Immunochemical Methods Research Articles

Modern View on the Role of Sex-Specific Levels of High-Sensitive Cardiospecific Troponins T and I in the Diagnosis of Myocardial Infarction.

It is well known that the molecules of cardiospecific troponins T and I are localized in the troponin-tropomyosin complex of the cytoplasm of cardiac myocytes and, due to the specific localization, these cardiospecific troponins are widely used as diagnostic biomarkers of myocardial infarction. Cardiospecific troponins are released from the cytoplasm of cardiac myocytes as a result of irreversible cell damage (for example, ischemic necrosis of cardiomyocytes in myocardial infarction or apoptosis of cardiac myocytes in cardiomyopathies and heart failure) or reversible damage (for example, intense physical exertion, hypertension, the influence of stress factors, etc.). Current immunochemical methods for determining cardiospecific troponins T and I have extremely high sensitivity to subclinical (minor) damage to myocardial cells and, thanks to modern high-sensitive methods, it is possible to detect damage to cardiac myocytes in the early (subclinical) stages of a number of cardiovascular pathologies, including myocardial infarction. So, recently, leading cardiological communities (the European Society of Cardiology, the American Heart Association, the American College of Cardiology, etc.) have approved algorithms for early diagnosis of myocardial infarction based on the assessment of serum levels of cardiospecific troponins in the first 1 - 3 h after the onset of pain syndrome. An important factor that may affect early diagnostic algorithms of myocardial infarction are sex-specific features of serum levels of cardiospecific troponins T and I. This manuscript presents a modern view on the role of sex-specific serum levels of cardiospecific troponins T and I in the diagnosis of myocardial infarction and the mechanisms of formation of sex-specific serum levels of troponins.

Expression of recombinant Omp18 and MOMP of Campylobacter jejuni and the determination of their suitability as antigens for serological diagnosis of campylobacteriosis in animals.

Campylobacteriosis causes gastrointestinal tract lesions in adults and children and may result in severe complications. The primary sources of infection are infected animals and animal products. Immunochemical methods effectively diagnose intestinal infections but require highly specific antigens to detect their antibodies. This study aimed to obtain two recombinant immunogenic antigens of Campylobacter jejuni, an outer membrane protein with a molecular weight of 18 kDa (Omp18) and the major outer membrane protein (MOMP) with a molecular weight of 45 kDa, and evaluate their suitability for the serological diagnosis of campylobacteriosis using immunochromatographic assay (ICA). The C. jejuni Omp18 and MOMP gene sequences were synthesized de novo (Macrogen, Korea) and cloned into the pET32 expression plasmid. Using these genetic constructs, electrocompetent cells of the Escherichia coli BL21 strain were transformed and cultured under various conditions. Antigens were purified and refolded using metal affinity chromatography. The properties of the purified proteins were studied by western blotting, liquid chromatography with tandem mass spectrometry, and enzyme-linked immunosorbent assay (ELISA). We developed two recombinant E. coli BL21 cells producing rOmp18 and Recombinant MOMP (rMOMP) antigens with molecular weights of 36 and 64 kDa, respectively. Amino acid sequence analysis of the obtained antigens showed complete homology with the reference sequences in the PubMed NCBI database. Western blotting using positive-control sera demonstrated the specificity of the recombinant antigens. The results of ELISA with 94 bovine sera showed the interaction of recombinant antigens with specific antibodies. The obtained rOmp18 and rMOMP antigens can detect antibodies in the serum of infected or recovered animals and can be used to develop ICA.

Differences between inflammatory cells infiltrated into tunica intima, media, and adventitia of ascending aortic aneurysms within diabetic and hypertensive patients.

The risk factors that are the most significant for the development of most cardiovascular diseases are arterial hypertension (AH), type 2 diabetes (DM), and inflammation. However, for the development of aortic aneurysms, DM is not one of them. Our study aimed to evaluate the difference between inflammatory infiltration in three individual layers of the ascending aortic aneurysm within diabetic and hypertensive patients. Forty-five patients aged 36 to 80 were divided into a group with diabetic patients without AH (group DM, N=8) and hypertensive patients without DM (group AH, N=37). For the histological analysis, aortic aneurysms were stained with hematoxylin eosin and Movat. We used immunochemical methods to detect pro- (M1), anti-inflammatory (M2) macrophages, T-helper, T-killer cells, B cells, and plasma cells. Statistical analysis was done by independent-samples Kruskal-Wallis test adjusted by Bonferroni correction for multiple tests (P<0.05). We found no difference in the volume density of collagen, elastin, vascular smooth muscle cells (VSMC), and ground substance between groups. In the DM group, there were significantly fewer M2, T-helpers, and T-killers in the media than in the intima and the adventitia (P<0.05). There were no significant differences in the number of M1, B, and plasma cells between all three vascular layers (P<0.05). In the AH group, there were significantly fewer B and plasma cells, T-helper, T-killer cells, M1, and M2 in the media than in the intima and adventitia (P<0.05). Our results conclude that the tunica media in the aneurismal wall of the AH group retained immune privilege. In contrast, in the DM group, all three layers were immune-privileged.

Differences between inflammatory cells infiltrated into tunica intima, media, and adventitia of ascending aortic aneurysms within diabetic and hypertensive patients.

The risk factors that are the most significant for the development of most cardiovascular diseases are arterial hypertension (AH), type 2 diabetes (DM), and inflammation. However, for the development of aortic aneurysms, DM is not one of them. Our study aimed to evaluate the difference between inflammatory infiltration in three individual layers of the ascending aortic aneurysm within diabetic and hypertensive patients. Forty-five patients aged 36 to 80 were divided into a group with diabetic patients without AH (group DM, N=8) and hypertensive patients without DM (group AH, N=37). For the histological analysis, aortic aneurysms were stained with hematoxylin eosin and Movat. We used immunochemical methods to detect pro- (M1), anti-inflammatory (M2) macrophages, T-helper, T-killer cells, B cells, and plasma cells. Statistical analysis was done by independent-samples Kruskal-Wallis test adjusted by Bonferroni correction for multiple tests (P<0.05). We found no difference in the volume density of collagen, elastin, vascular smooth muscle cells (VSMC), and ground substance between groups. In the DM group, there were significantly fewer M2, T-helpers, and T-killers in the media than in the intima and the adventitia (P<0.05). There were no significant differences in the number of M1, B, and plasma cells between all three vascular layers (P<0.05). In the AH group, there were significantly fewer B and plasma cells, T-helper, T-killer cells, M1, and M2 in the media than in the intima and adventitia (P<0.05). Our results conclude that the tunica media in the aneurismal wall of the AH group retained immune privilege. In contrast, in the DM group, all three layers were immune-privileged.

Therapeutic Drug Monitoring of Tacrolimus Based on Volumetric Absorptive Microsampling Technique (VAMS) in Renal Transplant Pediatric Recipients-LC-MS/MS Method Development, Hematocrit Effect Evaluation, and Clinical Application.

Tacrolimus (TAC) is post-transplant pharmacotherapy's most widely used immunosuppressant. In routine clinical practice, frequent uncomfortable venipuncture is necessary for whole-blood (WB) collection to check trough TAC levels. Volumetric absorptive microsampling (VAMS) is an alternative strategy to WB collection. In this study, we aimed to validate and develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for TAC quantification in WB and VAMS samples. After extraction with water and protein precipitation, the samples were directly analyzed using LC-MS/MS. Whole-blood and VAMS capillary-blood samples were collected from 50 patients treated with TAC during the follow-up visits. The cross-correlation between the developed methods was evaluated using Passing-Bablok regression and a Bland-Altman bias plot. The matrix effect (ME) and carry-over were insignificant for both scenarios. There was a high correlation between the processes and no significant clinical deviation. LC-MS/MS methods were successfully developed and validated in the 0.5-60 ng/mL calibration range. This study demonstrated and confirmed the utility of VAMS-based TAC monitoring in the pediatric population. This is the first study to directly develop and validate the VAMS LC-MS/MS method for evaluating the hematocrit effect in the pediatric population. The statistical correlation between immunochemical and VAMS-based methods was satisfactory.

Phosphorylation and Dephosphorylation of Beta-Amyloid Peptide in Model Cell Cultures: The Role of Cellular Protein Kinases and Phosphatases.

Phosphorylation of beta-amyloid peptide (Aβ) at the Ser8 residue affects its neurotoxicity, metal-dependent oligomerisation, amyloidogenicity, and other pathogenic properties. Phosphorylated Aβ (pS8-Aβ) was detected in vivo in AD model mice and in the brains of patients with AD. However, the pS8-Aβ production and the regulation of its levels have not been previously studied in detail. In this paper, immunochemical methods together with radioactive labelling were used to study the Aβ phosphorylation by intracellular and surface protein kinases of HEK293 cells and brain endothelial cells (bEnd.3). It was found that HEK293 robustly phosphorylated Aβ, likely with contribution from casein kinase 2 (CK2), whereas in bEnd.3, the activity of Aβ phosphorylation was relatively low. Further, the study showed that both HEK293 and bEnd.3 could dephosphorylate pS8-Aβ, mainly due to the activity of protein phosphatases PP1 and PP2A. The Aβ dephosphorylation efficiency in bEnd.3 was three times higher than in HEK293, which correlated with the reduced abundance of pS8-Aβ in vascular amyloid deposits of patients with AD compared to senile plaques. These data suggest an important role of CK2, PP1, and PP2A as regulators of Aβ phosphorylation, and point to the involvement of the blood-brain barrier in the control of Aβ modification levels.

Ефективність та безпечність підготовки до вакцинації проти вірусу SARS-COV-2 у пацієнтів із хронічною рецидивуючою кропив’янкою

Abstract. The aim: to develop a personalized algorithm for vaccination against COVID-19 for patients with severe chronic urticaria and to evaluate its effectiveness and safety. Methods. Patients with severe (according to the UAS7 scale) chronic spontaneous urticaria and concomitant gastroenterological pathology, who were observed at the City Allergology Center, were included in a prospective single-center clinical study. All patients underwent a complete physical examination, evaluation of complaints, history of life and illness, presence of concomitant pathology. Patients with a history of COVID19 or complete or partial vaccination against COVID-19 were excluded from the study. Blood tryptase level was determined before vaccination using immunofluoroenzymatic analysis (ImmunoCAP). Total serum IgE was determined using an immunochemical method. Preparation for vaccination against the SARS-COV-2 virus was carried out according to the developed algorithm: the appointment of 5 mg of desloratadine orally 30 minutes before vaccination with a normal level of tryptase (&lt; 11 ng/l); and 20 mg of desloratadine 30 minutes before vaccination at an elevated level of tryptase (≥ 11 ng/L) with further observation of the condition of patients during the day after vaccination. Results. The study included 30 patients aged 24 to 63 years, median age 52 (39-59) years, of which 66.7 % were women. The duration of the disease was, on average, 6 years, and 63.3 % of patients suffered from urticaria for five or more years. All 100 % of patients had a severe course of chronic urticaria: from 28 to 42 points on the UAS7 scale, median 42 (42-42) points. Among patients with severe chronic urticaria and concomitant gastrointestinal pathology included in the study, 60 % had elevated serum tryptase levels, were older and had a higher prevalence of H. pylori infection, but a lower prevalence of concomitant allergic pathology. The level of tryptase did not correlate with the level of total IgE. Conclusions The developed individual algorithm for pre-vaccination preparation of patients with severe chronic urticaria has shown its effectiveness and safety in preparing such a difficult category of patients for vaccination against SARS-CoV-2 infection, which is vital today.

System analysis of surface CD markers during the process of granulocytic differentiation.

Plasma membrane proteins with extracellular-exposed domains are responsible for transduction of extracellular signals into intracellular responses, and their accessibility to therapeutic molecules makes them attractive targets for drug development. In this work, using omics technologies and immunochemical methods, we have studied changes in the content of markers of clusters of differentiation (CD markers) of neutrophils (CD33, CD97, CD54, CD38, CD18, CD11b, CD44, and CD71) at the level of transcripts and proteins in NB4, HL-60 and K562 cell lines, induced by the treatment with all-trans-retinoic acid (ATRA). Transcriptomic analysis revealed the induction of CD38, CD54, CD11b, and CD18 markers as early as 3 h after the addition of the inducer in the ATRA-responsive cell lines HL-60 and NB4. After 24 h, a line-specific expression pattern of CD markers could be observed in all cell lines. Studies of changes in the content of CD antigens by means of flow cytometry and targeted mass spectrometry (MS) gave similar results. The proteomic profile of the surface markers (CD38, CD54, CD11b, and CD18), characteristic of the NB4 and HL-60 lines, reflects different molecular pathways for the implementation of ATRA-induced differentiation of leukemic cells into mature neutrophils.

GLUCOCORTICOIDS AND MINERALOCORTICOIDS IN BLOOD PLASMA AS MARKERS FOR THE EARLY DIAGNOSIS OF ADRENAL CORTEX TUMORS

Summary: Aim: assessment of the pituitary-adrenal and renin-aldosterone systems state in patients with various types of adrenocortical adenomas. Object and methods: in 35 patients with adrenocortical adenomas, the hormonal status was evaluated by a direct quantitative immunoenzymatic analysis. The levels of glucocorticoids (cortisol) and mineralocorticoids (aldosterone) were determined. The levels of adrenocorticotropic hormone (ACTH) and renin were assessed by immunochemical methods using monoclonal antibodies specific for ACTH and renin. Results: increasing level of the cortisol against the background of a decreasing level of the ACTH in patients with cortisol-producing adrenocortical adenomas was revealed. In patients with aldosterone-producing adenomas, aldosteronism develops against the background of an unchanged level of renin and an increase in the aldosterone-renin ratio. Conclusions: the occurrence of hormone-dependent adenomas in the adrenal cortex disrupts the functioning of the pituitary-adrenal and renin-aldosterone systems, which serves as a criterion for the early diagnosis of adrenocortical adrenal adenomas.

INVESTIGATION OF THE MECHANISMS OF IMMUNE REACTIVITY AND DISORDERS OF BONE METABOLISM IN MULTIPLE MYELOMA

In order to study the mechanisms of immune reactivity (IR) and bone metabolism (BM) disorders in groups of patients with initially diagnosed multiple myeloma (MM), with MM after standard therapy, with bone pathology of other etiology and elderly patients without bone pathology, markers of bone metabolism (MBM) were determined by immunochemical methods: Type 1 collagen N-terminal propeptide (P1NP), osteocalcin (fragment with amino group, N-MID), and β-aspartic acid of type I collagen C-telopeptides (β-crosslaps), immunoreactivity markers (IRM): C-reactive protein, β2-microglobulin, interleukin-6 and hormones (parathyroid hormone, free thyroxine (T4c.), total testosterone). It was revealed that in patients with primary MM, as a rule, there is a significant change in the levels of MBM and IRM. These changes in primary MM were detected much more often and were more pronounced than in patients with MM treated according to the standard program, patients without bone neoplasia and patients without bone pathology. There is a definite relationship between the intensity of IR and the severity of BM disorders. All this may indicate that in MM, bone tissue damage occurs as a result of a pronounced predominance of bone resorption over remodeling processes in conditions of the development of a chronic systemic inflammatory process with significant metabolic disorders. The result of these processes is the manifestation of a syndrome of bone and mineral disorders preceding the development of osteoporosis, which can be diagnosed using MBM, which therefore become significant indicators in the diagnosis of MM, assessment of bone tissue lesions before the appearance of clinical and instrumental signs of osteoporosis. It is also advisable to consider the possibility of using MBM for differential diagnosis, predicting the outcome of the disease, monitoring MM therapy and as indicators of the need to include drugs normalizing bone metabolism in the standard MM therapy regimen.

Analysis of total aflatoxins in commercially available cereal products in the Republic of North Macedonia

Aflatoxins, as naturally occurring toxins, can accumulate in maturing crops or in grain during transportation and represent serious threat for contamination of cereal processed products. Consumption of aflatoxin contaminated foodstuffs can cause acute or chronic toxicity in humans. With an aim to protect public health, maximum residue levels (MRLs) for these toxins are defined by the regulation authorities. To control the compliance of aflatoxin residues in cereal-based products to legislation, a convenient analytical technique that will provide rapid, specific, accurate and costly effective analysis such as ELISA method is required. In this study, concentration of total aflatoxins in twelve cereal processed products supplied under random selection from several retail outlets in Republic of North Macedonia was determined using commercially available RIDASCREEN Aflatoxin total kit. Analyses have shown that none of the examined samples contained aflatoxins above the MRLs (4 μg/kg). According to the obtained results, the ELISA method can be used for routine detection and quantification of total aflatoxins in cereal products due to simplicity, specificity and sensitivity. In addition, this study may initiate the use of immunochemical methods for analysis of potential aflatoxin contamination in order to establish data collection for the presence of these concerning toxins in cereal-based products available on the national market. Keywords: aflatoxins, ELISA, immunochemical, cereals

The effect of an epigenetic diet on migraine patients

Migraine is a common type of primary headache disorder, distinguished by recurrent attacks of moderate to severe unilateral throbbing pain, often accompanied by symptoms such as nausea, photophobia, and phonophobia.&#x0D; Objective — to study the effect of epigenetic diet on frequency and intensity of migraine headache attacks, indicators of psycho‑emotional status and quality of life of migraine patients.&#x0D; Methods and subjects. 130 people aged from 18 to 55 years (average age — 38.6 ± 8.3 years) were examined, of which 15 (11.5 %) were men and 115 (88.4 %) were women. The patients were distributed into two groups: group A — patients who followed an epigenetic diet enriched with 5 mg of folic acid, group B — patients who did not follow any diet. The diagnosis of migraine was confirmed using the criteria of the International Headache Society. Before the study, all participants were assessed using the Work Disability Assessment Scale (MIDAS). Also, the frequency of headache was determined in all patients. Headache intensity was assessed using a visual analog scale (VAS). Psychoemotional status at the beginning and at the end of the study was assessed using the Hamilton Anxiety Scale and the Beck Depression Scale. In all patients, the content of folic acid and homocysteine in the blood was determined using an immunochemical method with electrochemiluminescence detection.&#x0D; Results. When analyzing the data before the start of the study, it was found that the average concentration of folic acid in group A was 2.8 ± 3.6 ng/ml and was significantly lower than in group B (p = 0.003). The average level of homocysteine was different in both groups (р = 0.04). In group A, against the background of using an epigenetic diet, the average level of folic acid remarkably increased (p = 0.001), and the average level of homocysteine significantly decreased (p = 0.003). Also, in group A, a notable decrease in the intensity and frequency of headache was found (р = 0.02; р = 0.04), a decrease in the average level of anxiety according to the Hamilton scale (from 15.0 ± 3.5 points to 9 ± 2 points) (p = 0.03). A direct relationship between the level of homocysteine and the score on the Hamilton Anxiety Scale and the MIDAS scale was established, as well as a negative correlation between the level of folic acid and the score on the Beck Depression Scale.&#x0D; Conclusions. Evidence of the influence of changes in the content of homocysteine and folic acid in the blood of patients with migraine against the background of an epigenetic diet on the severity of headache and quality of life was revealed. The use of an epigenetic diet in the prevention and treatment of migraine needs further study, as it is difficult to predict the final effect of the diet due to its low specificity to the epigenome and the large number of interactions between the active components of the diet.&#x0D;

Biological properties of domestic strain vRub-Ant of rubella virus

Introduction. Rubella is a mild infectious disease affecting mainly children and is caused by the rubella virus, part of the Matonoviridae family, genus Rubivirus. Rubella causes congenital rubella syndrome (CRS) and is the main cause of developmental abnormalities, especially blindness and deafness.&#x0D; There is no specific treatment for rubella and CRS. In order to avoid possible complications from rubella infection, a live attenuated rubella vaccine based on the foreign strain of Wistar RA 27/3 rubella virus is used. However, the actual, more effective and preferred vaccine strain the rubella virus for the Russian Federation is considered to be a viral strain of rubella circulating on its territory.&#x0D; The aim of the study was to study the biological properties of the developed domestic cold-adapted strain vRub-Ant circulating in the territory of the Russian Federation.&#x0D; Materials and methods. Following cell cultures were used in the study human embryo lung diploid cell strain LECH-3, transferable cell line from embryonic kidney cells of green monkeys Vero CCL-81 and Vero ECC, human mesenchymal stem cells, human peripheral blood mononuclear cells (PBMC). Cell cultures were grown on a DMEM/F12 nutrient medium with the addition of 5% fetal bovine serum. Swabs from the pharynx and nasal passages from a child with rubella were used as clinical virus-containing material. Monoclonal anti-idiotypic antibodies m(anti-ID)Ab were used to assess the expression level of alpha/beta and gamma interferon receptors (/ and IFN-R)Ab, imitating the biological effects of alpha/beta and gamma interferons (/ and IFN) of humans. The cultural, virological, immunochemical and serological research methods were applied in the study.&#x0D; Results. Attenuation of the vRub-Ant clinical isolate of rubella virus was carried out for 20 consecutive passages on LECH-3 diploid cells at a reduced temperature of 30C. The main biological markers of attenuation were determined to be ts and ca phenotypes. The avirulence of the attenuated viral strain (att-phenotype) was assessed by the level of expression of / and IFN-R. A lower level of / and IFN-R expression was found on the membranes of human PBMC induced by the vaccine strain vRub-Ant in comparison with the parent wild variant of the rubella virus. This trait,the att phenotype, is characteristic of attenuated viral strains. It has been shown that the vaccine strain vRub-Ant has lost neurotropism and was unable to bind to the membrane receptors of the brain (MRB) of guinea pig embryos, unlike its parent rubella virus strain. The high immunogenicity of the domestic cold-adapted strain vRub-Ant was confirmed by high titers of neutralizing rubella antibodies observed in guinea pigs immunized subcutaneously with one vaccination dose of the virus.&#x0D; Conclusion. A domestic attenuated vaccine strain vRub-Ant of the rubella virus that has the main biological markers of attenuation (ts-ca and att phenotypes) has been developed. The vaccine strain vRub-Ant induces a high levels of neutralizing antibodies in guinea pigs following the immunization with a single vaccination dose of the vaccine. The viral strain vRub-Ant has lost its tropism to the MRB of guinea pig embryos, unlike its parent variant.

Influence of Label and Solid Support on the Performance of Heterogeneous Immunoassays.

Conventional immunochemical methods used in clinical analysis are often not sensitive enough for early-stage diagnosis, resulting in the need for novel assay formats. Here, we provide a detailed comparison of the effect of different labels and solid supports on the performance of heterogeneous immunoassays. When comparing three types of streptavidin-modified labels─horseradish peroxidase, carboxyfluorescein, and photon-upconversion nanoparticles (UCNPs)─UCNPs led to the most sensitive and robust detection of the cancer biomarker prostate-specific antigen. Additionally, we compared the immunoassay formats based on conventional microtiter plates and magnetic microbeads (MBs). In both cases, the highest signal-to-background ratios and the lowest limits of detection (LODs) were obtained by using the UCNP labels. The MB-based upconversion-linked immunosorbent assay carried out with a preconcentration step provided the lowest LOD of 0.46 pg/mL in serum. The results demonstrate that the use of UCNPs and MBs can significantly improve the sensitivity and working range of heterogeneous immunoassays for biomarker detection.

Оценка экспрессии гена ctxA штамма Vibrio cholerae 569В при производстве холерной вакцины

Cultivation of production strains of Vibrio cholerae is one of the most important stages in the production of cholera bivalent chemical vaccines. In the production of immunobiological preparations, it is necessary to use producer strains with stable properties that persist in a number of generations. The aim of this study was to evaluate the expression of the ctxA gene using molecular genetic methods of the V. cholerae 569B producing strain. By PCR with electrophoretic accounting of the results, the presence of this strain of the ctxA gene in the chromosome was established. The expression of this gene in all hourly samples of culture fluid was recorded by the method of PCR with reverse transcription, taking into account the results in real time and digital drip PCR. Immunochemical methods confirmed the presence of cholera toxin in the samples, with a maximum mark at the ninth hour, which corresponded to the maximum indicators of the “concentration of microbial cells”. Thus, molecular genetic methods have shown the stability of the production strain V. cholerae 569B in samples of 4 independent cultivations.

The dark side of current analytic methods for Bence Jones Proteinuria.

Bence Jones proteinuria (BJP) refers to monoclonal free immunoglobulin light chains detected in urine, deriving from the clonal expansion of plasma cells in the bone marrow in patients with plasma cell dyscrasias, associated with monoclonal gammopathies of uncertain origin. This review summarizes routinely diagnostic procedures to assess BJP highlighting critical steps of pre-analytical, analytical, and post-analytical phases. The best option for BJP detection is the first morning void urine sample and immunofixation electrophoresis detection technique (IFE) the recommended method, with the employment of specific polyvalent antisera. Other qualitative tests for a quick evaluation of BJP are currently available. Densitometric analysis performed on the 24-hour urine is the recommended method to quantify BJP. To overcome the 24-hour collection, it is possible to use morning urine sample and correlate the assessed value of BJP to creatininuria. In addition to the traditional ones, we here reviewed screening methods currently used to avoid false negatives and reduce the time around test (TAT), together with immunochemical quantification methods for increased sensitivity, after checking BJP by IFE. Mass spectrometry emerges as a new challenge in the determination of BJP. The employment of different based-assays methods may be useful for diagnostic purposes to improve the accuracy of BJP monitoring in monoclonal gammopathies.

Cancer glycomics offers potential biomarkers and therapeutic targets in the framework of 3P medicine.

Glycosylation is one of the most important post-translational modifications (PTMs) in a protein, and is the most abundant and diverse biopolymer in nature. Glycans are involved in multiple biological processes of cancer initiation and progression, including cell-cell interactions, cell-extracellular matrix interactions, tumor invasion and metastasis, tumor angiogenesis, and immune regulation. As an important biomarker, tumor-associated glycosylation changes have been extensively studied. This article reviews recent advances in glycosylation-based biomarker research, which is useful for cancer diagnosis and prognostic assessment. Truncated O-glycans, sialylation, fucosylation, and complex branched structures have been found to be the most common structural patterns in malignant tumors. In recent years, immunochemical methods, lectin recognition-based methods, mass spectrometry (MS)-related methods, and fluorescence imaging-based in situ methods have greatly promoted the discovery and application potentials of glycomic and glycoprotein biomarkers in various cancers. In particular, MS-based proteomics has significantly facilitated the comprehensive research of extracellular glycoproteins, increasing our understanding of their critical roles in regulating cellular activities. Predictive, preventive and personalized medicine (PPPM; 3P medicine) is an effective approach of early prediction, prevention and personalized treatment for different patients, and it is known as the new direction of medical development in the 21st century and represents the ultimate goal and highest stage of medical development. Glycosylation has been revealed to have new diagnostic, prognostic, and even therapeutic potentials. The purpose of glycosylation analysis and utilization of biology is to make a fundamental change in health care and medical practice, so as to lead medical research and practice into a new era of 3P medicine.

Anabolic Steroids in Fattening Food-Producing Animals-A Review.

Simple SummaryAnabolic steroids significantly affect animal tissues and cause morphological and histological changes, which are often irreversible. This issue is currently a very hot topic, as the answers to the questions concerning the health of endangered animals and humans vary greatly from country to country. There is a need to further investigate whether the use of anabolic steroids in animal fattening threatens consumer health and to develop new tools for the detection of anabolic steroids in meat. One possibility for detection could be to observe histological changes in the tissues, which form a typical pattern of anabolic abuse. This review gathered information on the anabolic steroids most commonly used in animal fattening, the legislation governing this issue, and the main effects of anabolics on animal tissues.Anabolic steroids are chemically synthetic derivatives of the male sex hormone testosterone. They are used in medicine for their ability to support muscle growth and healing and by athletes for esthetic purposes and to increase sports performance, but another major use is in fattening animals to increase meat production. The more people there are on Earth, the greater the need for meat production and anabolic steroids accelerate the growth of animals and, most importantly, increase the amount of muscle mass. Anabolic steroids also have proven side effects that affect all organs and tissues, such as liver and kidney parenchymal damage, heart muscle degeneration, organ growth, coagulation disorders, and increased risk of muscle and tendon rupture. Anabolic steroids also have a number of harmful effects on the developing brain, such as brain atrophy and changes in gene expression with consequent changes in the neural circuits involved in cognitive functions. Behavioral changes such as aggression, irritability, anxiety and depression are related to changes in the brain. In terms of long-term toxicity, the greatest impact is on the reproductive system, i.e., testicular shrinkage and infertility. Therefore, their abuse can be considered a public health problem. In many countries around the world, such as the United States, Canada, China, Argentina, Australia, and other large meat producers, the use of steroids is permitted but in all countries of the European Union there is a strict ban on the use of anabolic steroids in fattening animals. Meat from a lot of countries must be carefully inspected and monitored for steroids before export to Europe. Gas or liquid chromatography methods in combination with mass spectrometry detectors and immunochemical methods are most often used for the analysis of these substances. These methods have been considered the most modern for decades, but can be completely ineffective if they face new synthetic steroid derivatives and want to meet meat safety requirements. The problem of last years is the application of “cocktails” of anabolic substances with very low concentrations, which are difficult to detect and are difficult to quantify using conventional detection methods. This is the reason why scientists are trying to find new methods of detection, mainly based on changes in the structure of tissues and cells and their metabolism. This review gathered this knowledge into a coherent form and its findings could help in finding such a combination of changes in tissues that would form a typical picture for evidence of anabolic misuse.

Fecal occult blood testing in colorectal cancer screening programs

Colorectal carcinoma (CRC) is the third most common cancer worldwide and ranks second as a cause of cancer mortality. CRC screening is carried out in many countries for detection early-stage CRC and its prevention by removing precancerous lesions, and includes fecal occult blood testing and colonoscopy. Different countries use their own approach to screening, including methods of detection of fecal occult blood. The strategy for fecal occult blood testing is based on the fact that already in the early stages the tumor can bleed, and small traces of blood in the stool are detected before the onset of clinical symptoms of the disease. Different countries use their own approach to the CRC screening, including methods for fecal occult blood testing. Chemical and immunochemical methods are used to determine occult blood in the feces. The chemical method is based on the detection of heme peroxidase activity. The immunochemical method detects human globin using specific antibodies. The immunochemical method can be qualitative and quantitative. The sensitivity of a quantitative immunochemical test depends on the selected threshold and the stage of CRC. The lower the threshold value, the higher the sensitivity, but the lower the specificity due to false positive results. CRC screening programs use different thresholds for quantitative immunochemical testing in different countries, which is caused by the availability of colonoscopy in a positive test result, as well as the rate of CRC in this population. To increase the sensitivity of the immunochemical test, some programs suggest using it in combination with other methods: detection of DNA in stool, examination of other proteins in feces (transferrin, haptoglobin). This review presents the methods used in the world for fecal occult blood testing, their advantages and limitations; recommendations for reporting the results of a quantitative immunochemical test; thresholds recommended in screening programs in different countries for quantitative immunochemical tests. The results of pilot screening for fecal occult blood testing in some regions of the Russian Federation are also presented.

Interleukin 6 Concentration in Synovial Fluid of Patients with Inflammatory and Degenerative Arthritis

The present study aims to compare interleukin 6 concentration in synovial fluid in patients with known types of arthritis. Persistent synovitis without known markers, such as Rheumatoid Factor (RF), Anti- Citrullinated Protein Antibodies (ACPA), and genetic markers as HLA-B27, is not uncommon. It is valuable to determine the presence of chronic inflammation and put it in correlation with agerelated changes, which are especially relevant for middle-aged patients with mono- or oligoarthritis, when the dilemma to start disease-modifying drugs for inflammatory disease often is present. Interleukin 6 (IL-6) plays a significant role in chronic inflammation. IL-6 concentration in synovial fluid reflects the presence and activity of joint inflammation. Synovial fluid was obtained from 101 patients with chronic synovitis. IL-6 concentration was determined by the immunochemical luminescence method. The median IL-6 concentration in synovial fluid in patients with osteoarthritis (OA) was 138.0 pg/ml (interquartile range (IQR) 43.4 to 296.0); in patients with rheumatoid arthritis was 2516.5 pg/ml, (IQR 1136.0 to 25058.0); in reactive arthritis 2281.0 pg/ml (IQR 1392.0 to 8652.0); psoriatic arthritis 1964.0 pg/ml (IQR 754.0 to 7300.0); ankylosing spondylitis 2776.0 pg/ml (IQR 514.7, 3944.0); in a group with negative RF, ACPA and HLA-B27 inflammatory arthritis 2163.0 pg/ml (IQR 822.0 to 7875.0). There is statistically significant difference of IL-6 concentration comparing OA and each inflammatory arthritis group, p<0.0001. IL-6 detection in the synovial fluid is helpful in arthritis evaluation. The results show that an IL-6 level over 1000 pg/ml suggests the diagnosis of inflammatory arthritis.