Solid-phase Assay Research Articles

Дослідження цитокінового спектру ротової рідини хворих із генералізованим пародонтитом на тлі метаболічного синдрому

The purpose of the study was to study the state of cytokine regulation of oral fluid in patients with generalized periodontitis and metabolic syndrome. Materials and methods. For this study, 3 groups of surveys were formed. The main group included 30 people with generalized periodontitis on the background of metabolic syndrome; 30 people with generalized periodontitis, without somatic pathology, formed a comparison group. The obtained results were compared with the data of 20 practically healthy individuals with intact periodontium who were included in the control group. The content of pro-inflammatory cytokines IL-1β, IL-6, TNF-α and anti-inflammatory cytokines IL-4, TGF-β1 in the oral fluid of the study groups was determined by solid-phase enzyme-linked immunosorbent assay. Results and discussion. According to the research, on average, the highest levels of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) were observed in patients with periodontal disease on the background of metabolic syndrome. We investigated increase in the concentration of proinflammatory IL-1β. The average value of IL-6 in the oral fluid of patients with metabolic syndrome exceeded this figure in persons not burdened with somatic pathology by 1.3 times, the difference with healthy individuals was more significant: the indicators differed by 2 times. That can be considered an immune response to the inflammatory process in periodontal tissues. The next stage is the beginning of the cytokine cascade, which is characterized by increased production of IL-6 and TNF-α – inducers of acute phase protein synthesis. TNF-α causes an increase in the number of free radicals and can lead to intensification of apoptosis. Due to the fact that anti-inflammatory IL-4 blocks the induced expression of pro-inflammatory IL-6 and TNF-α, a decrease in its level in oral fluid can be considered an unfavorable factor in the course of inflammatory-dystrophic periodontal lesions in patients with syndrome X. Given that TGF-β1 is an immunosuppressive factor, a decrease in its concentration indicates a deficiency of local factors of immune protection in patients with periodontal disease on the background of metabolic syndrome. Conclusion. Patients with metabolic syndrome and periodontal disease have significant disorders of cytokine regulation, which are complicated by age: expression of proinflammatory IL-1β, IL-6, TNF-α on the background of reduced anti-inflammatory cytokines IL-4 and TGF-β1. Such changes in cytokine homeostasis indicate chronic inflammation, insufficient efficiency of regenerative processes in tooth-retaining tissues, and, as a consequence, lead to a more severe course of periodontal disease in people with metabolic syndrome

Characterization of two chicken origin highly pathogenic H7N9 viruses isolated in northern China

Since the national vaccination program was implemented with the H5/H7 bivalent vaccine in poultry in September 2017, the prevalence of H7N9 avian influenza viruses (AIVs) has been controlled effectively in China. However, highly pathogenic H7N9 viruses still exist, causing sporadic outbreaks especially in some regions of northern China. During our routine surveillance in poultry in 2020, we isolated two strains of H7N9 subtype AIV from breeder layer farms in northern China. We found that these two chicken-origin H7N9 isolates were both highly pathogenic (HP) with a four-amino-acid (KRTA) insertion and an I326V mutation (H3 numbering) in the cleavage site of HA to make the motif PEVPKRKRTAR↓GLF. Molecular markers associated with antigenic drift and enhanced pathogenicity in mammals and interspecies transmission were detected in both isolates. Remarkably, both strains gained the F102V and N157D mutations in their HA genes, which have never been reported before. Solid-phase direct binding assay showed that these two isolates both had dual-receptor binding characteristics, while thermal and acid stability assays indicated that they were relatively stable in high-temperature or acidic conditions. In addition, the animal experiments demonstrated that both strains were highly pathogenic to chickens but low pathogenic to mice. These results suggested that the evolution of H7N9 subtype AIV is still continuing, and they pose a potential threat to poultry and public health. Thus, attention should be paid to the importance of continual surveillance of the H7N9 AIVs.

The Past, Present, and Future in Antinuclear Antibodies (ANA).

Autoantibodies are a hallmark of autoimmunity and, specifically, antinuclear antibodies (ANAs) are the most relevant autoantibodies present in systemic autoimmune rheumatic diseases (SARDs). Over the years, different methods from LE cell to HEp-2 indirect immunofluorescence (IIF), solid-phase assays (SPAs), and finally multianalyte technologies have been developed to study ANA-associated SARDs. All of them provide complementary information that is important to provide the most clinically valuable information. The identification of new biomarkers together with multianalyte platforms will help close the so-called “seronegative gap” and to correctly classify and diagnose patients with SARDs. Finally, artificial intelligence and machine learning is an area still to be exploited but in a next future will help to extract patterns within patient data, and exploit these patterns to predict patient outcomes for improved clinical management.

Clinical Application of a Solid-Phase Assay for SARS-CoV-2 IgG to Predict a Neutralizing Antibody Titer.

To assess protective immunity among a general population against severe acute respiratory syndrome coronavirus 2, the correlation of the commercially available solid-phase assay (SPA) for SARS-CoV-2 IgG with a neutralization assay must be investigated. Both the neutralization assay and SPA were performed on samples of 143 recovered coronavirus disease 2019 (COVID-19) patients. SARS-CoV-2 IgG was measured using two SPAs for the chemiluminescence immunoassay principle with different target proteins: nucleocapsid and spike protein (Architect i2000SR [Abbott] and Liaison XL [DiaSorin], respectively). The plaque reduction neutralization test (PRNT) was conducted to obtain titers for the neutralizing antibody. All patients had PRNT titers ranging from 10 to 2,560. Spike Ab SPA had greater sensitivity than nucleocapsid Ab SPA (81.1% [116/143] and 70.6% [101/143], respectively, p = 0.003). The values measured for both SPAs had a positive correlation with the PRNT titers (both R = 0.77, p < 0.001). To predict a high PRNT titer (≥ 160), cutoff values of two SPAs were adjusted based on receiver-operating characteristics curve analysis. The nucleocapsid Ab SPA (cutoff index of 4.17) attained 90.3% sensitivity and 75.9% specificity, whereas the spike Ab SPA (cutoff value of 109 unit/mL) attained 87.1% sensitivity and 89.3% specificity. Therefore, the spike Ab SPA had greater specificity than the nucleocapsid Ab SPA (p = 0.003). The qualitative SPA for nucleocapsid Ab, as well as the quantitative SPA for spike Ab, had a modest positive correlation with the neutralization assay. However, spike Ab SPA was more suitable for neutralizing capacity.

Impact of donor-specific anti-human leukocyte antigen antibodies in haploidentical hematopoietic stem-cell transplantation: A single-center retrospective study

Introduction: While donor-specific anti-human leukocyte antigen (HLA) antibodies (DSA) have been implicated in graft rejection in solid organ transplantation, their role in hematopoietic stem-cell transplantation (HSCT) remains unclear. Aim: The aim of this study is to study the role of DSA for proper donor selection and its effect in the setting of allogeneic HSCT. Methodology: HLA A, B, C, DRB1, and DQB1 high-resolution typing, and DSA cross match (XM) of patients (n = 73) and their prospective donors (n = 74) were assessed. A case–control study was designed retrospectively to evaluate the effect of pre- existing DSAs on engraftment. Thirty-five cases with 5/10 HLA alleles mismatches and 38 cases with full HLA matched, these two controls were selected for comparison. These were matched for disease, graft type, conditioning regimen, age, gender, blood group, and sensitizing events. DSAs were tested with solid-phase assay (Luminex 100/200 platform). Results: DSAs were detected in six of 35 patients (17%); however, donors selected for transplantation were all negative for DSA crossmatch. These six patients who underwent haploidentical (HI) transplants had antibodies against Class I and II. One patient carried antibodies against both classes. A patient who experienced primary graft failure had a second HI transplant. No other known factors that could negatively influence engraftment were associated with the development of graft failure in this patient. Conclusions: DSAs are not associated with graft rejection in patients undergoing HI stem-cell transplantation. Anti-HLA sensitization should be evaluated routinely in HSCT with HLA mismatched donors for a better outcome.

Isolation of synchronized E. coli elongation complexes for solid-phase and solution-based in vitro transcription assays.

Synchronized transcription elongation complexes (TECs) are a fundamental tool for investigating the biochemical properties of RNA polymerases (RNAPs) and nascent RNA. We recently developed a standardized system for isolating high-purity synchronized E. coli RNAP TECs from an in vitro transcription reaction. Our system uses a custom 5' leader sequence, called C3-SC1 to immobilize synchronized TECs on magnetic beads so that free DNA and non-productive transcription complexes can be depleted. The synchronized elongation complexes isolated by our procedure, called C3-SC1TECs, are >98% active, >95% pure, and can be used in both solid-phase and solution-based transcription assays. The yield of the procedure relative to input DNA is ~11% when C3-SC1TECs are isolated for solid-phase assays and ~8% when C3-SC1TECs are isolated for solution-based assays. Here we describe protocols for purifying C3-SC1TECs, and for assessing the activity, homogeneity, and yield of C3-SC1TEC preparations.

A cross-sectional study to estimate the serum level of C-reactive protein among pediatric meningitis with bacterial etiology

Background: Bacterial meningitis is a global public health issue. C-reactive protein (CRP) has suitable diagnostic value in distinguishing between bacterial and aseptic meningitis especially in cases of negative bacterial culture of the blood and spinal fluid. Aim and Objective: The objective of this study was to estimate the serum level of CRP among pediatric meningitis cases with bacterial etiology. Materials and Methods: The hospital-based descriptive cross-sectional study was performed in a tertiary care pediatric hospital in Eastern India from June to August, 2021 with 150 samples. Patients fulfilling the inclusion criteria were selected for this study after obtaining informed consent. Cerebrospinal fluid sample was collected as per standard guidelines. Phenotypic identification of bacteria including antimicrobial susceptibility testing was done by automation (Vitek 2 compact, bioMerieux). Quantitative estimation of CRP was performed in a solid phase and sandwich-format immunometric assay using a gold antibody conjugate. Human rights, welfare, and autonomy were protected as per national ethical guidelines. Results: Median age (Inter Quartile Range) of 150 cases was 3(1–4.5) year. Escherichia coli (60.52%, 23/38) was the commonest isolate (P &lt; 0.00000001 by Binomial test calculation) followed by Klebsiella pneumoniae (34.24%, i.e., 13/38). A total of 43/150 (28.66%) participants had higher serum CRP. Serum CRP was raised more in Gram-negative bacterial etiology (36 out of 38, 94.73%). Mean serum CRP was higher in Gram-negative cases (P &lt; 0.05). Conclusion: Serum CRP was found significantly higher in meningitis caused by Gram-negative bacteria.

Characterization of Influenza Virus Binding to Receptors on Isolated Cell Membranes.

An interplay between receptor-binding properties of influenza viruses (IVs) and spectrum of sialic acid-containing receptors on target cells in birds and mammals determine viral host range, tissue tropism, and pathogenicity. Here, we describe method that allows to characterize binding of IVs to biologically relevant cellular receptors using a conventional solid-phase enzyme-linked assay. In this method, we isolate plasma membranes from respiratory and intestinal epithelial cells of animal origin (Subheading 3.2). We adsorb the membranes in the wells of 96-well ELISA plates, incubate the membrane-coated wells with serially diluted IVs, and determine amounts of IVs attached to the membranes using viral ability to bind peroxidase-labeled sialoglycoprotein fetuin. Based on the concentration dependence of IV binding to the membrane, we estimate binding avidity and number of binding sites. We describe two variants of the assay in Subheadings 3.6 and 3.7 and provide examples.

Prozone phenomenon in pretransplant testing: An interesting conundrum involving solid-phase and cell-based assays.

Human leukocyte antigen (HLA) is a major determinant in deciding upon solid organ histocompatibility. Donor-specific anti-HLA antibodies (Donor-specific anti-HLA antibodies (DSAs)) are always a contraindication for solid organ transplantation, and identification of DSA becomes very crucial before transplantation to provide long-term graft survival. For identification of DSA, usually, either cell-based or HLA bead-based assay is being used in laboratories. However, both cell-based and bead-based assays have certain limitations. One such common limitation is "prozone effect," which can give false-negative results. Here, we would like to present a small pilot study to analyze the effect of the prozone phenomenon in the cell-based and HLA bead-based assays and its utility in histocompatibility testing. In a series of four experiments, cell-based assay, flow cytometric cross-match (FCXM), and HLA bead-based flow cytometric panel reactive antibodies (PRAs) were performed. Single-antigen bead (SAB) testing was conducted as a first experiment on four known positives samples for anti-HLA antibody-antibodies. In the second experiment, these four samples were pooled together (called pooled sera in the text) and tested for FCXM and PRA. In the third experiment, known commercially available positive control sera were mixed with pooled positive sera (positive control sera + pooled sera) to prepare, what we have called "positive concoction" in the text. In the fourth experiment, the positive concoction was diluted serially (1:2, 1:4, 1:8, and 1:16) and FCXM and PRA were performed again to analyze and compare the prozone effect. Pooled sera did not have the expected median fluorescence intensity (MFI) values in FCXM assay, whereas the PRA was showing >90% positivity. In positive concoction, the MFI of FCXM assay was observed to be declining; however, PRA values remained almost constant. Dilutions of the pooled sera showed that MFI values of FCXM assays were increased suddenly after dilution. The highest MFI values were observed in 1:4 dilution of the sera, and then, it declined gradually, but the PRA values remained almost constant even after serial dilutions. In our experimental findings, it was clear that cell-based assay (FCXM) was more severely affected by the prozone, whereas solid-phase (flow PRA) assay remained resistant to prozone.

Evaluation of the Glycan-Binding and Esterase Activities of Hemagglutinin-Esterase-Fusion Glycoprotein from Influenza D Virus.

Influenza D virus (IDV) is a new member of influenza virus that uses cattle as the primary reservoir and infects multiple agricultural animals. Similar to influenza C virus (ICV), IDV also has seven segments in its genome and has only one major surface glycoprotein, called the hemagglutinin-esterase-fusion (HEF) protein, for receptor-binding, receptor-destroying, and membrane fusion. HEF utilizes 9-O-acetylated sialic acids as its receptor and has both receptor binding and esterase activities, thus is a critical determinant of host tropism. Here, we summarize the methods to evaluate the glycan-binding and esterase activities of HEF in vitro. The glycan-bind property is monitored through glycan microarray, MDCK cell-binding assay, Hemagglutination assay, solid-phase lectin binding assay, and immunofluorescence of tissue sections, and its esterase property is analyzed via esterase enzymatic activity assay.

USE OF BACTERIAL LYSATE IN COMPLEX TREATMENT OF ACUTE BRONCHITIS COMBINED WITH EUTHYROID SICK SYNDROME IN PRESCHOOLERS

Objective. This study's objective was to determine bacterial lysate's effect on the state of humoral immunity (IgA, IgM, IgG) in preschool children with acute bronchitis in combination with euthyroid sick syndrome. Materials and methods. We examined 135 children of preschool age (3 to 6 years) with acute bronchitis (main group) and 28 healthy children (control group). Group Ia included 45 children with acute bronchitis who were treated according to the standard scheme. Preschoolers with acute bronchitis in Group Ib (47 patients) additionally received bacterial lysate. Group IIa included 21 children with acute bronchitis and euthyroid sick syndrome (ESS) manifestations who received standard therapy. Group IIb consisted of 22 patients with acute bronchitis and ESS who received standard therapy and were additionally given an immunomodulator. The bacterial lysate consisted of Staphylococcus aureus 6×109 colony-forming units (CFU), Streptococcus pyogenes 6×109 CFU, Streptococcus viridans 6×109 CFU, Klebsiella pneumoniae 6×109 CFU, Klebsiella ozaenae 6×109 CFU, Haemophilus influenzae B 6×109 CFU, Neisseria catarrhalis 6×109 CFU, Streptococcus pneumoniae 6×109 CFU (the latter included the following types in amount of 1×109 CFU each: TY1/EQ11, TY2/EQ22, TY3/EQ14, TY5/EQ15, TY8/EQ23, TY47/EQ24). The effectiveness of bacterial lysate in children with acute bronchitis and with or without ESS was determined by comparing the humoral immunity parameters (IgA, IgM, IgG) in the acute period (1 to 2 days after the onset) and convalescence period (7 to 10 days after the onset). Levels of immunoglobulins (IgA, IgM, and IgG) were measured by solid-phase enzyme-linked immunosorbent assay. All results were statistically processed using the SPSS 26 software. Results. No statistically significant difference was found between the values of immunoglobulins A, M, and G in children with acute bronchitis and with or without ESS manifestations in the acute period of the disease. At the same time, the above parameters were significantly higher than the corresponding values in the control group. A statistically significant difference was observed in the humoral immunity parameters of patients who were given the bacterial lysate vs. patients who were not. In children who were additionally given the bacterial lysate, IgA levels practically equaled the levels of the control group in the convalescence period. The serum levels of IgM and IgG in children who additionally received the immunomodulator tended to approach the values of the control group. Conclusions. The use of bacterial lysate positively affected the restoration of humoral homeostasis, especially on the level of immunoglobulin A, in children with acute bronchitis and with or without the euthyroid sick syndrome. The bacterial lysate demonstrated high efficiency against this disease in this group of patients.

Examination of Whole-Cell Galectin Binding by Solid Phase and Flow Cytometric Analysis.

We have utilized simple flow cytometric and fluorescence-based solid phase assays to study the interaction of glycan binding proteins (GBP) to cell surface glycoconjugates. These methods utilize commonly employed flow cytometry techniques and commercially available streptavidin-coated microplates to immobilize various biotinylated ligands, such as glycopeptides, oligosaccharides, and whole cells. Using this approach, fluorescently labeled GBPs, in particular, members of the galectin family, can be interrogated for potential interactions with cell surface carbohydrates, including elucidation of the potential impact of alterations in glycosylation on carbohydrate recognition. Using these approaches, we present examples of flow cytometric and fluorescence-based solid phase assays to study galectin-carbohydrate interactions.

Machine-learning assisted antibiotic detection and categorization using a bacterial sensor array

With the extensive global use of antibiotics, the problems associated with environmental and food antibiotic residues have significantly increased, necessitating new methods for rapid detection and categorization of compounds with antibiotic activity. In an answer to this need, we report a new platform, bacterial array solid-phase assay (BacSPA), based on monitoring the responses of 15 stress-responsive Escherichia coli sensor strains. These bioreporters, genetically modified by fusing bioluminescence (luxCDABE) reporter genes upstream of stress-induced gene promoters, were inoculated on solidified agar slabs individually mixed with 11 different antibiotics, belonging to 7 mode of action classes. The antibiotic-induced bioluminescence by the different strains generated a distinct response pattern for each antibiotic class. This luminescence pattern was monitored by time-lapse photography, and a machine learning algorithm, Multiclass Decision Forest, was applied to train categorization models that either identified the compound or categorized its class. The best model displayed a 65% accuracy for compound identification and 90% for class classification, within three hours of exposing the sensor array to the tested compound. The method also effectively categorized antibiotics at different concentrations: the trained model categorized eight antibiotics at concentrations ranging from 125 ppb to 1000 ppb, with accuracies mostly higher than 70%. The method was further successfully applied for categorizing antibiotics not included in the training. With a more extensive future database, encompassing a broader range of existing antibiotics, this method may be turned into a powerful tool for detecting and categorizing both known and new antibiotic residues in food or environmental samples.

Treatment forms of house dust mites allergens intended for allergen-specific immunological therapy

BACKGROUND: The growth of allergic diseases dictates the necessity to develop new forms of therapeutic allergens since therapy with native water-salt extracts of allergens is unsafe and convenient. The risk of systemic side effects during allergen-specific immunotherapy sets the task for researchers to create modern therapeutic forms of allergens with high immunogenic and low allergenic activity. Our laboratory has been developing preparations for the diagnosis and treatment of house dust mites allergies for many years.&#x0D; AIMS: To create modern therapeutic forms of allergen preparations from house dust mites of the genus Dermatophagoides that are intended for allergen-specific immunotherapy&#x0D; MATERIALS AND METHODS: Physicochemical and immunobiological methods, such as electrophoresis in polyacrylamide gel, micropoint immunoblot, solid-phase enzyme-linked immunosorbent assay, and the reaction of binding inhibition of allergen-specific immunoglobulin E in the sera of patients were used to study the obtained preparations.&#x0D; RESULTS: A technology has been developed to obtain a granular dosage form of a mixed allergen from Dermatophagoides pteronyssinus and Dermatophagoides farinae mites for sublingual use. This treatment form has successfully passed preclinical tests, has a pronounced immunogenic activity, and reduced allergenicity, and is convenient for pediatric practice. Allergoids, which are chemically modified preparations, are obtained to reduce allergenicity and increase therapeutic allergen immunogenicity. A succinylated monomeric house dust mites allergoid Dermatophagoides pteronyssinus was created and studied with the National Research Center, Institute of Immunology Federal Medical-Biological Agency of Russia. The study of the immunobiological properties of the obtained preparation showed that the monomeric allergoid has increased immunogenic and decreased allergenic activity contrary to the native water-salt extract.&#x0D; CONCLUSIONS: The created forms of mite allergens can be used to treat patients who are sensitized to house dust mites of the genus Dermatophagoides after clinical trials.

Diagnostic Utility of High Dose Heparin Confirmation Step in Heparin Induced Thrombocytopenia ELISA Assay

Introduction:Heparin-induced thrombocytopenia (HIT) is a potentially life-threatening condition that could occur following exposure to heparin. Accurate and timely diagnosis is critical for appropriate clinical management. Laboratory testing for suspected cases is based on screening for the presence of serum anti-PF4/heparin antibodies using solid-phase enzyme-linked immunosorbent assay (ELSIA) which is known to be sensitive but less specific. A positive ELISA test is followed by functional testing to demonstrate the platelet activating properties and heparin dependence of the pathogenic antibodies. Serotonin release assay (SRA) is considered the gold standard functional test for the diagnosis of HIT. In most anti-PF4/heparin ELISA assays, a high-dose heparin buffer (100U/mL) confirmation step is recommended to demonstrate heparin dependence of detected antibodies and increase the specificity of the assay. The necessity of this confirmation step is controversial with some reports suggesting it could lead to misinterpreting positive ELISA results as negative or indeterminate, especially in cases of very strong and high titer HIT activating antibodies. We hence aimed to investigate the utility of applying this confirmation step as part of an inhouse validation study of a mass spectrometry-coupled SRA (Mayo-SRA).Materials:Three hundred archived serum samples were tested using anti-PF4/heparin IgG antibody ELISA (Immucor Diagnostics, GA, USA). High-dose heparin (100U/mL) confirmation step was performed on all samples with OD units ≥0.4 as recommended by the manufacturer. Samples with OD ≥ 0.4 and ≥50% OD inhibition in the high dose heparin confirmation step are interpreted positive. Mayo-SRA results were compared to a reference 14C SRA method. The 4T clinical score was retrospectively calculated for all patient (range 0-8 points).Results:Of the 300 tested samples, 57 samples were interpreted positive by the anti-PF4/heparin screening ELISA. 33 of the 57 samples were positive using the reference 14C SRA method, whereas 43 samples were positive by Mayo-SRA assay (≥20% serotonin release). Three additional samples were positive by Mayo-SRA, but negative by both screening ELISA and the reference 14C SRA method. All samples with OD units ≥0.4 displayed >50% inhibition in the high-dose heparin regardless of the intensity of the initial OD value or the HIT 4T score, with the exception of one that was negative by both SRA methods and of 1.35 OD value and 6 4T HIT score (Fig-1A). Importantly, thirteen samples were anti-PF4/heparin positive, but SRA negative (by Mayo-SRA and reference method). These samples also displayed positive %heparin inhibition (≥50% OD inhibition) (Fig-1B). Lastly, there were no differences in the degree of %inhibition in samples positive by both reference and Mayo-SRA or Mayo-SRA only (Fig-1B).Conclusion:In our patient cohort, addition of the high dose heparin inhibition confirmation step to the screening anti-PF4/heparin ELISA assay was of no additional diagnostic utility. We hence propose eliminating the heparin inhibition step which would improve laboratory turnaround time, reduce costs, and importantly speed up urgent clinical management decisions.Figure Legend:Fig-1A. No correlation between initial OD values and %OD inhibition using high dose heparin. Scatter plot of all ELISA positive samples (OD ≥0.4) grouped according to OD values. Samples include all SRA-positive and thirteen SRA-negative ELISA-positive samples. Fig-1B. Scatter plot of %OD inhibition comparing samples positive by Mayo-SRA and reference SRA method, positive by Mayo-SRA only, and ELISA-positive SRA-negative by both methods. [Display omitted] DisclosuresPruthi: Bayer Healthcare AG: Honoraria; CSL Behring: Honoraria; Merck: Honoraria; Genentech: Honoraria; HEMA Biologics: Honoraria; Instrumentation Laboratory: Honoraria. Padmanabhan: Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Serum osteoprotegerin levels, endothelial function and carotid intima-media thickness in type 2 diabetic patients

ObjectivesOsteoprotegerin (OPG), a well-known protein that inhibits osteoclast formation and activity, might also be a potential marker for identifying patients with high cardiovascular risk. This study aimed to compare OPG levels, FMD, and CIMT measurements in subjects with vs. without diabetes and investigate the association of serum osteoprotegerin level with the early atherosclerotic markers, endothelial function, and carotid intima-media thickness (CIMT) in patients with type 2 diabetes mellitus (DM2). MethodsForty-nine patients with DM2 (F/M: 26/23, 49.3 ± 10.0 years) and 45 healthy volunteers (F/M: 26/19, 48.3 ± 7.5 years) were included in this cross-sectional study. Serum OPG levels were measured by solid-phase enzyme-linked immunosorbent assay (ELISA). Fasting plasma glucose (FPG) and HbA1c levels were measured. CIMT was measured by B-mode ultrasound, and endothelial function was evaluated via flow-mediated dilation (FMD) of the brachial artery with Doppler ultrasonography. ResultsSerum OPG levels were significantly higher in patients with DM2 (617.0 ± 111.0 pg/mL) compared to controls (481.0 ± 96.0 pg/mL, p < 0.001). While CIMT in diabetic patients (0.65 + 0.13 mm) was higher than controls (0.54 ± 0.10 mm, p = 0.009), FMD measurement was lower in DM2 group (4.2% ± 3.1 mm vs. 7.6% ± 4.1 mm, p = 0.01). Univariate analysis showed that OPG was associated with the presence of diabetes (OR: 6.999, p = 0.001, R2: 15.1%) and hypertension (OR = 6.925, p = 0.001, R2: 13.2%). There was no relationship between OPG levels and CIMT or FMD. ConclusionOsteoprotegerin and CIMT levels were increased, and FMD measurements were decreased in patients with DM2. No association between CIMT, FMD, and OPG measurements was observed. The presence of DM and hypertension were associated with circulating OPG levels.

Immune response and phagocytosis of children with streptococcal tonsillitis and infected with the herpes virus type 6.

The aim of the work is to study the indicators of cellular and humoral immune response and FAN of patients with tonsillitis in children infected with HHV-6 type.&#x0D; Materials and methods of research. In 72 children aged 3–15 years (mean age 8.42±1.44 years), patients with streptococcal tonsillitis of moderate severity, the levels of the main subpopulations of blood lymphocytes were determined using monoclonal antibodies by solid-phase enzyme-linked immunosorbent assay, phagocytic activity of neutrophils (FAN) using the nitrosine tetrazolium reduction test (HCT test) and serum IgA, IgM, IgG levels by radial immunodiffusion. The studies were carried out in the acute period and in the period of convalescence. Mathematical and statistical processing of the data obtained was carried out using Microsoft Excel 2003 and Statistica 6.0 programs. The significance of differences between the mean values was determined using the Student's test (t).&#x0D; Results. Different mechanisms of formation of pathological process at streptococcal tonsillitis at children depending on the concomitant HHV-6 type infection are revealed. In patients with mixed infection in the acute period of tonsillitis determine the signs of deficiency of the cellular immune system with the maximum deviation of CD-lymphocytes (t=3.13 vs. t=2.16 in mono-infection), the tension in the humoral link of the immune response Ig deviation (t=1.63 against t=0.91 in mono-infection) on the background of phagocytosis violation in terms of FAN deviation (t=3.22 against t=2.01 in mono-infection). By the period of convalescence, the restoration of the imbalance of specific and non-specific protection factors does not occur. Increased IgG content in the acute period is one of the features of streptococcal tonsillitis in children on the background of HHV-6t infection.&#x0D; Conclusions. Concomitant VGL-6 type infection in children with streptococcal tonsillitis affects the mechanisms of formation of the inflammatory process and the reactivity of patients, forming the preconditions for adverse outcomes of the disease. The obtained results can be the basis for early detection of herpes virus infection in patients with tonsillitis, will help to optimize the management of patients in different periods of the disease.

Predictive value of immunological markers after bacille Calmette-Guérin induction in bladder cancer.

To investigate the predictive value of different immunological markers on treatment outcomes after bacille Calmette-Guérin (BCG) induction in high-risk non-muscle-invasive bladder cancer (NMIBC). Patients who underwent transurethral resection of bladder tumour for NMIBC were assessed for study eligibility. Urine and blood samples were taken from patients at baseline (immediately before first dose of induction) and after induction (4 h after last [sixth] dose). Urine samples were evaluated for interleukin (IL)-2 and IL-10 by solid-phase enzyme-linked immunosorbent assay. Blood samples were evaluated for tumour necrosis factor α (TNF-α), cytotoxic T-lymphocyte antigen 4 (CTLA-4) and transcription factors (TFs) (GATA-binding protein 3 [GATA3], T-box expressed in T cells [T-bet], and forkhead box protein 3 [FoxP3]) using quantitative reverse transcriptase-polymerase chain reaction analysis. Change pattern and fold change of each evaluable marker was assessed in relation to different treatment outcomes (initial complete response [ICR]/recurrence/progression). Between July 2013 and May 2019, 204 patients were included. Among evaluable markers, urinary IL-2 and serum TNF-α increased in all patients, serum CTLA-4 and FoxP3+ showed a predominant decreased pattern in 188 (92.2%) and 192 (94.1%) patients, respectively. An ICR was achieved in 186 (91.2%) patients. Serum TNF-α fold change and urinary IL-10 change pattern were significantly associated with an ICR (P = 0.001 and P = 0.03, respectively). At a median (range) follow-up of 37 (20-88) months, 104 (56%) patients developed recurrence. Urinary IL-10, serum CTLA-4, T-bet+ , FoxP3+ change patterns and GATA3+ /T-bet+ ratio were significantly associated with tumour recurrence (P = 0.001, P = 0.001, P = 0.02, P = 0.009 and P = 0.001, respectively). Tumour progression occurred in 34 (18.3%) patients. Urinary IL-10, serum CTLA-4, serum T-bet+ change patterns and GATA3+ /T-bet+ ratio were independent predictors of tumour progression (P = 0.001, P = 0.001, P = 0.02 and P = 0.001, respectively). Urinary IL-10 and serum TNF-α can significantly predict ICR. Moreover, change pattern of urinary IL-10, serum CTLA-4, TFs (GATA3, T-bet and FoxP3) and GATA3+ /T-bet+ ratio after BCG induction can independently predict further BCG response. These markers could be implemented in clinical practice when management options are discussed or in systems with severe BCG shortage.

Appearance of new CDC-reactive antibodies in patients waiting for kidney transplantation

BackgroundPatients awaiting kidney transplantation are regularly screened for HLA-antibodies, but there is scarce data about the optimal interval. MethodsResults from Complement-dependent cytotoxicity testing (CDC) for waitlisted patients were reviewed for increases in panel reactive antibodies (PRA) by at least 10%-points. Clinical records were screened for historic immunizing events and possible trigger factors preceding the PRA-increase. Additionally, non-pretransplanted men tested negative for HLA antibodies by solid-phase assays (SPA) out of their first two samples on the waiting list (“non-immunized men”) were evaluated for detection of HLA antibodies by SPA during their further stay on the waiting list. Results15,360 samples from 1928 patients tested by CDC were analyzed for changes in PRA. PRA-increases occurred most frequently in patients waitlisted recently for retransplantation (annual incidence 6%). Removal of previous transplants, severe infections and/or reduced immunosuppression triggered 65% of PRA-increases during the first year after waitlisting. Transfusions accounted for 55% of PRA-increases in later years. Leucocyte-reduced red blood cell units not only boosted historic antibodies, but even induced primary immunization. In the second part of the study, 6780 samples tested by SPA from 703 non-immunized men were evaluated for development of HLA-antibodies. Only 9 men (1.3%) turned HLA antibody-positive (annual incidence 0.4%). ConclusionA uniform screening interval does not fit all: Frequencies should be highest in patients newly waitlisted for re-transplant and lowest in non-immunized men. Transfused patients should be monitored closely for development of HLA-antibodies even if leukoreduced products are used.

High-Throughput Solid-Phase Assay for Substrate Profiling and Directed Evolution of Transketolase.

Thiamine diphosphate-dependent enzymes, and specifically transketolases, form one of the most important families of biocatalytic tools for enantioselective carbon-carbon bond formation yielding various hydroxyketones of biological interest. To enable substrate profiling of transketolases for acceptance of different donors and acceptors, a simple, direct colorimetric assay based on pH reaction variation was developed to establish a high-throughput solid-phase assay. This assay reduces the screening effort in the directed evolution of transketolases, as only active variants are selected for further analysis. Transketolase activity is detected as bicarbonate anions released from the α-ketoacid donor substrate, which causes the pH to rise. A pH indicator, bromothymol blue, which changes color from yellow to blue in alkaline conditions, was used to directly detect, with the naked eye, clones expressing active transketolase variants, obviating enzyme extraction.