Serum Of Patients Research Articles

The Relationship Between Serum IgE Level and IL-4 and IL-13 Cytokines in Colorectal Cancer Patients

ABSTRACT Background Colorectal cancer (CRC) is the most common malignancy of the digestive system in the world. Immune cells and molecules in tumor microenvironment are crucial.Identifying immune system components in cancer aids in biomarker discovery. This study investigated the serum IgE levels and expression of IL-4 and IL-13 in the tissue and serum of CRC patients and explored their possible association with pathological and clinical factors. Materials and methods Thirty-six patients with CRC and 36 healthy individuals were involved in the study. Tissues and blood samples were collected. Serum levels of IgE and IL-4 and IL-13 were analyzed using the ELISA method. The quantitative Real-Time PCR (qRT-PCR) technique was used to assess the expression levels of the cytokines in CRC tissue samples in comparison with the adjacent control tissue. Results Our results revealed that the serum level of IL-4 and IL-13 and also their gene expression levels were significantly decreased in CRC patients compared to the controls. The results of this study revealed that there is no significant difference in the serum levels of IgE between CRC patients and the control group. Conclusion All in all, the results of the current research suggest that the expression levels of IL-13, IL-4, and IgE vary between CRC tissue.

Comparative targeted lipidomics between serum and cerebrospinal fluid of multiple sclerosis patients shows sex and age-specific differences of endocannabinoids and glucocorticoids

Multiple sclerosis (MS) is a complex chronic neuroinflammatory disease characterized by demyelination leading to neuronal dysfunction and neurodegeneration manifested by various neurological impairments. The endocannabinoid system (ECS) is a lipid signalling network, which plays multiple roles in the central nervous system and the periphery, including synaptic signal transmission and modulation of inflammation. The ECS has been identified as a potential target for the development of novel therapeutic interventions in MS patients. It remains unclear whether ECS-associated metabolites are changed in MS and could serve as biomarkers in blood or cerebrospinal fluid (CSF). In this retrospective study we applied targeted lipidomics to matching CSF and serum samples of 74 MS and 80 non-neuroinflammatory control patients. We found that MS-associated lipidomic changes overall did not coincide between CSF and serum. While glucocorticoids correlated positively, only the endocannabinoid (eCB) 2-arachidonoyl glycerol (2-AG) showed a weak positive correlation (r = 0.3, p < 0.05) between CSF and serum. Peptide endocannabinoids could be quantified for the first time in CSF but did not differ between MS and controls. MS patients showed elevated levels of prostaglandin E2 and steaorylethanolamide in serum, and 2-oleoylglycerol and cortisol in CSF. Sex-specific differences were found in CSF of MS patients showing increased levels of 2-AG and glucocorticoids in males only. Overall, arachidonic acid was elevated in CSF of males. Interestingly, CSF eCBs correlated positively with age only in the control patients due to the increased levels of eCBs in young relapsing-remitting MS patients. Our findings reveal significant discrepancies between CSF and serum, underscoring that measuring eCBs in blood matrices is not optimal for detecting MS-associated changes in the central nervous system. The identified sex and age-specific changes of analytes of the stress axis and ECS specifically in the CSF of MS patients supports the role of the ECS in MS and may be relevant for drug development strategies.

Silencing LncRNA SNHG14 alleviates renal tubular injury via the miR-483-5p/HDAC4 axis in diabetic kidney disease.

This study explored the clinical value of long non-coding RNA small nucleolar RNA host gene 14 (SNHG14) in diabetic kidney disease (DKD) and the mechanism of renal tubular injury. Patients with DKD, type 2 diabetes mellitus (T2DM) and healthy individuals (HVs) were included, as well as the human proximal tubular epithelial cell line (HK-2) induced by high glucose was also included. The mRNA levels of SNHG14 in the serum and cells were detected using RT-qPCR. Diagnostic significance was examined using receiver operating characteristic (ROC) analysis. A commercial test kit, flow cytometry, and enzyme-linked immunosorbent assays were employed to assess reactive oxygen species (ROS) production, apoptosis, inflammatory factor secretion, and extracellular matrix protein levels in HK-2 cells. The dual-luciferase reporter assay and RNA immunoprecipitation were used to validate miR-483-5p concerning SNHG14 or histone deacetylase 4 (HDAC4). SNHG14 and HDAC4 levels were elevated in the serum of DKD patients and HG-induced HK-2 cells, while miR-483-5p levels were decreased (P < 0.001). SNHG14 increased HDAC4 levels by sponging miR-483-5p. Elevated SNHG14 levels significantly differentiated DKD patients from HVs (AUC = 0.944) and T2DM (AUC = 0.867). Silencing of SNHG14 alleviated HG-induced ROS production and apoptosis as well as the over-secretion of inflammatory factors and extracellular matrix proteins; however, this alleviation was typically suppressed by low expression of miR-483-5p (P < 0.001). Elevated miR-483-5p alleviates HG-induced renal tubular injury, but this alleviation is suppressed by HDAC4 overexpression. In summary, suppression of SNHG14 has been shown in our study to mitigate renal tubular injury in DKD by regulating apoptosis, oxidative stress, inflammation, and fibrosis through the miR-483-5p/HDAC4 axis.

Efficacy of 100% autologous platelet-rich plasma and 100% autologous serum in dry eye disease: a randomised controlled trial

ObjectiveThe 0bjective is to compare treatment effects between undiluted autologous platelet-rich plasma (APRP) and autologous serum (AS) in patients with moderate-to-severe dry eye disease (DED).Methods and analysisA single-centre, randomised, double-masked,...

Type 1 diabetes mellitus; ovarian reserve; anti-Müllerian hormone; ovarian function

BACKGROUND: Prolonged course of diabetes mellitus often leads to the development of vascular complications, insufficiency of ovarian function. The occurrence of diabetes mellitus in women before pregnancy (pregestational diabetes mellitus) it can have various adverse consequences for the mother, fetus, child and the course of pregnancy. Pregnancy planning in women with pregestational diabetes significantly improves the outcomes of pregnancy and childbirth. AIM: The aim of this study was to analyze gonadotropic and ovarian function parameters in patients with type 1 diabetes mellitus depending on the age of onset, duration and compensation level of the disease. MATERIALS AND METHODS: The patients included into this clinical prospective study were divided into two groups. The main group consisted of 216 patients with type 1 diabetes mellitus aged 18 to 39 years (mean age: 27.1 ± 3.1 years). The control group consisted of 30 healthy women of reproductive age. Chemiluminescent immunoassay was used to determine blood serum levels of anti-Müllerian hormone, follicle-stimulating hormone, luteinizing hormone, prolactin, estradiol, testosterone, free testosterone, and sex hormone-binding globulin from day 2 to day 5 of the menstrual cycle. Serum progesterone levels were measured on days 20–23 of the menstrual cycle for three consecutive cycles. Glycated hemoglobin levels were also studied in the blood serum of patients with type 1 diabetes mellitus. All the patients underwent pelvic ultrasound examination to assess the cohort of antral follicles and ovarian volume. RESULTS: The average age of menarche in patients with type 1 diabetes mellitus who developed the disease at the age of under 10 years was 14.2 ± 1.2 years. This was higher than in the group of girls with the onset of the disease over the age of 10 years (13.5 ± 1.2 years) and in women of the control group (11.9 ± 1.1 years). When assessing the ovarian reserve in patients with type 1 diabetes mellitus, we found a decrease in anti-Müllerian hormone level and antral follicle count in the group of women with disease onset at the age of under 10 years. We did not find differences in the ovarian reserve parameters in patients depending on disease duration. Normogonadotropic normoprolactinemic ovarian insufficiency was detected in 154 (71.3%) of 216 patients, while the rest 62 (28.7%) women had an ovulatory menstrual cycle. In patients with anovulation, luteal phase deficiency and ovulatory menstrual cycle, we found no differences between the age of development of type 1 diabetes mellitus, its duration, glycated hemoglobin levels, total daily insulin dose and insulin dose per kilogram of body mass. In patients receiving intensive insulin therapy with continuous subcutaneous insulin infusion, glycated hemoglobin level was lower than in those receiving multiple dose insulin injection therapy. CONCLUSIONS: The average age of menarche in patients with type 1 diabetes mellitus is higher than in healthy women. The ovarian reserve parameters in patients with early onset of the disease, before 10 years of age, are lower than in patients who developed type 1 diabetes mellitus after 10 years of age. Duration of pregnancy planning period depends on compensation level of the disease and lasts from six to 12 months. This requires the doctor and patient to prepare for the implementation of reproductive tasks as early as possible, especially in patients whose disease onset occurred in pre-pubertal period.

Elevated serum mtDNA in COVID-19 patients is linked to SARS-CoV-2 envelope protein targeting mitochondrial VDAC1, inducing apoptosis and mtDNA release.

Mitochondria dysfunction is implicated in cell death, inflammation, and autoimmunity. During viral infections, some viruses employ different strategies to disrupt mitochondria-dependent apoptosis, while others, including SARS-CoV-2, induce host cell apoptosis to facilitate replication and immune system modulation. Given mitochondrial DNAs (mtDNA) role as a pro-inflammatory damage-associated molecular pattern in inflammatory diseases, we examined its levels in the serum of COVID-19 patients and found it to be high relative to levels in healthy donors. Furthermore, comparison of serum protein profiles between healthy individuals and SARS-CoV-2-infected patients revealed unique bands in the COVID-19 patients. Using mass spectroscopy, we identified over 15 proteins, whose levels in the serum of COVID-19 patients were 4- to 780-fold higher. As mtDNA release from the mitochondria is mediated by the oligomeric form of the mitochondrial-gatekeeper-the voltage-dependent anion-selective channel 1 (VDAC1)-we investigated whether SARS-CoV-2 protein alters VDAC1 expression. Among the three selected SARS-CoV-2 proteins, small envelope (E), nucleocapsid (N), and accessory 3b proteins, the E-protein induced VDAC1 overexpression, VDAC1 oligomerization, cell death, and mtDNA release. Additionally, this protein led to mitochondrial dysfunction, as evidenced by increased mitochondrial ROS production and cytosolic Ca2+ levels. These findings suggest that SARS-CoV-2 E-protein induces mitochondrial dysfunction, apoptosis, and mtDNA release via VDAC1 modulation. mtDNA that accumulates in the blood activates the cGAS-STING pathway, triggering inflammatory cytokine and chemokine expression that contribute to the cytokine storm and tissue damage seen in cases of severe COVID-19.

Diagnostic value and clinical significance of lncRNA &lt;i&gt;NEAT1 &lt;/i&gt;combined with miR-425-3p in children with viral myocarditis

Background. Viral myocarditis (VMC) is common in children. Previous studies have reported the clinical value of nuclear paraspeckle assembly transcript 1 (NEAT1) and microRNA-425-3p (miR-425-3p) in certain diseases, but not in VMC. This article was designed to investigate the expression of long noncoding RNA (lncRNA) NEAT1 and miR-425-3p in the serum of patients with VMC and their clinical significance. Methods. We assessed VMC and healthy patients and analyzed differences in the expression levels of NEAT1 and miR-425-3p. The correlation and targeting relationship between the two were reported by Spearman correlation analysis and luciferase reporter assay. ROC curves were plotted to reflect the diagnostic effect of both. In addition, according to the 12-month prognostic effect grouping, patients with VMC were separated into a group with good vs. poor prognosis, and the difference in the expression levels of NEAT1 and miR-425-3p between the two groups were analyzed. The ability of the two markers in the prognosis of VMC was further analyzed by multiple logistic regression. Results. NEAT1 expression was up-regulated in VMC and miR-425-3p expression was down-regulated, and there was a negative correlation and targeting link between the two. The diagnostic efficacy of both NEAT1 and miR-425-3p was higher than that of a single indicator. High expression of NEAT1 and low expression of miR-425-3p were found in VMC patients with poor prognosis. Both were independent influencers of VMC prognosis. Conclusion. NEAT1 and miR-425-3p expressions were affected by VMC and had important clinical implications for VMC, indicating for the first time the clinical function of NEAT1 and miR-425-3p in VMC.

Inflammatory priming of human mesenchymal stem cells induces osteogenic differentiation via the early response gene IER3.

Mesenchymal stem cells (MSCs) have gained tremendous interest due to their overall potent pro-regenerative and immunomodulatory properties. In recent years, various invitro and preclinical studies have investigated different priming ("licensing") approaches to enhance MSC functions for specific therapeutic purposes. In this study, we primed bone marrow-derived human MSCs (hMSCs) with an inflammation cocktail designed to mimic the elevated levels of inflammatory mediators found in serum of patients with severe injuries, such as bone fractures. We observed a significantly enhanced osteogenic differentiation potential of primed hMSCs compared to untreated controls. By RNA-sequencing analysis, we identified the immediate early response 3 (IER3) gene as one of the top-regulated genes upon inflammatory priming. Small interfering RNA knockdown experiments established IER3 as a novel positive regulator of osteogenic differentiation. Mechanistic analysis further revealed that IER3 deletion significantly downregulated bone marrow stromal cell antigen 2 (BST2) expression and extracellular signal-related kinase 1/2 (ERK1/2) phosphorylation in hMSCs, suggesting that IER3 regulates osteogenic differentiation through BST2 and ERK1/2 signaling pathway activation. On the basis of these findings, we propose IER3 as a novel therapeutic target to promote hMSC osteoblastogenesis, which might be of high clinical relevance, for example, in patients with osteoporosis or compromised fracture healing.

Association between the Val66Met (rs6265) polymorphism of the brain-derived neurotrophic factor (BDNF) gene, BDNF protein level in the blood and the risk of developing early‑onset Parkinson's disease.

Brain-derived neurotrophic factor (BDNF) is involved in the maintenance of dopamine level and the survival of dopaminergic neurons, which may affect the functionality of brain structures responsible for motor and cognitive function. The aim of the study was to assess the association of individual and combined single nucleotide polymorphism (SNP) in the rs6265 BDNF (Val66Met), rs397595 DAT (SLC6A3), and rs4680 COMT (Val158Met) genes with early‑onset of Parkinson's disease (PD) patients. Moreover, we assessed the association between the BDNF Val66Met polymorphism and the level of BDNF protein in the serum of patients with PD and controls. The study involved 163 patients with idiopathic PD divided into early onset (<55 years) and late‑onset (>55 years) groups and 91 healthy age‑matched people (Control). The SNP were determined using the TaqMan Real‑Time PCR method. Serum BDNF levels were determined by ELISA assay. The risk of developing early PD in people with the BDNF genotype AG increases threefold in comparison with the carriers of the BDNF genotype GG. In PD patients and healthy people with the BDNF genotypes AG and AA, a lower serum BDNF level was found compared to those with the BDNF genotype GG in both groups. The results of our study indicate that the presence of the Val66Met BDNF gene polymorphism is associated with reduced blood BDNF levels and an elevated risk of developing early‑onset PD. This effect appears to be more pronounced in men.

9330 Atumelnant (crn04894) Induces Rapid And Sustained Reductions In Serum And Urine Cortisol In Patients With Acth-Dependent Cushing Syndrome During A Phase 1b/2a, Single Center, 10-day, Inpatient, Open-Label Study

Abstract Disclosure: H. Elenius: Grant Recipient; Self; Crinetics Pharmaceuticals, Inc. R. McGlotten: Grant Recipient; Self; Crinetics Pharmaceuticals, Inc. C. Moore: Grant Recipient; Self; Crinetics Pharmaceuticals, Inc. Y.B. Omotosho: Grant Recipient; Self; Crinetics Pharmaceuticals, Inc. A. Ayala: Employee; Self; Crinetics Pharmaceuticals, Inc.. Stock Owner; Self; Crinetics Pharmaceuticals, Inc. Y. Wu: Employee; Self; Crinetics Pharmaceuticals, Inc.. Stock Owner; Self; Crinetics Pharmaceuticals, Inc. P.J. Trainer: Employee; Self; Crinetics Pharmaceuticals, Inc.. Stock Owner; Self; Crinetics Pharmaceuticals, Inc. R.S. Struthers: Employee; Self; Crinetics Pharmaceuticals, Inc.. Stock Owner; Self; Crinetics Pharmaceuticals, Inc. A. Krasner: Employee; Self; Crinetics Pharmaceuticals, Inc.. Stock Owner; Self; Crinetics Pharmaceuticals, Inc. S.F. Betz: Employee; Self; Crinetics Pharmaceuticals, Inc.. Stock Owner; Self; Crinetics Pharmaceuticals, Inc. L.K. Nieman: Grant Recipient; Self; Crinetics Pharmaceuticals, Inc.. Atumelnant is a potent, once-daily oral, nonpeptide, first-in-class competitive and selective melanocortin type 2 receptor (MC2R) antagonist that blocks ACTH-mediated G-protein activation and subsequent signaling. It is being developed for the treatment of ACTH-dependent Cushing Syndrome (ADCS) and classic congenital adrenal hyperplasia. We report preliminary data from the first-in-disease, dose-finding study of atumelnant in patients with ADCS (NCT05804669). Inpatient participants with active ADCS (24h urine free cortisol (UFC) &amp;gt;1.3xULN, ACTH &amp;gt;10 pg/mL) received atumelnant 80 mg once daily at 08.00 for 10 days (D1-10) followed by a 4-day washout. Endpoints included changes in UFC (upper limit of normal (ULN) 45 ug/d), early morning cortisol (EMC), and early morning ACTH (EMA, ULN 46 pg/mL). Daily questionnaires assessed adverse events (AEs) and signs and symptoms of adrenal insufficiency (AI) and ADCS. Data are shown as median (range). We report 5 participants (4 men; 4 Cushing disease, 1 ectopic ACTH; age 47 years (34-55)); each completed the study. All participants developed biochemical evidence of AI (EMC &amp;lt;5 mcg/dL) after 2 days (1-10) of atumelnant administration and then commenced physiologic hydrocortisone (HC) add-back. Compared to D1, biochemical disease control (normal UFC plus EMC &amp;lt;5 mcg/dL) was shown on D11 in all participants, while receiving HC replacement: UFC 252 mcg (99-293) vs 24 mcg (3.9-51); EMC 14 mcg/dL (10.7-18.1) vs 1.4 (1.0-4.7). This was associated with an increase in EMA: 52.1 pg/mL (33-1088) vs 78 pg/mL (48.8-4045). HC was discontinued when EMC was ≥7 mcg/dL, on D13 (D12-18). Many pre-existing features of ADCS improved during atumelnant administration, including poor concentration, insomnia (n=3 each); brain fog, anxiety, irritability (n=2 each); fatigue, poor memory, low libido, depression, edema and bloating (n=1 each). Low testosterone, neutrophilia (n=3 each) and leukocytosis (n=2) observed at baseline resolved during atumelnant administration. Anti-hypertensive therapy was paused or reduced in 3 subjects. New fatigue, headache, nausea, anorexia, edema (n=2 for each) all coincided with development of biochemical AI; most symptoms improved with HC add-back. Serious adverse events included AI and one non-treatment related transient GI bleed 19 days after the last dose. All other AEs were mild or moderate: transient increases of creatinine (n=2), and ALT (&amp;lt;3xULN, n=2). In conclusion, the first 5 patients with ADCS to receive once daily, oral atumelnant experienced rapid lowering of serum and urine cortisol and improvement or resolution of some signs and symptoms of ADCS. Atumelnant was generally well-tolerated. The observed compensatory increase in ACTH was not associated with loss of efficacy or clinical sequelae. This ongoing study will further explore the relationship between atumelnant dose, including lower doses, and therapeutic response. Presentation: 6/3/2024

7908 GPNMB Promotes Tumor Growth And Is A Biomarker For Lymphangioleiomyomatosis (LAM)

Abstract Disclosure: E. Gibbons: None. M. Taya: None. H. Wu: None. S. Lopa: None. J. Moss: None. E.P. Henske: None. F.X. McCormack: None. S.R. Hammes: None. Lymphangioleiomyomatosis (LAM) is a rare, progressive cystic lung disease affecting almost exclusively female-sexed individuals. The cysts represent regions of lung destruction caused by smooth muscle tumors containing mutations in one of the two tuberous sclerosis (TSC) genes. mTORC1 inhibition slows but does not stop LAM advancement. Furthermore, monitoring disease progression is hindered by insufficient biomarkers. Therefore, new treatment options and biomarkers are needed. LAM cells express melanocytic markers, including Glycoprotein Non-Metastatic Melanoma Protein B (GPNMB). The function of GPNMB in LAM is currently unknown; however, GPNMB’s unique cell surface expression on tumor versus benign cells makes GPNMB a potential therapeutic target, and persistent release of its extracellular ectodomain suggests potential as a serum biomarker. Here we establish that GPNMB regulates TSC2-null tumor cell invasion as well as TSC2-null xenograft tumor growth, and that GPNMB expression is dependent on mTORC1 signaling. Further, we show that GPNMB’s ectodomain is released from the cell surface of TSC2-null cells by ADAM10 and 17 proteases, and we identify the protease target sequence on GPNMB. In fact, mutating this cleavage sequence so that the GPNMB ectodomain can no longer be shed results in cessation of TSC2-null tumor growth. Finally, we demonstrate that GPNMB’s ectodomain is present at higher levels in LAM patient serum compared to healthy controls, and that ectodomain levels decrease with mTORC1 inhibition, making it a potential LAM biomarker. Presentation: 6/3/2024

7908 GPNMB Promotes Tumor Growth and is a Biomarker for Lymphangioleiomyomatosis (LAM)

Abstract Disclosure: E. Gibbons: None. M. Taya: None. H. Wu: None. S. Lopa: None. J. Moss: None. E.P. Henske: None. F.X. McCormack: None. S.R. Hammes: None. Lymphangioleiomyomatosis (LAM) is a rare, progressive cystic lung disease affecting almost exclusively female-sexed individuals. The cysts represent regions of lung destruction caused by smooth muscle tumors containing mutations in one of the two tuberous sclerosis (TSC) genes. mTORC1 inhibition slows but does not stop LAM advancement. Furthermore, monitoring disease progression is hindered by insufficient biomarkers. Therefore, new treatment options and biomarkers are needed. LAM cells express melanocytic markers, including Glycoprotein Non-Metastatic Melanoma Protein B (GPNMB). The function of GPNMB in LAM is currently unknown; however, GPNMB’s unique cell surface expression on tumor versus benign cells makes GPNMB a potential therapeutic target, and persistent release of its extracellular ectodomain suggests potential as a serum biomarker. Here we establish that GPNMB regulates TSC2-null tumor cell invasion as well as TSC2-null xenograft tumor growth, and that GPNMB expression is dependent on mTORC1 signaling. Further, we show that GPNMB’s ectodomain is released from the cell surface of TSC2-null cells by ADAM10 and 17 proteases, and we identify the protease target sequence on GPNMB. In fact, mutating this cleavage sequence so that the GPNMB ectodomain can no longer be shed results in cessation of TSC2-null tumor growth. Finally, we demonstrate that GPNMB’s ectodomain is present at higher levels in LAM patient serum compared to healthy controls, and that ectodomain levels decrease with mTORC1 inhibition, making it a potential LAM biomarker. Presentation: 6/3/2024

9308 GPNMB Promotes Tumor Growth And Is A Biomarker For Lymphangioleiomyomatosis (LAM)

Abstract Disclosure: E. Gibbons: None. M. Taya: None. H. Wu: None. S. Lopa: None. J. Moss: None. E.P. Henske: None. F.X. McCormack: None. S.R. Hammes: None. Lymphangioleiomyomatosis (LAM) is a rare, progressive cystic lung disease affecting almost exclusively female-sexed individuals. The cysts represent regions of lung destruction caused by smooth muscle tumors containing mutations in one of the two tuberous sclerosis (TSC) genes. mTORC1 inhibition slows but does not stop LAM advancement. Furthermore, monitoring disease progression is hindered by insufficient biomarkers. Therefore, new treatment options and biomarkers are needed. LAM cells express melanocytic markers, including Glycoprotein Non-Metastatic Melanoma Protein B (GPNMB). The function of GPNMB in LAM is currently unknown; however, GPNMB’s unique cell surface expression on tumor versus benign cells makes GPNMB a potential therapeutic target, and persistent release of its extracellular ectodomain suggests potential as a serum biomarker. Here we establish that GPNMB regulates TSC2-null tumor cell invasion as well as TSC2-null xenograft tumor growth, and that GPNMB expression is dependent on mTORC1 signaling. Further, we show that GPNMB’s ectodomain is released from the cell surface of TSC2-null cells by ADAM10 and 17 proteases, and we identify the protease target sequence on GPNMB. In fact, mutating this cleavage sequence so that the GPNMB ectodomain can no longer be shed results in cessation of TSC2-null tumor growth. Finally, we demonstrate that GPNMB’s ectodomain is present at higher levels in LAM patient serum compared to healthy controls, and that ectodomain levels decrease with mTORC1 inhibition, making it a potential LAM biomarker. Presentation: 6/3/2024

Analysis of changes in the levels of soluble molecules CD50, CD54 and CD95 in the blood serum of patients with uterine fibroids depending on the characteristics of the disease (pilot study)

Background. Currently, the search for circulating immunological and inflammatory markers that play a significant role in the pathogenesis of uterine fibroids (UF) is relevant.Aim. Changes in the serum levels of soluble molecules CD50 (sCD50), CD54 (sCD54) and CD95 (sCD95) in patients with UF depending on the characteristics of the disease.Materials and methods. The study involved 78 patients with UF aged 31–59 years and 45 clinically healthy women of comparable age. The patients were divided into the following groups depending on the localization of the myomatous node: interstitial-subserous localization (n = 17), subserous localization (n = 16), submucosal localization (n = 15), interstitial-submucosal localization (n = 15), interstitial localization (n = 15). In 34.6 % patients, the number of myoma nodes was 4–6, in 46.2 % women there were 2–3 nodes, in 19.2 % – 1 node.Determination of the serum concentration of sCD50, sCD54 and sCD95 molecules was performed using a two-site enzyme immunoassay and expressed in conventional units (U / ml).Results. The average serum concentration of sCD95, sCD54 and sCD50 in all patients with UF was significantly higher than in the control group (p &lt;0.05). The maximum level of the tested molecules was found in UF patients with submucosal localization and the presence of one myomatous node.Conclusions. The detected increase in sCD50, sCD54 and sCD95 levels in UF patients indicates the participation of these proteins in the immunopathogenesis of this pathology. The imbalance in studied proteins levels is most pronounced in submucosal localization of fibroids, which may indicate an unfavorable course of the disease and serve as an additional criterion for selecting patients in the preoperative period.

Microarray-based evaluation of selected recombinant timothy grass allergens expressed in E. Coli and N. Benthamiana

BackgroundTimothy grass (Phleum pratense) is a significant source of allergens, and recombinant allergens are increasingly used for diagnostic purposes. However, the performance of different recombinant allergen production systems in diagnostic assays needs further investigation to optimize their use in clinical settings.ObjectiveThe main objective of this study was to analyze and compare the diagnostic performance of recombinant timothy grass allergens produced in E. coli and N. benthamiana using a custom-made microarray chip.MethodsRecombinant timothy grass allergens Phl p 1, Phl p 2, Phl p 5, Phl p 6, Phl p 11, and Phl p 12 were produced in E. coli and/or N. benthamiana. A total of 113 patient serum samples were tested to evaluate the diagnostic sensitivity, specificity, inter-assay variability, and correlation of allergen-specific IgE detection compared to commercial multiplex tests (ALEX and ISAC). Additionally, the prevalence of sIgE to these allergens was assessed.ResultsPhl p 1, Phl p 2, Phl p 5, Phl p 6 and Phl p 11 showed high or very high positive correlation in immunoreactivity with other commercial multiplex tests. Notably, Phl p 11 fused with maltose-binding protein (MBP) demonstrated high diagnostic specificity and sensitivity, with a 0.3 arbitrary cut-off value. However, a high intra-assay variation was observed. The study also assessed specific IgE prevalence to timothy grass allergens within the tested patient cohort.ConclusionsRecombinant allergens from both E. coli and N. benthamiana demonstrated strong diagnostic potential on the microarray platform, with Phl p 11 (MBP-fused) showing particularly high performance. High intra-assay variation highlights the need for further optimization in allergen formulation and microarray storage conditions. These results highlight the potential of recombinant allergens for diagnostic applications, despite challenges with allergen stability in microarray formats. Specific IgE prevalence to timothy allergens revealed a sensitization profile consistent with findings from multiple studies.Graphical

Bringing PERT to Pediatrics: Initial Experience and Outcomes of a Pediatric Multidisciplinary Pulmonary Embolism Response Team (PERT)

IntroductionMultidisciplinary Pulmonary Embolism Response Teams (PERTs) streamline care of adults with life-threatening pulmonary embolism (PE). Given rarity of pediatric PE, developing a clinical, educational, and research PERT paradigm is a novel and underutilized concept in pediatrics. Research QuestionIs PERT feasible in pediatrics, and does it improve PE care? Study Design and MethodsA strategy-to-execution proposal to launch a pediatric PERT was developed for institutional buy-in. Key stakeholders collectively implemented PERT. Data were collected for the two-year pre- and post-PERT eras, and outcomes were compared. ResultsPERT implementation took 12 months. Our PERT, led by hematology, comprises of pediatric experts in emergency medicine, critical care, interventional cardiology, anesthesiology, and interventional radiology. Data on 30 patients pre-PERT and 31 post-PERT were analyzed. Pre-PERT, 10%(3/30), 13%(4/30), 20%(6/30), and 57%(17/30), and post-PERT, 3%(1/31), 10%(3/31), 16%(5/31), and 71%(22/31) were categorized as high-risk, intermediate-LOW risk, intermediate-HIGH risk, and low-risk PE, respectively. Post-PERT, there were 13 unique PERT activations. PERT was activated on all eligible PE patients and, additionally, on four low-risk PEs. Time-to-echocardiogram was shorter post-PERT (4.7 hrs vs 2 hrs, P=0.0147). Anticoagulation was ordered (90 min vs 54 min, P=0.003) and given sooner (154 min vs 113 min, P=0.049) post-PERT. There were no differences in time-to-reperfusion therapies (12 hrs pre-PERT vs 8.7 hrs post-PERT, P=0.1). Five (83.3%) of six eligible (intermediate-HIGH and high-risk) patients received reperfusion therapies in the post-PERT era compared to three (37.5%) of eight eligible patients in the pre-PERT era (P=0.0001). There were no differences in major bleeding, mortality, or length of stay in either era. InterpretationThe pediatric PERT paradigm was successfully created and adopted locally. Our PERT enhanced access to experts, facilitated timely advanced therapies, and held value for low-risk PE. The University of Texas Southwestern Medical Center (UTSW) and Children’s Health System of Texas pediatric PERT may serve as a best-practice model for streamlining care for pediatric PE.

Expression of Serum DCLK1 in Gastric Cancer Patients and Its Relationship with CEA, CA19-9, and CA72-4

Objective: To investigate the expression of Doublecortin-like kinase-1 (DCLK1) in the serum of gastric cancer patients and its relationship with carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), and carbohydrate antigen 72-4 (CA72-4). Methods: Fifty patients diagnosed with gastric cancer at the hospital from January to December 2021 were selected as the gastric cancer group, and 50 patients diagnosed with chronic atrophic gastritis during the same period were selected as the control group. The serum concentrations of DCLK1, CEA, CA19-9, and CA72-4 were measured in both groups. Cut-off values and AUC (Area Under the Curve) were determined based on the ROC curve, and the expression of DCLK1 and its relationship with CEA, CA19-9, and CA72-4 were analyzed. Results: The average concentrations of DCLK1, CEA, CA19-9, and CA72-4 in the serum of gastric cancer patients were significantly higher than those in the control group (P &lt; 0.05). CA72-4 had the highest sensitivity (62%), CEA had the highest specificity (98%), and DCLK1 had the largest AUC (0.709). The combined diagnosis of gastric cancer using DCLK1, CEA, and CA19-9 resulted in the largest AUC (0.826), with a sensitivity of 82% and a specificity of 76%. Conclusion: The expression of DCLK1, CEA, CA19-9, and CA72-4 in the serum of gastric cancer patients is significantly higher than that in the control group. The combined detection of DCLK1, CEA, and CA19-9 offers better sensitivity and specificity for the diagnosis of gastric cancer.

B-266 Therapeutic Drug Monitoring of Natalizumab and Anti-Natalizumab Antibodies

Abstract Background Natalizumab (NTZ) is a monoclonal antibody directed against the α4-integrin subunit of α4β1 and α4β7 integrins. NTZ is used to treat relapsing and remitting forms of multiple sclerosis. Although a majority of patients on standard dosing (intravenous 300 mg, every 4 weeks) reach therapeutic drug concentrations, about 10% of patients will eventually fail NTZ therapy due to the development of anti-NTZ antibodies. NTZ and anti-NTZ antibody concentrations in serum are used together to provide patient-specific pharmacokinetic and immunogenic assessment to aid in patient management. Methods Serum measurements of NTZ and anti-NTZ antibody levels are performed by lab developed electro-chemiluminescent immunoassays where assay design and principle are equivalent to biologic therapeutic drug monitoring described in our previous publication. The natalizumab drug assay is an immunoassay validated to measure free drug (pharmacodynamically active, ADA-unbound) in serum. The anti-natalizumab antibody assay utilizes a solution phase bridging method designed to measure total binding antibodies (including IgM &amp; IgG subtypes). All anti-natalizumab antibody positive samples are confirmed for drug-specificity by an additional signal suppression step. The anti-natalizumab antibody assay is quantitative and drug-tolerant, meaning that circulating drug in patient serum does not interfere with anti-NTZ detection and quantitation. Results Here, we report concomitant measurement of NTZ and anti-NTZ antibody in 160 patient serum samples. Positive anti-NTZ levels from 24 to 235 ng/mL were found in 7.5% (12/160) patients. Anti-NTZ-free samples (148/160) had NTZ drug levels ranging from undetectable (&amp;lt;1.0) to 85 ug/mL. The mean and median drug level in 90 samples (results &amp;gt;1.0) was 10.8 and 2.7, respectively. 61/90 samples had therapeutic drug concentrations (at least 2.0 ug/mL). In the presence of anti-NTZ, 67% (8/12) had undetectable drug level and (4/12) had mean and median drug levels of 1.2 and 1.3, respectively, which is 9- or 2-fold lower than the mean and median drug levels of the anti-NTZ Ab -free group. Conclusions Low serum NTZ and high anti-NTZ are associated with loss of clinical efficacy, while elevated NTZ may increase progressive multifocal leukoencephalopathy risk. Assays to measure biologic drugs and their anti-drug antibodies in patient serum may be useful tools in dose optimization and patient management.

A-083 Performance evaluation of Sentinel Diagnostics’ ACE assay on the Abbott Alinity c system and ARCHITECT c16000 System

Abstract Background The analytical performances of the Angiotensin Converting Enzyme (ACE) assay for quantitative determination of ACE activity in human serum and plasma have been evaluated on the Abbott Alinity c system and ARCHITECT c16000 System. ACE (dipeptidyl carboxypeptidase) is a glycoprotein peptidyldipeptide hydrolase that cleaves histidylleucine dipeptide from angiotensin I, a relatively inactive decapeptide. The latter is converted to the potent vasoconstrictor, angiotensin II. ACE also inactivates bradykinin. Elevated levels of ACE activity occur in serum of patients with active sarcoidosis, and occasionally in premature infants with respiratory distress syndrome, in adults with tuberculosis, Gaucher’s disease, leprosy, and in many other pathologic conditions involving lung and liver diseases. Methods ACE hydrolyses urylacryloylphenylalanine-glycylglycine (FAPGG) to furylacryloylphenylalanine (FAP) and glycylglycine. Hydrolysis of FAPGG results in a decrease in absorbance at 340 nm. The ACE activity in the sample is determined by comparing the sample reaction rate to that obtained with the Sentinel ACE calibrator. Performances evaluation have been done following the latest CLSI guidelines protocols available. Results Summary of the analytical performances for the tested assay is reported in table below. Conclusions Analytical performances of Sentinel Diagnostics’ ACE assay demonstrate that quantitative determination of ACE activity in human serum and plasma are suitable for the routine measurement of the analyte on Abbott Alinity c system and ARCHITECT c16000 System.

B-080 Development and Validation of a GQ1b IgG ELISA as a Diagnostic Aid for Subacute Demyelinating Polyneuropathies

Abstract Background Autoimmunity targeting GQ1b is associated with group of disorders that includes Miller-Fisher Syndrome (MFS), Bickerstaff Brainstem Encephalitis (BBE) and classic Guillain Barre Syndrome (GBS) with ophthalmoplegia. Collectively these are referred to as GQ1B-IgG related syndromes. The prevalence of GQ1b-IgG in this population of patients is high and has been reported to be &amp;gt;80% in well-defined clinical cohorts. Clinical phenotypic presentation is diverse and requires highly accurate antibody testing. The objective of this study was to evaluate a newly developed custom GQ1b-IgG assay, using a semi-quantitative ELISA-based method. Methods A GQ1b-IgG ELISA was developed and validated. Patient sera along with assay controls and a single-point calibrator are diluted and incubated in duplicate wells in a 96-well plate coated with GQ1b antigen. The plate is washed and incubated with anti-human-IgG secondary antibody conjugated with HRP. After a colorimetric reaction, optical densities (OD) are read. The OD of patient samples are compared against the calibrator, generating a cut-off index (COI). Samples ≥1.0 COI are considered positive. During the validation phase, analytical and clinical performance characteristics were evaluated. Studies included assessment of imprecision at multiple concentrations of GQ1b-IgG, accuracy using method comparison, analytical sensitivity and specificity, reference range, common interferences, plate drift, and clinical performance in relevant disease and control cohorts. Results Intra-assay imprecision (coefficient of variation; %CV) ranged from 1.0 - 16% across all concentrations of GQ1b-IgG. Inter-assay imprecision (%CV) ranged from 12.4 - 44.5% but was &amp;lt;20% at or above the assay cut-off. We found perfect qualitative agreement with an independent ELISA reference method (20 positive and 20 negative samples selected based on reference method results [reference interval &amp;lt;1:100]). Limit of detection (LOD) was calculated at a COI of 0.22. To assess analytical specificity, 40 samples with high concentrations of immunoglobulins (hypergammaglobulinemia [N=15], IgG M-protein [N=15] and systemic lupus erythematosus [N=10] were tested and all were negative. A reference interval study was performed on 140 healthy donor serums, all were negative. Interference studies were conducted on 2 positive and 2 negative serum samples, spiked for highly hemolytic (1000 mg/dL), lipemic (2000 mg/dL) and icteric (60 mg/dL) conditions. There was no impact on the assay even with high levels of a given interferent. Plate drift on 2 distinct patient pools tested across the plate (COI range 0.7 - 1.3) had overall plate CV’s ≤10%. ROC analysis was performed on a cohort of 278 patient samples (15 MFS cases and 263 controls [suspected demyelinating neuropathy or disease mimics]). A cut-off value of 1.0 (COI) was selected based on the Youden index. At this cut-off, clinical sensitivity for MFS was 87% while clinical specificity was 99.2%. Conclusions The observed performance of the GQ1b-IgG ELISA was acceptable for clinical laboratory implementation. A COI of ≥ 1.0 was selected for clinical use.