Blood Plasma Of Patients Research Articles

Disturbances in Muscle Energy Metabolism in Patients with Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease type of motor neuron disorder characterized by degeneration of the upper and lower motor neurons resulting in dysfunction of the somatic muscles of the body. The ALS condition is manifested in progressive skeletal muscle atrophy and spasticity. It leads to death, mostly due to respiratory failure. Within the pathophysiology of the disease, muscle energy metabolism seems to be an important part. In our study, we used blood plasma from 25 patients with ALS diagnosed by definitive El Escorial criteria according to ALSFR-R (Revised Amyotrophic Lateral Sclerosis Functional Rating Scale) criteria and 25 age and sex-matched subjects. Aside from standard clinical biochemical parameters, we used the NMR (nuclear magnetic resonance) metabolomics approach to determine relative plasma levels of metabolites. We observed a decrease in total protein level in blood; however, despite accelerated skeletal muscle catabolism characteristic for ALS patients, we did not detect changes in plasma levels of essential amino acids. When focused on alterations in energy metabolism within muscle, compromised creatine uptake was accompanied by decreased plasma creatinine. We did not observe changes in plasma levels of BCAAs (branched chain amino acids; leucine, isoleucine, valine); however, the observed decrease in plasma levels of all three BCKAs (branched chain alpha-keto acids derived from BCAAs) suggests enhanced utilization of BCKAs as energy substrate. Glutamine, found to be increased in blood plasma in ALS patients, besides serving for ammonia detoxification, could also be considered a potential TCA (tricarboxylic acid) cycle contributor in times of decreased pyruvate utilization. When analyzing the data by using a cross-validated Random Forest algorithm, it finished with an AUC of 0.92, oob error of 8%, and an MCC (Matthew's correlation coefficient) of 0.84 when relative plasma levels of metabolites were used as input variables. Although the discriminatory power of the system used was promising, additional features are needed to create a robust discriminatory model.

Increased expression of miR-320b in blood plasma of patients in response to SARS-CoV-2 infection

Coronavirus disease 2019 (COVID-19) is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Recent research has demonstrated how epigenetic mechanisms regulate the host–virus interactions in COVID-19. It has also shown that microRNAs (miRNAs) are one of the three fundamental mechanisms of the epigenetic regulation of gene expression and play an important role in viral infections. A pilot study published by our research group identified, through next-generation sequencing (NGS), that miR-4433b-5p, miR-320b, and miR-16–2-3p are differentially expressed between patients with COVID-19 and controls. Thus, the objectives of this study were to validate the expression of these miRNAs using quantitative real-time polymerase chain reaction (qRT-PCR) and to perform in silico analyses. Patients with COVID-19 (n = 90) and healthy volunteers (n = 40) were recruited. MiRNAs were extracted from plasma samples and validated using qRT-PCR. In addition, in silico analyses were performed using mirPath v.3 software. MiR-320b was the only miRNA upregulated in the case group com-pared to the control group. The in silico analyses indicated the role of miR-320b in the regulation of the KITLG gene and consequently in the inflammatory process. This study confirmed that miR-320b can distinguish patients with COVID-19 from control participants; however, further research is needed to determine whether this miRNA can be used as a target or a biomarker.

Diagnostic Significance Vasoendothelial Growth Factor Depending on the Severity and Duration of Atopic Dermatitis, as Well as Taking into Account the Presence of Markers of Herpes Virus Infection

Currently, the problem of diagnosis and treatment of diseases associated with disorders of the angiogenesis process, as well as regenerative processes, is acute. Factors regulating the processes of angiogenesis in allergic diseases, including atopic dermatitis, play a key role in maintaining chronic inflammation and can have a significant impact on the course of the disease. Materials and methods: The study is analytical cross-sectional and presented by a comprehensive examination of 140 patients with AtD aged 2 to 12 years (median age 4.2 years), divided into 2 groups: 70 children with an established diagnosis of AtD; 70 children with atopic dermatitis infected with herpes simplex virus (AtD+HVI). The control group consisted of 70 somatically healthy children. A special laboratory examination included the determination of specific IgM and/or IgG class antibodies to herpes simplex virus type 1-2 antigens by enzyme immunoassay (ELISA); determination of the DNA of the studied herpesviruses in blood samples by polymerase chain reaction; determination of vascular endothelial growth factor A (VEGF-A) in the blood plasma of patients by ELISA. The results of our own research: A statistically significant (p <0.001) increase in the level of vascular endothelial growth factor A in blood serum was found in children with AtD compared with the control group. Against the background of infection with the herpes simplex virus, an increase in the level of vascular endothelial growth factor A in blood serum was revealed compared with patients with atopic dermatitis (p < 0.001). There was also a statistically significant increase in serum VEGF-A levels in patients with AtD (p < 0.001) and AtD+HVI (p < 0.001) with an increase in the severity of AtD. This was confirmed by the results of a correlation analysis that revealed the relationship between the level of VEGF-A and the severity of clinical symptoms of the disease.The addition of herpesvirus infection to AtD worsens the clinical symptoms of this disease.

Efficacy of blood plasma spectroscopy for early liver cancer diagnostics in obese patients

Introduction and ObjectivesHepatocellular carcinoma (HCC) represents one of the most common cancers worldwide. A considerable proportion of HCC is caused by cirrhosis related to metabolic dysfunction-associated steatohepatitis (MASH). Due to the increasing prevalence of metabolic syndrome, it is estimated that MASH-related HCC will become the most prevalent etiology of HCC. Currently, HCC screening is based on liver ultrasonography; however, the sensitivity of ultrasonography for early HCC stages in obese patients only reaches 23 %. To date, no studied biomarker shows sufficient efficacy for screening purposes. Nevertheless, the usage of spectroscopic methods offers a new perspective, as its potential use would provide cheap, fast analysis of samples such as blood plasma. Material and MethodsWe employed a combination of conventional and chiroptical spectroscopic methods to study differences between the blood plasma of obese cirrhotic patients with and without HCC. We included 20 subjects with HCC and 17 without evidence of liver cancer, all of them with body mass index ≥ 30. ResultsSensitivities and specificities reached values as follows: 0.780 and 0.905 for infrared spectroscopy, 0.700 and 0.767 for Raman spectroscopy, 0.840 and 0.743 for electronic circular dichroism, and 0.805 and 0.923 for Raman optical activity. The final combined classification model based on all spectroscopic methods reached a sensitivity of 0.810 and a specificity of 0.857, with the highest area under the receiver operating characteristic curve among all models (0.961). ConclusionsWe suggest that this approach can be used effectively as a diagnostic tool in patients who are not examinable by liver ultrasonography. Clinical trial registrationNCT04221347

LGALS3 cfDNA methylation in seminal fluid as a novel prostate cancer biomarker outperforming PSA.

The unmet challenge in prostate cancer (PCa) management is to discriminate it from benign prostate hyperplasia (BPH) due to the lack of specific diagnostic biomarkers. Contemporary research on potential PCa biomarkers is directed towardmethylated cell-free DNA (cfDNA) from liquid biopsies since epigenetic mechanisms are strongly involved in PCa development. In the present research, cfDNA methylation of the LGALS3 gene in blood and seminal plasma of PCa and BPH patients was assessed using pyrosequencing, as well as LGALS3 DNA methylation in tissue biopsies. Liquid biopsy samples were taken from patients with clinical suspicion of PCa, who were subsequently divided into two groups, that is, 42 with PCa and 55 with BPH, according to the histopathological analysis. Statistically significant higher cfDNA methylation of LGALS3 in seminal plasma of BPH than in PCa patients was detected by pyrosequencing. ROC curve analysis showed that it could distinguish PCa and BPH patients with 56.4% sensitivity and 70.4% specificity, while PSA did not differ between the two patient groups. In contrast, there was no statistically significant difference in LGALS3 cfDNA methylation in blood plasma between the two patient groups. In prostate tumor tissue, there was a statistically significant DNA hypermethylation of LGALS3 compared to surrounding nontumor tissue and BPH tissue. The DNA hypermethylation of the LGALS3 gene represents an event specific to PCa development. In conclusion, LGALS3 cfDNA methylation in seminal fluid discriminates early PCa and BPH presenting itself as a powerful novel PCa biomarker highly outperforming PSA.

The effect of atorvastatin on mevalonate levels in blood plasma in patients with coronary heart disease and muscle side effects

The effect of atorvastatin on mevalonate levels in blood plasma in patients with coronary heart disease and muscle side effects

Comparative analysis of the profile of circulating microRNAs in the blood plasma of patients with gliomas

Against the background of modest successes in the development of new diagnostic and therapeutic tools to improve the survival of patients with glial brain tumors, early diagnosis of this pathology remains relevant. Endogenous noncoding miRNAs that regulate the expression of target mRNAs have become attractive targets for the development of circulating biomarker-based assays, because sample acquisition does not require invasive sampling such as biopsy.Purpose of the study. To determine the levels of circulating microRNAs in the blood plasma of patients with glial tumors, meningiomas and apparently healthy donors, using high-output sequencing.Material and methods. 26 blood plasma samples were selected from the biobank data base of the National Medical Research Center for Oncology, and the total RNA was studied using the NGS sequencing method. The sample included: 2 cases of oligodendroglioma (grades 2–3), 6 – astrocytomas of 2–4 degrees of malignancy, 7 – glioblastomas of 4 degrees of malignancy, 7 – benign neoplasms (meningiomas), 4 – control (conditionally healthy donors).Results. During the primary analysis, a pool of 71 differentially expressed microRNAs was identified, the expression of which was tumor-specific: 20 microRNAs for glioblastoma, 4 microRNAs for astrocytoma, 23 microRNAs for oligodendroglioma, 24 microRNAs for meningioma. At the same time, 47 microRNAs showed increased levels in the blood plasma compared to the control group, 15 showed a corresponding decrease in levels. A comparative analysis identified microRNAs that specifically differentiate each tumor type.Conclusion. The results obtained seem promising and set the vector for further research, which will include expanding the sample and validating the identified biomarkers to determine their diagnostic value.

Aberrant baseline cytokine profile in patients with newly diagnosed acquired aplastic anaemia correlates with disease severity and the treatment response

BackgroundImmune dysregulation is crucial in the pathogenesis of acquired aplastic anaemia (aAA). There is paucity of data regarding correlation of baseline cytokine profile with treatment response in aAA. ObjectivePresent prospective case-control study aimed to correlate the baseline cytokines in patients with aAA with the treatment response. MethodsFifty-one patients with newly-diagnosed aAA > 13 years of either sex were enrolled over 1.5 years. Twenty age-and sex-matched healthy controls (HC) were also included. The cytokine profile (IL-2, 4, 6, 8, 10, 17, IFN-γ and TNF-α) in the peripheral blood plasma of aAA patients was performed at the baseline using cytometric bead analysis. The cytokine levels were compared with HC and correlated with response to immunosuppressive therapy (IST) at 3-months. ResultsThe median age of cases was 29 years (range,13–74). The cases had higher mean levels of IL2 (p = 0.326), IL4 (p = 0.038), IL6 (p = 0.000), IL10 (p = 0.002), TNF-α (p = 0.302), IFN-γ (p = 0.569) and IL-17 (p = 0.284) than the HC. The baseline levels of all the cytokines were higher (statistically non-significant) among responders (n = 13) than the non-responders (n = 14) to IST. ConclusionsBaseline cytokine profile in patients with aAA might predict response to the IST. Larger studies are needed to validate our results.

СВЯЗЬ ЦИРКУЛИРУЮЩИХ УРОВНЕЙ АМИНОКИСЛОТ С РАЗВЕТВЛЕННОЙ ЦЕПЬЮ У ПАЦИЕНТОВ С ХРОНИЧЕСКОЙ СЕРДЕЧНОЙ НЕДОСТАТОЧНОСТЬЮ С НАЛИЧИЕМ НАРУШЕНИЙ УГЛЕВОДНОГО ОБМЕНА

The importance of the problem of chronic heart failure (CHF) is hard to overestimate: this syndrome is diagnosed in millions of patients around the world and its prevalence continues to grow. The proportion of patients suffering from carbohydrate metabolism disorders (CMD) is also steadily increasing. The close connection between carbohydrate metabolism disorders and the progression of CHF allowed such patients to be classified into a separate phenotype cluster - the “metabolic phenotype of CHF”. The pathophysiology of the “metabolic phenotype of CHF” is based on significant metabolic disorders, which determines the characteristic features of the metabolome of this cohort of patients. Therefore, the purpose of this work is to compare and evaluate the relationship between metabolites that occupy one of the most important places in the metabolic processes of the body - the levels of branched chain amino acids (BCAA) and the phenotypic characteristics of patients with CHF. The study included 218 patients: 133 patients of the main group suffering from CHF and having CMD; comparison group - 85 patients with CHF without CMD. The groups were comparable by gender (p=0,324) and age (p=0,936). The levels of BCAAs - leucine, isoleucine, valine - in the blood plasma of patients were assessed by high-performance liquid chromatography combined with mass spectrometry (HPLC-MS). Based on the results of the analysis, the following data were obtained: plasma levels of valine and leucine in patients with CHF and CMD were higher (p=0,022 and p<0,001, respectively) than in patients with CHF and normal carbohydrate metabolism. A direct relationship between the level of leucine + isoleucine and the concentration of glucose (r=0,3, p<0,001) and an inverse relationship between the plasma concentration of valine and the level of N-terminal propeptide of natriuretic hormone (NTproBNP) (r=-0,388, p<0,001) were found. It has been established that a high level of tissue insulin resistance in patients is associated with increased concentrations of BCAA. The results indicate the important role of BCAA in the pathogenesis of the “metabolic phenotype of CHF,” which requires further research in this direction.

DNA-Binding Proteins and Passenger Proteins in Plasma DNA-Protein Complexes: Imprint of Parental Cells or Key Mediators of Carcinogenesis Processes?

Knowledge of the composition of proteins that interact with plasma DNA will provide a better understanding of the homeostasis of circulating nucleic acids and the various modes of interaction with target cells, which may be useful in the development of gene targeted therapy approaches. The goal of the present study is to shed light on the composition and architecture of histone-containing nucleoprotein complexes (NPCs) from the blood plasma of healthy females (HFs) and breast cancer patients (BCPs) and to explore the relationship of proteins with crucial steps of tumor progression: epithelial-mesenchymal transition (EMT), cell proliferation, invasion, cell migration, stimulation of angiogenesis, and immune response. MALDI-TOF mass spectrometric analysis of NPCs isolated from blood samples using affine chromatography was performed. Bioinformatics analysis showed that the shares of DNA-binding proteins in the compositions of NPCs in normal and cancer patients are comparable and amount to 40% and 33%, respectively; in total, we identified 38 types of DNA-binding motifs. Functional enrichment analysis using FunRich 3.13 showed that, in BCP blood, the share of DNA-binding proteins involved in nucleic acid metabolism increased, while the proportion of proteins involved in intercellular communication and signal transduction decreased. The representation of NPC passenger proteins in breast cancer also changes: the proportion of proteins involved in transport increases and the share of proteins involved in energy biological pathways decreases. Moreover, in the HF blood, proteins involved in the processes of apoptosis were more represented in the composition of NPCs and in the BCP blood-in the processes of active secretion. For the first time, bioinformatics approaches were used to visualize the architecture of circulating NPCs in the blood and to show that breast cancer has an increased representation of passenger proteins involved in EMT, cell proliferation, invasion, cell migration, and immune response. Using breast cancer protein data from the Human Protein Atlas (HPA) and DEPC, we found that 86% of NPC proteins in the blood of BCPs were not previously annotated in these databases. The obtained data may indirectly indicate directed protein sorting in NPCs, which, along with extracellular vesicles, can not only be diagnostically significant molecules for liquid biopsy, but can also carry out the directed transfer of genetic material from donor cells to recipient cells.

LEVELS OF MICRO RNA-21-3R IN THE BLOOD OF HYPERTENSIVE PATIENTS WITH NORMAL BODY WEIGHT AND WITH ABDOMINAL OBESITY

Objective: The combination of arterial hypertension (AH) with abdominal obesity (AO) significantly increases the severity of hypertension and accelerates the development of hypertension-mediated organ damage, in particular, left ventricular hypertrophy (LVH). MicroRNAs are small non-coding RNA molecules that are involved in the regulation of post-transcriptional gene expression. A number of experimental studies have revealed the antihypertensive, antihypertrophic and antifibrosing properties of microRNA-21-3p. The goal is to study the plasma levels of microRNA-21-3p in hypertensive patients with normal body weight (NBW) and with abdominal obesity (AO). Design and method: 27 patients (16 men) with stage 2-3 hypertension (13 with NBW and 14 with stage II AO) aged from 42 to 60 years were examined. The control group consisted of 12 practically healthy individuals. All patients underwent general clinical laboratory and instrumental examination. Circulating plasma microRNA-133a levels were obtained by polymerase chain reaction using the CFX96 Touch System, “TaqMan microRNA Assay” and “TaqMan® Universal PCR Master Mix” reagent kits. Results: A significant decrease in the levels of micoRNA-21-3p in the blood plasma was established in the whole group of patients with hypertension (with NBW and with AO) (1.43±0.08), p<0.05) in comparison with practically healthy individuals (3.24 ±0.07). At the same time, the levels of this miRNA in the blood plasma of patients with hypertension and AO (1.13±0.06) were significantly lower (p<0.05) than in patients with hypertension and NBW (1.67±0.07). A correlation analysis revealed negative correlations between the levels of micoRNA-21-3p in the blood plasma and the levels of “office” systolic blood pressure (R=-0.44; p=0.04), as well as the left ventricular myocardial mass index (LVMMI) (R= -0.38; p=0.03) in the whole group of patients with AH (with NBW and AO). Conclusions: The results of this study may indicate the presence of a significant deficiency in the production of mircRNA-21-3p in patients with hypertension, especially when it is combined with AO, as well as the involvement of this miRNA in the mechanisms of progression of hypertension and the development of LVH.

HYPERCOAGULATION OF BLOOD AND THROMBOCYTOPENIA AFTER SARS-COV-2 – PNEUMONIA AS PREDICTORS OF AN UNFAVORABLE PROGNOSIS IN PATIENTS WITH ARTERIAL HYPERTENSION

Objective: To evaluate the dynamics of hypercoagulation parameters of blood plasma and platelets in patients with arterial hypertension (AH) in the early and late post-covid period. Design and method: The prospective study included 340 AH patients aged 41 to 89 years (mean 69; m/f 175/165) infected with SARS-CoV-2 diagnosed by PCR. Of these, 177 people (group 1) were diagnosed with acute pneumonia with the lung damage volume according to CT ranging from 15% to 85%; 163 patients (group 2) had an acute respiratory viral disease. The groups were comparable in age, gender, and concomitant comorbid pathology. Coronary heart disease (CHD) was detected in 85.6% of patients; diabetes mellitus (DM) in 31.5%. The patients were examined three times: on the 30th day from the onset of the disease, at the end of the 3rd month and after 9 months. C-reactive protein (CRP) (0-5 mg/l), ferritin (20-250 mg/l), D-dimer (0-230 ng/ml), fibrinogen (2-4 g/l), lymphocytes, and blood platelets were analyzed. Results: On day 30, group 1 patients showed signs of inflammation: ferritin 258±61.2; CRP 12.3±5.1; lymphocytes 27±8.3%; platelets 160±70.4∗10^9/l; ESR 17±13.7 mm/h. The tendency to hypercoagulation was detected by the indicators of D-dimer (715±241.3) and fibrinogen (3.7±0.9). By the end of the 3rd month, the levels of D-dimer (585±159.4; p=0.015); ferritin (177±59.1; p=0.0002) and CRP (7±5.6; p=0.001) remained elevated. Only after 9 months, D-dimer (100±9.1; p=0.0001) and CRP (2.6±0.2; p=0.0001) were normalized; lymphocyte (32±5.1%; p=0.00001) and platelets (305±69.2∗10^9/l; p=0.00001) increased with a persistent elevated ferritin level (213±124.8; p=0.047). In group 2, the indicators of hypercoagulation and inflammation were moderately elevated. Only after 9 months CRP and D-dimer levels were normalized and the number of platelets increased. Conclusions: In the early and late post-covid period, AH patients with comorbid pathology retain an inflammatory reaction and a procoagulant state, which, in combination with endothelial dysfunction, is a trigger for venous and arterial thrombosis.

Level of glial cell derived neurotrophic factor in the blood plasma of rheumatoid arthritis patients and its relationship with alexithymia.

Glial cell derived neurotrophic factor (GDNF) has an important role in the pathogenetic mechanisms and clinical manifestations of rheumatoid arthritis (RA). Alexithymia is associated with a severe clinical course and worse prognosis, while the relationship between alexithymia and GDNF in RA patients has not been investigated before. The aims of the study were to investigate the GDNF level in blood plasma in RA patients depending on the presence of alexithymia and to evaluate the relationship of GDNF level with clinical manifestation and quality of life. Fifteen men and 73 women with RA were examined using the Disease Activity Score with 28-joint count (DAS28) with erythrocyte sedimentation rate (ESR) index, the Simple Disease Activity Index (SDAI), the Rheumatoid Arthritis Clinical Disease Activity Index (CDAI), the Visual Analogue Scale (according to the assessment of the patient - VAS-P and the assessment of the doctor - VAS-D), the Health Assessment Questionnaire (HAQ), the Toronto Alexithymia Scale (TAS-20), the Disability Rating Index (DRI) and SF-36 indexes. Glial cell derived neurotrophic factor level in the blood plasma was determined by enzyme-linked immunosorbent assay (ELISA). Forty percent of RA patients had alexithymia. Glial cell derived neurotrophic factor level in the examined patients was 3.73 ±2.59 pg/ml, in patients with alexithymia 4.08 ±2.87 pg/ml, without alexithymia 3.48 ±2.37 pg/ml (p = 0.295). Patients with alexithymia had a higher erythrocyte sedimentation rate (ESR) and index scores than patients without alexithymia - ESR: 34.29 ±14.22 vs. 22.73 ±12.03 mm/h (p = 0.017), DAS28: 6.53 ±0.66 vs. 6.09 ±0.55 (p = 0.017), VAS-D: 7.19 ±0.81 vs. 6.53 ±0.83 (p = 0.020), HAQ: 1.78 ±0.58 vs. 1.51 ±0.54 (p = 0.040). Also they had worse SF-36 indicators - physical functioning: 39.52 ±13.78 vs. 51.00 ±14.90 (p = 0.019), role functioning due to physical condition: 30.95 ±20.77 vs. 46.67 ±24.76 (p = 0.041), physical component of health: 31.47 ±11.44 vs. 41.61 ±15.88 (p = 0.028). In patients with alexithymia, a correlation was found between the GDNF level and severity of pain according to VAS-P: rS = 0.338, p = 0.044, and VAS-D: rS = 0.446, p = 0.006. Alexithymia was found in 40% of RA patients. Rheumatoid arthritis patients with alexithymia had a nonsignificantly higher GDNF level compared to patients without alexithymia. In RA patients with alexithymia, an association of GDNF level in the blood plasma with RA activity, loss of functional capacity and reduced quality of life was established. Alexithymia in RA patients is an important factor in the clinical manifestation of RA and modification of the pathophysiological role of GDNF.

NMR-based metabolomic analysis of plasma from elderly patients with CVD before and after using contrast media

Contrast-induced acute kidney injury (CI-AKI) is a growingly common kidney problem caused by medical procedures involving contrast media (CM), especially in older patients with existing health issues. It is crucial to pinpoint potential biomarkers for the early detection of CI-AKI. Previously, we observed that iodixanol affects glucose, choline, and glutathione metabolism in endothelial cells under laboratory conditions. In this study, we used 1H NMR-based metabolomics to examine the metabolic changes in the blood plasma of elderly patients with cardiovascular disease (CVD) before and after receiving iodixanol. We identified altered metabolites in plasma 24 and 48 h after iodixanol injection compared to levels before injection. Notably, metabolites such as glucose, unsaturated fatty acids (UFA), low-density lipoprotein (LDL)/very low-density lipoprotein (VLDL), pyruvate, choline, and glycine showed potential as biomarkers at 24 h post-injection compared to levels before injection. Similarly, glucose, pyruvate, lactate, choline, and glycine in plasma could serve as potential biomarkers at 48 h post-injection. Iodixanol notably affected pathways related to glycolysis, fatty acid breakdown, and amino acid metabolism according to our metabolic pathway analysis. The altered levels of specific metabolites in plasma could be indicative of CM-induced kidney injury. Overall, this research aids in understanding the physiological mechanisms involved and in identifying early biomarkers and prevention strategies for CI-AKI.

DSC investigation of IgG thermostability in blood plasma of patients with myeloma G

DSC investigation of IgG thermostability in blood plasma of patients with myeloma G

Changes in the level of some tryptophan metabolites in the blood of patients with type 2 diabetes mellitus complicated by diabetic retinopathy

Aim: To study the content of tryptophan and the intermediate metabolites of kynurenine and serotonin pathways of its metabolism in the blood plasma of patients with type 2 diabetes mellitus (DM) and non-proliferative diabetic retinopathy as candidates for markers of the early stage of the pathological process.Material and Methods. Three groups of people were analyzed: the first group of 10 patients with type 2 diabetes mellitus and without diabetic retinopathy; the second group of 10 people with type 2 diabetes mellitus and non-proliferative stage of diabetic retinopathy; and the control group of 10 healthy people. The features of the first group were a decrease of macula’s photosensitivity in the fundus and a slight increase in the central thickness of the retina. Patients with diabetic retinopathy tended to have a moderate number of microaneurysms and microhemorrhages, moderately presented intraretinal microvascular anomalies in one quadrant, vein dilatation, clearness of the central retinal vein and its branches. In the macular zone there was an edema with hard exudates in the center and lateral to the fovea centralis. All participants of the study gave blood on an empty stomach in the morning, and after that in we measured the content of tryptophan (TRP), kynurenins ((kynurenine (KYN), 3-hydroxykynurenine (3-HKYN), kynurenic acid (KYNA)) and the level of L-5-hydroxytyrptophan (5HTrp) in blood plasma by HPLC method with fluorimetric and spectrophotometric detection.Results. The groups of people with type 2 diabetes mellitus showed the increasing of TRP level in blood relatively to healthy individuals: by 15.1% (p = 0.032) and 17.9% (p = 0.030) in the first and second groups, respectively. As for the patients of the first group, the content of their KYN was increased by 57.7% (p = 0.012) and KYNA by 33.6% (p = 0.012) relatively to the control and the concentration of 3-НKYN decreased by 18.1% (p = 0.020) relatively to healthy people. As for the patients in the second group, the changes in their level of kynurenines had the same direction, but were more visible. Thus, the concentration of KYN exceeded the same parameters of healthy individuals by 84.5% (p = 0.001) and the parameters of the first group by 18.0% (p = 0.049); the KYNA level increased by 56.6% (p = 0.001) relatively to the control and by 17.3% (p = 0.049) from that of the first group. There was a decrease in the content of 3-HKYN amounted to 18.6% of the control (p = 0.038) and an increase in the concentration of 5HTrp – 193,9% (p < 0.001).

Increased interleukin-9 and Th9 cells in patients with refractory Graves' disease and interleukin-9 polymorphisms are associated with autoimmune thyroid diseases.

Autoimmune thyroid diseases (AITDs) are prevalent disorders, primarily encompassing Graves' disease (GD) and Hashimoto's thyroiditis (HT). Despite their common occurrence, the etiology of AITDs remains elusive. Th9 cells, a new subset of CD4+T cells with immunomodulatory properties, have been linked to the development of various autoimmune diseases. However, research on the role of Th9 cells in AITDs is limited. We investigated the expression of Th9 cells,their functional cytokine IL-9, and transcription factor IRF4 in peripheral blood mononuclear cells (PBMCs) and plasma of AITD patients and healthy controls. Additionally, we explored the genetic association between four loci polymorphisms (rs31564, rs2069879, rs1859430, and rs2069868) of the IL-9 gene and AITDs. We reported, for the first time, that refractory GD patients exhibited elevated mRNA levels of IL-9 and IRF4 in PBMCs, increased IL-9 protein levels in plasma, and a higher proportion of Th9 cells in peripheral blood when compared to normal controls. Furthermore, human recombinant IL-9 protein was found to enhance IFN-g secretion in PBMCs from both GD patients and normal controls. At the genetic association level, after adjusting for age and sex, the rs2069879 polymorphism exhibited a significant association with AITDs under an additive model (P<0.001, OR= 0.05, 95% CI=0.03-0.08). Our results reveal that Th9 cells may exert a pivotal role in the pathogenesis and progression of refractory GD and HT, and IL-9 holds promise as a novel therapeutic target for the management of AITDs.

Oxidative modification products of lipids in the intraocular fluid of patients with cataracts associated with chronic non-infectious diseases

Introduction. To clarify the role of oxidative stress in the development of cataracts, it is necessary to determine the content of oxidative modification products directly in the eye tissue, but such studies are scarce, which is due to the difficulty of obtaining a sufficient amount of biomaterial for analysis and the need to use highly sensitive research methods as a result. Therefore, to assess the pathological changes in the lens, intraocular or tear fluid is used.Aim. To elucidate the role of oxidative stress in the development of cataracts by determining the content of lipid oxidation products and their comparative characteristics in intraocular fluid and in blood plasma in patients with cataracts associated with chronic non-infectious diseases.Materials and methods. The study involved 83 patients with cataracts associated with non-infectious diseases, who underwent surgical treatment. Of these, 30 patients had chronic obstructive pulmonary disease (COPD) as a comorbid condition, 45 had cardiovascular diseases (CVD), and 13 had diabetes mellitus (DM). The control group consisted of 30 practically healthy individuals without clinical signs of acute or chronic diseases. Lipid oxidation products were determined by ultraviolet spectroscopy. Absorption spectra of non-oxidized lipids (E204), diene conjugates (E233), conjugated trienes, and ketodiene (E278) were recorded. Additionally, the ratios of absorption spectra of diene conjugates to non-oxidized lipids (E233/E204), as well as conjugated dienes and ketodienes to non-oxidized lipids (E278/E204), were calculated. Tear and intraocular fluids, as well as blood plasma, served as materials for the study.Results. In the group of patients with cataracts associated with non-infectious diseases, the content of diene conjugates, conjugated trienes, and ketodienes was increased relative to similar indicators in the tear fluid of people in the control group. Moreover, more pronounced changes were observed in the group of patients with cataracts associated with COPD relative to those with CVD and DM. A comparative analysis confirmed that in the group of patients with cataracts associated with COPD, the content of oxidized lipids in intraocular fluid and in blood plasma was increased. Correlation-regression analysis showed a positive relationship between the indicators of diene conjugates in intraocular fluid and in blood plasma.Conclusions. The significant direct linear relationship between the indicators of oxidized lipids in intraocular fluid and in blood plasma in patients with cataracts and non-infectious diseases may indicate the possibility of using blood plasma for studying oxidative processes in the lens and its pathological changes for the purpose of selecting therapy and evaluating treatment efficacy. The results obtained confirm the role of oxidative stress in the development of cataracts in patients with chronic non-infectious diseases.

Abstract 1299: Cost-effectiveness analysis of circulating tumor DNA-based molecular profiling platforms for the detection of EGFR mutations in lung cancer

Abstract Background The detection of mutations in circulating tumor DNA (ctDNA) in blood plasma of lung cancer patients can be used to monitor the tumor-response to therapy and for the early detection of resistance mechanisms. In 2017, the Roche Cobas® EGFR Mutation Test v2 for the detection of the EGFR c.2369C&amp;gt;T p.(T790M) mutation in plasma-derived ctDNA was FDA-approved to identify patients eligible for osimertinib treatment. Today, different platforms are available for EGFR mutation testing in plasma-derived cell-free DNA (cfDNA). In this study we compared the clinical utility of five platforms for the detection of EGFR mutations in cfDNA offering insights into their operational costs, target coverage, hands-on-time and turn-around-time. Methods Four PCR-based platforms and one NGS-based approach for the detection of EGFR mutations were compared. Operational, maintenance, material and personnel costs were calculated for each platform and used as input variables for our recently developed ctDNA micro-costing framework (doi.org/10.1016/j.jmoldx.2022.10.004). This was compared to target coverage and turn-around-time of each platform in the context of therapy resistance. Results Per sample costs decreased for all platforms at maximal weekly throughput. The BioCartis Idylla™ demonstrated the shortest turn-around-time while the Roche Cobas® was the least expensive. Despite being the most expensive and time-consuming, the AVENIO ctDNA Expanded kit covered the most known resistance mechanisms to EGFR inhibitors, many of which are targetable with other TKIs. Conclusion EGFR ctDNA mutation analysis is only cost-effective when the throughput is high. This implies that centralization of cfDNA-testing is necessary to optimize cost-efficiency. With the advent of ctDNA analysis beyond EGFR in the context of targeted therapies for lung cancer, multigene detection assays will become more cost-effective with high throughput. Overview of results Platform Turn-around time at maximal weekly throughput Per sample cost at maximal weekly throughput Coverage of resistance mutations in EGFR 1Additional testing necessary Coverage of genome-wide resistance mutations to EGFR-TKI 1Additional testing necessary BioCartis Idylla™ ctEGFR Mutation Assay 2,5 hours €571 33% 12%1 Roche Cobas® EGFR Mutation Test v2 4 hours €497 33% 6% Bio-Rad QX200 droplet digital PCR (ddPCR) 7,5 hours €766 100%1 24%1 Agena MassARRAY® UltraSEEK® Lung Panel 2 days €613 53% 12% Roche AVENIO ctDNA Expanded kit 1 week €1551 100% 65% Citation Format: Pim Rozendal, Paul van der Leest, Elise M. van der Logt, Naomi Rifaela, Inge Platteel, Frank J. Scherpen, Harry J. Groen, Léon C. van Kempen, Ed M. Schuuring. Cost-effectiveness analysis of circulating tumor DNA-based molecular profiling platforms for the detection of EGFR mutations in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1299.

Tick-borne encephalitis virus transmitted singly and in duo with Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum bacteria by ticks as pathogens modifying lipid metabolism in human blood

BackgroundTicks are vectors of various pathogens, including tick-borne encephalitis virus causing TBE and bacteria such as Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum causing e.g. viral-bacterial co-infections (TBE + LB/HGA), which pose diagnostic and therapeutic problems. Since these infections are usually accompanied by inflammation and oxidative stress causing metabolic modifications, including phospholipids, the aim of the study was to assess the level of polyunsaturated fatty acids and their metabolism (ROS- and enzyme-dependent) products in the blood plasma of patients with TBE and TBE + LB/HGA before and after pharmacotherapy.MethodsThe total antioxidant status was determined using 2,20-azino-bis-3-ethylbenzothiazolin-6-sulfonic acid. The phospholipid and free fatty acids were analysed by gas chromatography. Lipid peroxidation was estimated by measuring small molecular weight reactive aldehyde, malondialdehyde and neuroprostanes. The reactive aldehyde was determined using gas chromatography coupled with mass spectrometry. The activity of enzymes was examined spectrophotometrically. An analysis of endocannabinoids and eicosanoids was performed using a Shimadzu UPLC system coupled with an electrospray ionization source to a Shimadzu 8060 Triple Quadrupole system. Receptor expression was measured using an enzyme-linked immunosorbent assay (ELISA).ResultsThe reduced antioxidant status as a result of infection was accompanied by a decrease in the level of phospholipid arachidonic acid (AA) and docosahexaenoic acid (DHA) in TBE, an increase in DHA in co-infection and in free DHA in TBE with an increase in the level of lipid peroxidation products. The enhanced activity of enzymes metabolizing phospholipids and free PUFAs increased the level of endocannabinoids and eicosanoids, while decreased 15-PGJ2 and PGE2 was accompanied by activation of granulocyte receptors before pharmacotherapy and only tending to normalize after treatment.ConclusionSince classical pharmacotherapy does not prevent disorders of phospholipid metabolism, the need to support treatment with antioxidants may be suggested.