggregation activity, renin, aldosterone, cortisol and endothelin.&#x0D; Results. The group of patients demonstrated a reliable elevation of PAI-1 plasma level. Compared with practically healthy people, the level of this biomarker was 1.6 times higher in patients with coronary artery disease. A significant elevation of PAI-1 level indicates an increased risk of endothelial and hemostasiological disorders in patients, which, in turn, increases the risk of thrombogenic complications. The study revealed the correlation of PAI-1 protein in CAD patients with such risk factors as mixed anxiety-depressive disorder, smoking, hypertension, obesity, and hypercholesterolemia.

Deciphering the chromatin organization and epigenomics involved in adipocyte differentiation and hypertrophy by multimodal nanoscopy.

The adipose tissue is the physiological energy reservoir for the whole body, but it also acts as a central metabolic organ in the regulation of body energy homeostasis. In particular, fat is stored in adipocytes inside cytosolic lipid droplets (LDs). Adipocyte hypertrophy occurring in obesity refers to an increase in adipocyte size due to LD enlargement. We aim to investigate the nuclear architecture and epigenetics during adipocyte differentiation and hypertrophy using an in vitro model of 3T3-L1 pre-adipocytes firstly differentiated into mature adipocytes, then cultured with fatty acids to induce hypertrophy. On pre-adipocytes, mature and hypertrophic adipocytes, we assessed lipid accumulation as a hypertrophy marker. Chromatin plays a central role in controlling gene expression under different physiological and pathological mechanisms. We employed optical nanoscale microscopy, such as confocal, super-resolution stimulation emission depletion (STED), and molecular biology analyses (ELISA), to assess the remodeling of nuclear architecture, chromatin organization, and epigenetic modifications associated with adipocyte differentiation and hypertrophy. Confocal and STED analyses showed appreciable differences in nuclear architecture, chromatin condensation, distribution, and chromatin methylation/acetylation among pre-adipocytes, mature and hypertrophic adipocytes. Moreover, we quantified the average changes in the DNA methylation (5-methylcytosine) by ELISA immunoassay. These preliminary results indicate epigenome and nuclear organization remodeling during adipocyte differentiation and hypertrophy. We can conclude that nuclear architecture remodeling is a significant and central factor in the genome function regulation.

Elucidating the mechanism of PI3K when interacting with TRPV1.

The interaction between Transient Receptor Potential Cation Channel Subfamily V member 1 (TRPV1), Phosphoinositide 3-Kinase (PI3K), and Receptor Tyrosine Kinase (RTK) provides us with a positive feedback loop that is responsible for recruiting Pi3K, a protein responsible for phosphorylating Phosphatidylinositol Bisphosphate (PIP2) into Phosphatidylinositol Trisphosphate (PIP3), and recruit TRPV1, an ion channel responsible for regulating heat and pain sensation, to specific areas in the plasma membrane (PM). In this study, we use ELISA immunoassay, microscopy, and other techniques to elucidate PI3K's functionality under the influence of TRPV1. Ultimately, we want to investigate this positive feedback loop and determine if we can interfere with excess TRPV1 activation and potentially stop excess pain and inflammation.

P514 Serum levels of Klotho in inflammatory bowel disease: an exploratory study

Abstract Background Alpha klotho (α-Klotho) is an anti-aging protein recognized as a humoral factor with anti-oxidant and anti-inflammatory effects, showing a bidirectional relationship with inflammation, a main pathophysiologic factor in atherosclerosis. A decrease in Klotho has been associated to cardiovascular disease (CVD), but no previous studies have investigated the relationship between inflammatory bowel disease (IBD), a risk factor for CVD, and Klotho levels. Therefore, we assessed if the serum levels of Klotho differ between patients with IBD and controls, and how this protein is related to the clinical and laboratory characteristics of the disease, subclinical atherosclerosis and metabolic risk factors. Methods Cross-sectional multicentric study that included 251 individuals: 191 IBD patients and 60 healthy controls was conducted. Serum Klotho levels were determined by commercial ELISA immunoassay (IBL International GmbH Laboratories). In the IBD patients disease activity was evaluated using CDAI for Crohn’s disease, partial Mayo (pMayo) for ulcerative colitis, specific laboratory parameters, determination of the Systematic Coronary Risk Evaluation (SCORE), and the presence of subclinical atherosclerosis was assessed using carotid ultrasound for identifying carotid plaque and measurement of carotid intima-media thickness. Results Serum Klotho levels were significantly higher in the IBD group: 734.31 pg/ml vs. 484.17 pg/ml (p&amp;lt;0.001) compared with controls. In the IBD group, no significant differences in the serum Klotho levels were found between the type of IBD (p=0.911), time from diagnosis (p=0.126), disease activity: CDAI (p=0.277), pMayo (p=0.916), type of treatment (0.76), the presence of plaque (p=0.101) or the intima media thickness (p=0.429). However, a significant inverse correlation between serum Klotho and the SCORE was observed in IBD patients: r = -0.176 (p= 0.014) (figure 1) Conclusion Patients with IBD had higher Klotho levels than healthy controls, with an inverse and significant relationship with the SCORE in the IBD patients. Further studies are necessary to clarify the role of Klotho in IBD.

Level of oxytocin prior to rugby and handball matches: An exploratory study among groups of Polish players

The aim of the present exploratory study was to assess the changes in urinary oxytocin (OT) concentration during the period between five days before, and on the day of match, among rugby and handball players. Nine male rugby players with a mean age of 27.62 years (SD = 4.21) and 18 male handball players with a mean age of 17.03 years (SD = 0.57) participated. Urinary oxytocin level was measured by ELISA immunoassay as a ratio to the concentration of creatinine [mg/ml] measured through colorimetric detection. The relative level of OT to creatinine (OT/CRE) significantly differed between the type of player (rugby or handball) but not between times of measurements. Significant differences were only between OT/CRE level in a day of match in rugby players and in 5 days before match in handball players (p&lt;0.05). There was no change in oxytocin levels during the time periods between five days before and on the day of a match, in either of the two kinds of players. The change in oxytocin might be traceable during the match but not before a match and this perhaps depends on a more subtle context of competition, but not on the assumption of competition. Further studies are needed based on more homogenous group with higher number of matches.

Leishmania infantum infecting the carnivore Nasua nasua from urban forest fragments in an endemic area of visceral leishmaniasis in Brazilian Midwest.

The aim of the present study was to investigate the occurrence of Leishmania infantum in South American coatis inhabiting two forest fragments in Campo Grande, Mato Grosso do Sul, Midwest region of Brazil, an endemic area of human and canine visceral leishmaniasis (VL). A total of 110 South American coatis were sampled in the conservation unit "Parque Estadual do Prosa" (PEP) and in the residential area "Vila da Base Aérea" (VBA) from March 2018 to April 2019. As a longitudinal study that include up to six recaptures of the same individual, a total of 190 capture events were obtained. Blood, bone marrow and skin samples were obtained for parasitological (axenic culture), serological (Enzyme Linked Immunosorbent Assay - ELISA and Dual-path Platform immunoassay - DPP® CVL) and molecular diagnostic assays (targeting kDNA for Leishmania spp. and L. infantum; and HSP70 followed by sequence analysis). Seropositivity for L. infantum was found in 33 individuals, six in PEP and 27 in VBA. Furthermore, L. infantum was detected by molecular analysis in 16 individuals, seven from PEP and nine from VBA. We also isolated L. infantum from bone marrow of one individual and detected a single positive skin sample in molecular assay from other individual, both from VBA. An overall infection rate of 36.4% (40/110) was observed, significantly higher in the VBA (49.1%) than in the PEP (21.6%), probably because VBA presents: (i) a large number of resident dogs and chickens that would be attracting sandflies; (ii) a denser population of this wild mammal species; and (iii) physical barriers and a lack of functional connectivity in the surroundings, preventing these animals to disperse out. We conclude that South American coati populations living in urban forest fragments of Campo Grande are affected by the epidemiological scenario of VL, known to involve dogs, vectors and humans. We highlight the importance of investigate the parasitism by L. infantum in this and other potential L. infantum reservoirs that inhabit urbanized regions endemic to VL.

&amp;lt;i&amp;gt;In Vitro&amp;lt;/i&amp;gt; Evaluation of Two Tissue Substitutes for Gingival Augmentation

Three-dimensional collagen matrices of porcine origin are being used as substitutes for soft tissue grafts in periodontal plastic surgery in search of aesthetic and natural results. This in vitro study aimed to compare Fibro-Gide® (GeistlichBiomaterials) and Mucoderm® (BotissBiomaterials) matrices during the initial phase of soft tissue formation. For this purpose, samples of 5 × 5 mm were obtained, and then human fibroblasts were plated on them. After 24, 48 and 72 h, cell viability was assessed using an MTT assay, and the secretion of type I collagen, MMP-2, TIMP-1 and TIMP-2 was analyzed by ELISA immunoassay. The control group (C) consisted of cells plated on polystyrene without the matrices. The morphology of the surfaces was also examined using scanning electron microscopy (SEM), as was the average roughness (Ra) of the samples by a profilometer. Topographic analysis revealed that roughness was significantly higher on Mucoderm® than on Fibro-Gide® (p 0.05). The synthesis of type I collagen, MMP-2 and TIMP-1 were significantly higher from cells plated on Fibro-Gide® than on Mucoderm®, in all time points (p ® than on Mucoderm® (p ® induced an increase in type I collagen, MMP-2 and TIMP-1 and TIMP-2.

N-acetylcysteine acts as a potent anti-inflammatory agent altering the eicosanoid profile in the development of simple steatosis and its progression to hepatitis.

We aimed to examine the influence of N-acetylcysteine (NAC) on the development of metabolic dysfunction-associated steatotic liver disease (MASLD) in rats with a specific focus on the eicosanoid pathway. The experiment was conducted on male Wistar rats fed a standard diet or a high-fat diet (HFD) for eight weeks. In the entire experiment, half of rats from both groups received intragastrically NAC solution prepared in normal saline. H + E staining was used for the histological assessment of liver tissue. The gas-liquid chromatography (GLC) technique was used for the assessment of the activity of n-3 and n-6 polyunsaturated fatty acid (PUFA) pathways and arachidonic acid concentration. ELISA and multiplex immunoassay kits were applied for the measurement of eicosanoid, cytokine, and chemokine levels. The Western blot technique was applied to determine the expression of proteins involved in the inflammation pathway. NAC decreased hepatic n-6 PUFA activity in all examined lipid pools and decreased the hepatic content of arachidonic acid as a pro-inflammatory precursor in each lipid pool, especially in the phospholipid fraction in rats with fatty lipid disease. NAC administration abolished 5-LOX expression, leading to a decrease in the content of pro-inflammatory leukotriene B4 and leukotriene C4. In rats with steatosis, NAC weakened NF-κB expression and raised Nrf-2 expression, inhibiting the synthesis of pro-inflammatory cytokines and chemokines. NAC treatment significantly rate-limited the progression of simple hepatic steatosis to hepatitis in a rat model of MASLD.

The Relevance of Thrombo-Inflammatory Biomarkers and Their Relationship with Circulating Glycosaminoglycans in End-Stage Renal Disease Patients

En-stage renal disease (ESRD) is a growing public health problem. The atherosclerotic cardiovascular complications are the leading causes of mortality and morbidity in the ESRD. In this study, we sought to quantify the levels of thrombo-inflammatory biomarkers in an ESRD patients in comparison to healthy controls to determine their relevance in thrombo-inflammation and adverse outcomes. The levels of D-Dimer, C-reactive protein (CRP), plasminogen activator inhibitor 1 (PAI-1) antigen, functional PAI-1, thrombin activatable fibrinolysis inhibitor, tissue plasminogen activator, von Willebrand factor, and anti-PF4 IgG and microparticle (MP) activity were quantified by using commercially available ELISA immunoassays for each of the ESRD (n = 73) and control plasma samples (n = 10). The levels of endogenous glycosaminoglycans (GAGs) were quantified by utilizing a Heparin Red Probe (Redprobes UG, Germany). The collected data were analyzed to demonstrate the relationship between various parameters. All the tested biomarkers were increased in ESRD patients in comparison to healthy controls (p < 0.05). These biomarkers have shown significant correlations within each other except for anti-PF4 Ig G and MPs. The CRP levels were significantly higher in patients who had coronary artery disease (CAD) (p < 0.05), but there was no significant difference in other biomarkers according to the cardiovascular outcomes. In the multivariate analysis, the CRP (odds ratio: 1.19; 95% confidence interval: 1.01-1.41; p: 0.03) value was an independent predictor of CAD. In this study, we demonstrated increased levels of 10 different biomarkers in ESRD patients. The CRP levels can be a good predictor of CAD in ESRD patients.

Senescence and autophagy relation with the expressional status of non-canonical estrogen receptors in testes and adrenals of roe deer (Capreolus capreolus) during the pre-rut period

The roe deer bucks represent a spontaneous model to study the synchronized testicular involution and recrudescence cycles. However, cellular processes and hormonal control of steroidogenic glands are scarcely known. For the present study testes and adrenal glands obtained from roe deer during the pre-rut season were used. We aimed to determine (i) senescence and autophagy involvement in testis atrophy (immunohistochemical analysis for tumor suppressor protein encoded by the cyclin-dependent kinase inhibitor 2A; p16 and microtubule-associated protein 1A/1B-light chain 3; LC3, respectively), (ii) the size of the adrenal cortex and medulla (morphometric analysis), (iii) G-protein coupled estrogen receptor (GPER) and estrogen-related receptors (ERRs; type α, β, and Y) distribution and expression (qRT-PCR and immunohistochemical analyses) and (iv) serum testosterone and estradiol levels (immunoassay ELISA). This study revealed pre-rut characteristics of testis structure with the presence of both senescence and autophagy-positive cells and higher involvement of senescence, especially in spermatogenic cells (P < 0.05). In the adrenal cortex, groups of cells exhibiting shrinkage were observed. The presence of ERRs in cells of the seminiferous epithelium and interstitial Leydig cells and GPER presence distinctly in Leydig cells was revealed. In adrenals, these receptors were localized in groups of normal-looking cells and those with shrinkage. Morphometric analysis showed differences in cortex width which was smaller (P < 0.05) than that of the medulla. A weak immunohistochemical signal was observed for ERRβ when compared to ERRα and ERRγ. The mRNA expression level of ERRα and ERRγ was lower (P < 0.001 and P < 0.05, respectively) while ERRβ was higher (P < 0.001) in adrenals when compared to testes. mRNA GPER expression was similar in both glands. In the pre-rut season, the testosterone level was 4.89 ng/ml while the estradiol level was 0.234 ng/ml. We postulate that: (i) senescence and autophagy may be involved in both reinitiation of testis function and/or induction of abnormal processes, (ii) hormonal modulation of testis inactivity may affect adrenal cortex causing cell shrinkage, (iii) ERRs and GPER localization in spermatogenic cells and interstitial cells, as well as cortex cells, may maintain and control the morpho-functional status of both glands, and (iv) androgens and estrogens (via ERRs and GPER) drive cellular processes in the testis and adrenal pre-rut physiology.

An Electrochemical Immunosensor Based on Carboxylated Graphene/SPCE for IgG-SARS-CoV-2 Nucleocapsid Determination

The COVID-19 pandemic has emphasized the importance and urgent need for rapid and accurate diagnostic tests for detecting and screening this infection. Our proposal was to develop a biosensor based on an ELISA immunoassay for monitoring antibodies against SARS-CoV-2 in human serum samples. The nucleocapsid protein (N protein) from SARS-CoV-2 was employed as a specific receptor for the detection of SARS-CoV-2 nucleocapsid immunoglobulin G. N protein was immobilized on the surface of a screen-printed carbon electrode (SPCE) modified with carboxylated graphene (CG). The percentage of IgG-SARS-CoV-2 nucleocapsid present was quantified using a secondary antibody labeled with horseradish peroxidase (HRP) (anti-IgG-HRP) catalyzed using 3,3',5,5'-tetramethylbenzidine (TMB) mediator by chronoamperometry. A linear response was obtained in the range of 1:1000-1:200 v/v in phosphate buffer solution (PBS), and the detection limit calculated was 1:4947 v/v. The chronoamperometric method showed electrical signals directly proportional to antibody concentrations due to antigen-antibody (Ag-Ab) specific and stable binding reaction.

Immune Profile of Blood, Tissue and Peritoneal Fluid: A Comparative Study in High Grade Serous Epithelial Ovarian Cancer Patients at Interval Debulking Surgery.

High-grade serous epithelial ovarian carcinoma (HGSOC) is an immunogenic tumor with a unique tumor microenvironment (TME) that extends to the peritoneal cavity. The immunosuppressive nature of TME imposes the major challenge to develop effective treatment options for HGSOC. Interaction of immune cells in TME is an important factor. Hence, a better understanding of immune profile of TME may be required for exploring alternative treatment options. Immune profiling of peritoneal fluid (PF), tumor specimens, and blood were carried out using flowcytometry, ELISA, and Procartaplex immunoassay. The frequency of CD56BrightNK cells and expression of functional receptors were reduced in PF. Increased activating NKp46+CD56DimNK cells may indicate differential antitumor response in PF. Functional receptors on NK, NKT-like and T cells were reduced more drastically in tumor specimens. Soluble ligands MIC-B and PVR were reduced, whereas B7-H6 was increased in PF. Dissemination of tumor cells contributes to soluble ligands in PF. A differential cytokine profile was found in serum and PF as IL-2, IL-8, IL-15, IL-27, IFN-γ, and GM-CSF were elevated specifically in PF. In conclusion, the differential immune profile and correlation of soluble parameters and NK cell receptors with chemo response score may add knowledge to understand anti-tumor immune response to develop effective treatment modality.

Evaluating the biological variability of blood‐based biomarkers for use in clinical trials

AbstractBackgroundBlood‐based biomarkers offer the possibility of inexpensive, minimally invasive, and accessible diagnostic tools for Alzheimer’s Disease (AD). In the context of clinical trials, these biomarkers hold promise for subject stratification and monitoring the effects of therapeutic interventions. The day‐to‐day variability of these measures must be robustly characterized to determine which blood‐based biomarkers would be the most sensitive for detecting treatment‐related responses. In this study we evaluate biomarker performance in plasma and identify the most reliable candidates for clinical trials.MethodEDTA plasma samples were collected from healthy controls (n=20, 50% female, mean age 56.03 ± 9.43 years) at baseline, 2 weeks, and 4 weeks. Singleplex ELISA and multiplex MesoScale Discovery (MSD) immunoassays were used to measure over 25 biomarkers that were selected for their relevance to dysregulated processes in AD and previously validated in cerebrospinal fluid (CSF). Technical performance was assessed by calculating intra‐ and inter‐plate coefficients of variation (CVs), analyzing dilution linearity, and evaluating freeze‐thaw stability. Biological intra‐individual variability was examined by calculating a biotemporal CV for each subject across the three timepoints.ResultsMost biomarkers were quantifiable in plasma and exhibited technical performance within our thresholds (intra‐ and inter‐plate CV &lt; 15%). The biomarkers exhibited a range of freeze‐thaw stability, with mean CVs from 5.2% for Adiponectin, a regulator of glucose metabolism, to 30.2% for GRP78, an ER chaperone protein. Plasma levels were relatively stable across time (biotemporal CV &lt; 20%) for several biomarkers, including 24‐OHC, VEGF‐D, Flt‐1, PlGF, sST2, and sTREM2. Several proteins exhibited significant variability over time (biotemporal CV &gt; 20%), such as 8‐OHdG, GRP78, and YKL‐40.ConclusionKey metabolic, vascular, and inflammatory biomarkers were reliably measurable in the plasma of healthy individuals. Several markers exhibited substantial variability over a 2‐4 week time period, which should be taken into consideration when selecting appropriate markers for assessing drug‐target engagement. Designing future clinical trials with multiple baseline visits will allow us to establish biological variability metrics for each individual’s biomarker levels, providing greater confidence in determining treatment effects. Evaluating biomarker stability in other large‐scale subject populations will be necessary to best inform blood‐based biomarker selection for clinical trials.