Bead-by-bead normalization of single antigen assays: A necessary step for accurate detection of weak anti-HLA antibodies.

Ascertaining the presence of weakly positive anti-HLA donor-specific antibodies (DSA) in organ transplantation with multiplex single antigen beads assays may be challenging despite their high sensitivity due to technical variability issues. Through extensive datasets of Next-Generation Sequencing HLA typings and single antigen analyses, we reassessed the mean fluorescence intensity (MFI) positivity threshold of the assay to enhance accuracy. By showing that some beads were more prone to false positivity than others, we propose a nuanced approach that accounts for nonspecific intrinsic reactivities at the HLA antigen level, that is, on a bead-by-bead basis, as it enhances assay precision and reliability. This is substantiated by a comprehensive statistical analysis of MFI values and the implementation of the determination of a "Quantile Adjusted Threshold 500" (QAT500) value for each bead. Applied to DSA detection during patients' follow-up, this approach discriminated better and earlier low-strength DSA that would later raise their MFI above the clinically relevant threshold of 3000. Moving from a subjective interpretation to a more objective and precise methodology allows for standardizing HLA antibody and DSA detection. The study emphasizes the need for further research with real clinical data to validate and refine this approach.

Does the Use of the "Proseek® Multiplex Inflammation I Panel" Demonstrate a Difference in Local and Systemic Immune Responses in Endometriosis Patients with or without Deep-Infiltrating Lesions?

Endometriotic lesions are able to infiltrate surrounding tissue. This is made possible partly by an altered local and systemic immune response that helps achieve neoangiogenesis, cell proliferation and immune escape. Deep-infiltrating endometriosis (DIE) differs from other subtypes through the invasion of its lesions over 5 mm into affected tissue. Despite the invasive nature of these lesions and the wider range of symptoms they can trigger, DIE is described as a stable disease. This elicits the need for a better understanding of the underlying pathogenesis. We used the "Proseek® Multiplex Inflammation I Panel" in order to simultaneously detect 92 inflammatory proteins in plasma and peritoneal fluid (PF) of controls and patients with endometriosis, as well as in particular patients with DIE, in order to gain a better insight into the systemically and locally involved immune response. Extracellular newly identified receptor for advanced gycation end-products binding protein (EN-RAGE), C-C motif Chemokine ligand 23 (CCL23), Eukaryotic translation initiation factor 4-binding protein 1 (4E-BP1) and human glial cell-line derived neurotrophic factor (hGDNF) were significantly increased in plasma of endometriosis patients compared to controls, whereas Hepatocyte Growth factor (HGF) and TNF-related apoptosis inducing ligand (TRAIL) were decreased. In PF of endometriosis patients, we found Interleukin 18 (IL-18) to be decreased, yet Interleukin 8 (IL-8) and Interleukin 6 (IL-6) to be increased. TNF-related activation-induced cytokine (TRANCE) and C-C motif Chemokine ligand 11 (CCL11) were significantly decreased in plasma, whereas C-C motif Chemokine ligand 23 (CCL23), Stem Cell Factor (SCF) and C-X-C motif chemokine 5 (CXCL5) were significantly increased in PF of patients with DIE compared to endometriosis patients without DIE. Although DIE lesions are characterized by increased angiogenetic and pro-inflammatory properties, our current study seems to support the theory that the systemic immune system does not play a major role in the pathogenesis of these lesions.

Neuroinflammatory Dysfunction of the Blood-Brain Barrier and Basement Membrane Dysplasia Play a Role in the Development of Drug-Resistant Epilepsy.

Drug-resistance epilepsy (DRE) is a key problem in neurology. It is possible that damage to the blood-brain barrier (BBB) may affect resistance in DRE. The aim of this work was to assess the damage and dysfunction in the BBB in the area of epileptic foci in patients with DRE under conditions of neuroinflammation. The changes to the BBB in temporal lobe epilepsy (by immunohistochemistry and transmission electron microscopy), levels of neuroinflammatory proteins, and cytokine levels in the blood (by multiplex analysis) were studied. Increased levels of vascular endothelial growth factor (VEGF) and growth-regulated protein (GRO), and decreased levels of epidermal growth factor (EGF) in plasma, combined with overexpression of the VEGF-A receptor by endotheliocytes were detected. Malformation-like growths of the basement membrane of the capillaries of the brain complicate the delivery of antiepileptic drugs (AEDs). Dysplasia of the basement membrane is the result of inadequate reparative processes in chronic inflammation. In conclusion, it should be noted that damage to the microcirculatory network of the brain should be considered one of the leading factors contributing to DRE.

[Pleiomorphism of the cytokine profile in nasal polyp tissue depending on the phenotype of chronic rhinosinusitis with nasal polyps]. / Pleiomorfizm tsitokinovogo profilya v tkani polipov v zavisimosti ot fenotipa polipoznogo rinosinusita.

Clinical phenotypes of chronic rhinosinusitis with nasal polyps (CRSwNP) are characterized with different inflammation patterns of mRNA expression of cytokines and depend on presence of allergic rhinitis (AR), atopic bronchial asthma (aBA) or nonatopic bronchial asthma (nBA). OBJECTIVE: To compare inflammation response in patients with different phenotypes of CRSwNP according to level secretion of the key cytokines in nasal polyp tissue. MATERIAL AND METHODS: 292 patients with CRSwNP were divided into four phenotypes: group 1 - CRSwNP without respiratory allergy (RA) and without BA; group 2a - CRSwNP+ AR with aBA; group 2b - CRSwNP+AR without aBA; group 3 - CRSwNP+nBA. Control group (n=36) included patients with hypertrophic rhinitis without atopy or BA. Using multiplex assay we defined the level of IL-1ß, IL-4, IL-5, IL-6, IL-13, IFN-γ, TGF-ß1, TGF-ß2, TGF-ß3 in nasal polyp tissue. RESULTS: The evaluation of cytokines levels in nasal polyps in different CRSwNP phenotypes showed a pleiotropy of different cytokine secretion depending on different comorbid pathology. In control group we estimated the lowest levels of all detected cytokines in comparison with other CRS groups. High levels of local proteins IL-5 and IL-13 and low levels of all isoform of TGF-ß characterized CRSwNP without RA and BA. The combination of CRSwNP with AR showed high levels of proinflammatory cytokines IL-6 and IL-1ß, and high levels of TGF-ß1 and TGF-ß2. The combination of CRSwNP with aBA estimated low levels of proinflammatory cytokines IL-1ß, IFN-γ; in case of CRS+nBA we determined the highest levels of TGF-ß1, TGF-ß2 and TGF-ß3 in nasal polyp tissue. CONCLUSIONS: Each CRSwNP phenotype is characterized by different mechanism of local inflammation. This underlies the necessity to diagnose BA and respiratory allergy among these patients. The evaluation of local cytokine profile in different CRSwNP phenotypes can help to determine the target anticytokine therapy for patients who has low efficacy of basic corticosteroid therapy.

Enhancing of detection resolution via designing of a multi-functional 3D connector between sampling and detection zones in distance-based microfluidic paper-based analytical device: multi-channel design for multiplex analysis.

One of the problems in the distance-based microfluidic paper-based analytical device (DB-µPAD) that limits the detection resolution is the mixing of reagents from the detection to the sampling zone or vice versa due to spreading by capillary action. In the present paper, to overcome mixing of the reagents in the zones, a multi-functional connector using a three-dimensional (3D) design has been developed externally to connect the two zones. Using such a novel design, it is acertained that there is no any mixing due to the dispersion of the reagents in the two zones. Interestingly, the simple 3D connector has other functions, such as its potential to be used as a masking zone and/or reaction zone whenever is needed. Based on this proposed 3D connector-based DB-µPAD, three parallel microchannels were built as detection zones with one sampling zone for multiplex analysis for the detection of Fe2+, Ni2+, and hardness of water. In the Ni2+ channel, the connector piece worked as both masking part and connection part. In the Fe2+ line, the connector served as the connector and reducing pad (Fe3+ to Fe2+ ions). While in the third channel, the connector has connection function only. Satisfactory limit of detection (LoD), accuracy, and precision were obtained using this design. The LODs obtained with the proposed design were 1.13, 0.62, and 1.87 mg.L-1 for total hardness, Fe2+, and Ni2+, respectively.

Cytokine Profiling in Different SARS-CoV-2 Genetic Variants.

This study is a successor of our previous work concerning changes in the chemokine profile in infection that are associated with different SARS-CoV-2 genetic variants. The goal of our study was to take into account both the virus and the host immune system by assessing concentrations of cytokines in patients infected with different SARS-CoV-2 variants (ancestral Wuhan strain, Alpha, Delta and Omicron). Our study was performed on 340 biological samples taken from COVID-19 patients and healthy donors in the timespan between May 2020 and April 2022. We performed genotyping of the virus in nasopharyngeal swabs, which was followed by assessment of cytokines' concentration in blood plasma. We noted that out of nearly 30 cytokines, only four showed stable elevation independently of the variant (IL-6, IL-10, IL-18 and IL-27), and we believe them to be 'constant' markers for COVID-19 infection. Cytokines that were studied as potential biomarkers lose their diagnostic value as the virus evolves, and the specter of potential targets for predictive models is narrowing. So far, only four cytokines (IL-6, IL-10, IL-18, and IL-27) showed a consistent rise in concentrations independently of the genetic variant of the virus. Although we believe our findings to be of scientific interest, we still consider them inconclusive; further investigation and comparison of immune responses to different variants of SARS-CoV-2 is required.

Synthesis, Self-Assembly in Crystalline Phase and Anti-Tumor Activity of 2-(2-/4-Hydroxybenzylidene)thiazolo[3,2-a]pyrimidines.

A series of new thiazolo[3,2-a]pyrimidines different by aryl substituents in 2 and 5 positions are synthesized and characterized in solution as well as in the crystalline phase using 1H and 13C NMR-, IR-spectroscopies, mass-spectrometry methods, and single crystal X-ray diffraction (SCXRD). The SCXRD study revealed the role of intermolecular H-bonding in the formation of supramolecular architectures (racemic monomers, centrosymmetric racematic dimers, or homochiral 1D chains) of obtained thiazolo[3,2-a]pyrimidines derivatives depending on solvents (aprotic DMSO or protic EtOH) used upon the crystallization process. Moreover, the in vitro study of cytotoxicity toward different tumor cells showed their high or moderate efficiency with moderate cytotoxicity against normal liver cells which allows to consider the obtained thiazolo[3,2-a]pyrimidine derivatives as promising candidates for application as antitumor agents.

A Rebuilding-Free Nucleic Acid Detection Strategy Enables Ultrasensitive Genotyping, N-in-1 Logic Screening and Accurate Multiplex Assay of Dangerous Pathogens.

Sensitive, rapid and low-cost nucleic acid detection is critical for controlling infectious pathogens. Here, we develop a ready-to-use and multimodal detection based on a rebuilding-free, ultrasensitive and selective strategy named dual hairpin ligation-induced isothermal amplification pro (DHLApro). Taking influenza A, influenza B, MERS-CoV, SARS-CoV-2 as model targets, we demonstrate DHLApro provides ≈zM level ultra-sensitivity, being equaling to 0.45 copy/µL in original sample. By simply changing the recognition module, a set of DHLApro components can be applied to a new target without performance loss. Moreover, DHLApro innovatively allows flexible logic/multiplex assay using one set of primer, for example, the "N pathogens-in-1" OR gate screening and accurate multi-channel multiplex assay. Compared with traditional methods, the cost of this logic/multiplex assay has been largely reduced and the cross-interference between the multiple primer sets is also avoided.

A qualitative and quantitative comparison of IgE antibody profiles with two multiplex platforms for component-resolved diagnostics in allergic patients.

BACKGROUND: Clinically complex phenotypes require more and more sophisticated and comprehensive diagnostic approaches, able to discriminate genuine sensitizations from cross-reactivity. Interpretative complexity of multiplex diagnostic arrays has somewhat limited their diffusion. This study compares two currently available methods, namely ISAC® test and ALEX2® test. METHODS: In total, 140 allergic individuals, with a history of atopic dermatitis, adverse food reactions, allergic rhinitis and/or bronchial asthma were studied by Allergy Explorer-ALEX2® macroarray and ImmunoCAP ISAC112® . Lin's concordance correlation coefficient, intraclass correlation coefficient and Bland-Altman plots were used to verify the agreement between continuous values. Cohen's kappa coefficient (k) was assessed for the molecules available in both tests. The degree of relationship was analysed using Spearman's correlation (quantitative variables) and Pearson's χ2 or Fisher's exact test (categorical variables). RESULTS: A substantial agreement (κ = 0.795) was observed between the two methods with 94,3% concordant results when results were dichotomized as negative or positive, but if double-negative results were discarded, the agreement dropped to 71%. Conversely, little or no concordance was observed comparing raw data. Considering the 102 molecules shared by both systems, 28/102 (27%) showed an almost perfect agreement (k > 0.81), and concordance was good (k > 0.61) in a further 32 (31%) cases. A perfect to substantial agreement was observed by comparing species-specific aeroallergens. Heterogeneous results emerged comparing panallergens (co-recognition ranging from 30% for tropomyosin/serum albumins to 70% for PR-10/profilin). The correlation among LTP, profilin and PR-10 assayed with ISAC® was better than ALEX2® , but the latter identified more positive cases due to the wider number of molecules available. The CCD blocker provided by ALEX® test abolishes the carbohydrate determinants signal in 60% of the 33 cases reactive to MUXF3 on the ISAC® test. CONCLUSION: Despite the excellent concordance of the species-specific markers, the analysis of the panallergens provided in both methods suggests a better performance of the ISAC® test on those components, while the ALEX2® test, which includes a larger number of allergens, allowing a broader molecular detection.

Peanut components measured by ISAC: comparison with ImmunoCap and clinical relevance in peanut allergic children.

BACKGROUND: Specific IgE (sIgE) against the peanut component Arachis hypogaea (Ara h) 2 has been shown to be the most important allergen to discriminate between peanut allergy and peanut tolerance. Several studies determined sIgE cut off values for Ara h 2, determined by singleplex measurements. However, cut off values for Ara h 2 from multiplex arrays are less well defined. The aim of this study was to evaluate the correlation between Ara h 2 sIgE determined by singleplex versus multiplex measurements and to assess the diagnostic value of the different peanut components included in Immuno Solid-phase Allergen Chip (ISAC) multiplex analysis in children with a suspected peanut allergy. METHODS: In this retrospective study we analyzed Ara h 2 sIgE values with singleplex Fluorescence Enzyme Immunoassay (FEIA, ImmunoCap) and multiplex microarray (ISAC) measurements in 117 children with a suspected peanut allergy. Also, other peanut components measured by ISAC were analyzed. Double blinded placebo controlled oral food challenges were used as golden standard. RESULTS: Among all studied peanut components FEIA Ara h 2 sIgE showed the highest area under the curve (AUC, 0.922), followed by ISAC Ara h 6 and Ara h 2 sIgE with AUCs of respectively 0.906 and 0.902. Best cut off values to diagnose peanut allergy were 4.40 kU/l for FEIA Ara h 2 sIgE and, 7.43 ISU and 8.13 ISU for respectively Ara h 2 and Ara h 6 sIgE in ISAC microarray. Ara h 2 sIgE determined in FEIA and ISAC showed a good correlation (r = 0.88; p < 0.01). CONCLUSION: Ara h 6 and Ara h 2 sIgE in multiplex ISAC are both good predictors of clinical peanut allergy in Dutch children, and their performance is comparable to the use of Ara h 2 in singleplex FEIA. The simultaneous measurement of different peanut components using ISAC is an advantage and clinically useful to detect peanut allergic children that are Ara h 2 negative but sensitized to other peanut proteins such as Ara h 6.

DNA-Templated Timer Probes for Multiplexed Sensing.

Simultaneous analysis based on encoded fluorophores suffers from potential crosstalk between fluorophores and the limited number of colors that can be practically resolved. Inspired by nontrivial temporal patterns in living organisms, we developed a DNA-templated probe by utilizing DNA polymerase (DNAP) for multiplexed detection of nucleic acids. These probes use differential delay times of signaling by a DNAP-mediated extension to distinguish different targets, which serve as the primers. Taking advantage of the high processivity and the controllable kinetics of DNAP, we find that multiplexed detection can be achieved in homogeneous solution using a single fluorophore. As a proof of concept, we developed assays for genomic DNA from four different bacteria. In addition, we designed and implemented probes to undergo a single oscillation in signal as an alternative way for multiplexing. We anticipate this approach will find broad applications not only in sensing but also in synthetic DNA nanosystems.

[Evaluation of aqueous cytokine levels in eyes with Fuchs endothelial corneal dystrophy and bullous keratopathy]. / Opredelenie urovnya tsitokinov vo vnutriglaznoi zhidkosti pri endotelial'noi distrofii rogovitsy Fuksa i bulleznoi keratopatii.

PURPOSE: To evaluate cytokine levels in the aqueous humor (AH) of patients with Fuchs endothelial corneal dystrophy (FECD) and bullous keratopathy (BK). MATERIAL AND METHODS: The study included 74 patients (74 eyes). The first group consisted of 31 patients (72.7±9.2 years) with FECD; the second group included 35 patients (72.4±9.1 years) with BK. The control group comprised 8 patients (74.3±4.1 years) with immature cataract. Before surgery, patients underwent pachymetry of the central cornea (RTvue-100 OCT, Optovue, USA). Patients of groups 1 and 2 underwent endothelial keratoplasty (DSAEK or DMEK), or penetrating corneal transplantation. Patients of the control group underwent phacoemulsification with implantation of intraocular lens. The initial stage of the surgery involved AH sample collection for evaluation of cytokine levels (IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12(p70), IL-13, IL-17, G-CSF, GM-CSF, IFNγ, MCP-1, MIP-1ß and TNF-α) by fluorescent flow cytometry using the Bio-Plex Pro Human Cytokine Panel, 17-plex (Bio-Rad, USA). RESULTS: Multiplex analysis of the AH content did not show any statistically significant differences in cytokine levels between decompensated FECD and BK eyes. The levels of IL-6, IL-8, GM-CSF, IFNγ, MCP-1, MIP-1ß were significantly elevated in FECD and BK eyes compared with healthy control. An insignificant deviation of IL-4 and IL-13 levels was detected in FECD and BK eyes compared with healthy controls. There were no significant differences in IL-1ß and TNF-α (indicators of acute inflammation) between the study groups. CONCLUSION: The obtained data confirm that FECD and BK are associated with disruption of ocular immune privilege that leads to chronic local inflammation, which in turn causes remodeling of the corneal tissues resulting in fibrosis.

Multiplex testing for the screening of lysosomal storage disease in urine: Sulfatides and glycosaminoglycan profiles in 40 cases of sulfatiduria.

PURPOSE: To describe an efficient and effective multiplex screening strategy for sulfatide degradation disorders and mucolipidosis type II/III (MLII/III) using 3 mL of urine. METHODS: Glycosaminoglycans were analyzed by liquid chromatography-tandem mass spectrometry. Matrix assisted laser desorption/ionization-time of flight tandem mass spectrometry was used to identify free oligosaccharides and identify 22 ceramide trihexosides and 23 sulfatides, which are integrated by 670 calculated ratios. Collaborative Laboratory Integrated Reports (CLIR; https://clir.mayo.edu) was used for post-analytical interpretation of the complex metabolite profile and to aid in the differential diagnosis of abnormal results. RESULTS: Multiplex analysis was performed on 25 sulfatiduria case samples and compiled with retrospective data from an additional 15 cases revealing unique patterns of biomarkers for each disorder of sulfatide degradation (MLD, MSD, and Saposin B deficiency) and for MLII/III, thus allowing the formulation of a novel algorithm for the biochemical diagnosis of these disorders. CONCLUSIONS: Comprehensive and integrated urine screening could be very effective in the initial workup of patients suspected of having a lysosomal disorder as it covers disorders of sulfatide degradation and narrows down the differential diagnosis in patients with elevated glycosaminoglycans.

Identification of new urinary risk markers for urinary stones using a logistic model and multinomial logit model.

BACKGROUND: Risk assessment for urinary stones has been mainly based on urinary biochemistry. We attempted to identify the risk factors for urinary stones by statistically analyzing urinary biochemical and inflammation-related factors. METHODS: Male participants (age, 20-79 years) who visited Nagoya City University Hospital were divided into three groups: a control group (n = 48) with no history of stones and two stone groups with calcium oxalate stone experience (first-time group, n = 22; recurring group, n = 40). Using 25-µL spot urine samples, we determined the concentrations of 18 candidate urinary proteins, using multiplex analysis on a MagPix® system. RESULTS: In univariate logistic regression models classifying the control and first-time groups, interleukin (IL)-1a and IL-4 were independent factors, with significantly high areas under the receiver operating characteristic curve (1.00 and 0.87, respectively, P < 0.01 for both). The multivariate models with IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) showed higher areas under the receiver operating characteristic curve (0.93) compared to that for the univariate model with IL-4. In the classification of control, first-time, and recurrence groups, accuracy was the highest for the multinomial logit model with IL-4, GM-CSF, IL-1b, IL-10, and urinary magnesium (concordance rate 82.6%). CONCLUSIONS: IL-4, IL-1a, GM-CSF, IL-1b, and IL-10 were identified as urinary inflammation-related factors that could accurately distinguish control individuals from patients with urinary stones. Thus, the combined analysis of urinary biochemical data could provide an index that more clearly evaluates the risk of urinary stone formation.

[Multiplex analysis on the luminex platform in complex estimation of cardiovascular biomarker dynamics in patients with acute myocardial infarction.]

The multimarker approach more accurately reflects the key mechanisms of pathogenesis and biochemical interactions, compared with the use of individual indicators. It is a reason of steadily growing interest in the development and use of various combinations of biomarkers in assessing the prognosis and stratification of cardiovascular risk in patients with a wide range of cardiological profiles. Multiplex analysis technology on the Luminex platform is the best tool for the simultaneous quantitative determination of a complex of different biomarkers in a single. Using the MILLIPLEX® MAP Human Cardiovascular Disease Panel, a multiplefold increase of FABP, Troponin I, CK-MB, BNP, Nt-proBNP, BNP in the first 24 hours after MI, decreasing in 6 months with a high degree of confidence, was shown. There were no differences in the content of LIGHT between the stages of observation, as well as in comparison with the reference range. The content of LIGHT on the first day of MI showed strong positive associations with markers of damage of myocardium and myocardial stress. On the first day of MI, a significant increase in the content of ESM-1, decreasing in 6 months after MI to the reference values was found. Strong positive associations of ESM-1 with Troponin I and BNP levels were established. A significant increase of proinflammatory cytokine OSM on the first day of MI, decreasing in the late post-infarction period to reference values was shown. Correlation analysis revealed direct relationships of OSM with Troponin I, CK-MB, Nt-proBNP and BNP. The use of the MILLIPLEX® MAP Human Cardiovascular Disease Panel 1 diagnostic multimarker panel allowed for the simultaneous quantitative analysis of 11 biochemical parameters, associated with inflammation, atherogenesis, endothelial dysfunction, ischemia and myocardial necrosis. The results can be used to improve the effectiveness of complex diagnostics in patients with primary myocardial infarction with ST segment elevation.

[The multiplex analysis of drug medicinals on the basis of technology of immunochips Phosphan.]

The new technique of multiplex qualitative analysis of narcotic, psychotropic remedies is developed on the basis of technology Phosphan using immunochips in the format of standard 96-wells plates, monoclonal antibodies to narcotic compounds and Pt-coproporphyrin as a long luminescent marker. The multiplex analysis was implemented using 20 mkl of human biological fluid (urine, blood serum or saliva) of 2 discs of 3.2 mm in diameter made of dried urine spot on paper. No preliminary processing or dilution of analyzed sample is required. The large range of measured concentrations was demonstrated under high sensitivity of analysis: 1 ng/ml of morphine and methadone, 0.5 ng/ml of barbiturates, 2 ng/ml of benzoylecgonine, methamphetamine, cannabinoids and benzodiazepines, 8 ng/ml amphetamine at variability of results no more than 15%. The approbation of technique was implemented using valid samples of urine (n=197) and blood serum (n=98) demonstrated that the technique permits to detect properly opiates, cocaine, cannabinoids, methadone, benzodiazepine, barbiturates and amphetamines at absence of false positive results in case of analysis of samples containing non-narcotic medications. The results of study of samples of dried urine spot on paper (n=50) well coincided with the results of analysis of fluid samples for all analyzed analytes. On the basis of proposed multiplex analysis a test-system Narc-Phosphan was developed for quantitative studying simultaneously up to 96 samples of various biological fluids, including as dried spots on paper. The analysis demonstrated high sensitivity, specificity and exactness during detection of the most prevailed narcotic substances that permits to propose this technique as a primary test during mass check-ups of population with purpose of detection of drug abuse, especially at the earlier stage.

Multiplex analysis of serum hormone and cytokine in patients with cervical cOPLL: towards understanding the potential pathogenic mechanisms.

Cervical ossification of the posterior longitudinal ligament (cOPLL) is one of the major causes of myelopathy. However, the mechanism underlying remains elusive. In the present study, using MILLIPLEX magnetic bead panel, we investigated four serum hormones and six serum cytokines in cOPLL patients and healthy subjects. The results showed that tumor necrosis factore-α (TNF-α) were significantly increased, and DDK-1 was significantly decreased in the serum from male and female cOPLL patients compared with those from healthy controls, respectively. Osteopontin (OPN) and fibroblast growth factor-23 (FGF-23) were significantly increased in male cOPLL patients compared with that in healthy male controls. Further analysis showed that FGF-23 and OPN significantly increased, dickkopf-1 (DKK-1) decreased in the extensive cOPLL group. In addition, a significant positive correlation between the OPN and FGF-23 was observed in male cOPLL patients. The results are useful for understanding the mechanism underlying cOPLL.

[Immunogenicity of recombinant analog of antitumor protein lactaptin].

Therapeutic monoclonal antibodies and recombinant proteins including cytokines are commonly used in the treatment of cancer and inflammatory diseases. In most cases, these protein-based drugs exhibit a high therapeutic efficacy, which is unfortunately frequently associated with a variety of side effects. We have investigated the in vitro and in vivo immunogenicity of recombinant antitumor protein lactaptin (RL2). Based on the qRT-PCR analysis, we have shown that, in MDA-MB-231 human breast adenocarcinoma cells, RL2 suppresses the NF-kB signaling cascade that regulates the reactions of innate immunity. RL2 inhibits the expression of the CXCL1 protein and apoptosis inhibitor A20 and enhances expression of IkB, NF-kB repressor. The ELISA method has been used to evaluate the antibody titer in the blood of mice, which received single and triple intravenous or intraperitoneal injections of RL2. The multiplex immunoassay of 23 cytokines in the mice blood has shown that the RL2 injections lead to a slight increase in the levels of systemic pro-inflammatory cytokine interleukin-5 (IL-5) and keratinocyte chemoattractant (KC), a homologue of human macrophage inflammatory protein-1 (MIP-1). These observations indicate the low immunogenicity of the recombinant lactaptin analog, which can be considered to be a potential molecular drug candidate for further clinical development.

[Scanning for KRAS, NRAS, BRAF, and PIK3CA mutations by DNA melting analysis with TaqMan probes].

Scanning for mutations by DNA melting analysis (DMA) is based on asymmetric PCR followed by the melting of duplexes formed by single-stranded amplicons with TaqMan probes. The method is optimally suited for clinical genetic testing; it is easy to perform, high-throughput, and sensitive. The detection limit of mutant alleles by the DMA method is about 3%, which is much higher than the sensitivity of Sanger sequencing. In addition, the DMA method is realized in a closed-tube format, while 2-h assay is carried out in a single tube without any intermediate or additional procedures thereby minimizing the risk of cross contamination of the samples. The validation of the DMA method was performed by scanning for mutations of clinically significant genes KRAS, NRAS, BRAF, and   PIK3CA in 324 DNA samples from tumors of patients with melanoma, colorectal and lung cancer. DNA was isolated either directly from tumor tissues, or from formalin-fixed paraffin-embedded tumor tissues. The detected mutations were verified by Sanger sequencing. The spectra of mutations identified in each tumor type correspond to the literature data and, thus, validate the use of DMA.

An evaluation of multiplex bead-based analysis of cytokines and soluble proteins in archived lithium heparin plasma, EDTA plasma and serum samples.

OBJECTIVE: To assess the usability of archived plasma and serum by multiplex (Luminex) analysis of circulating proteins (analytes) by evaluating the day to day variation, the effect of several freeze-thaw cycles, and the influence of the media and choice of anticoagulant. METHODS: Nineteen analytes in plasma and serum from 86 head and neck cancer patients and 33 controls were evaluated: EGFR, leptin, OPN, VEGFR-1, VEGFR-2, IL-2, IL-13, PDGF-bb, TNF, PAI-1, SDF-1a, IL-4, IL-6, IL-8, eotaxin, G-CSF, VEGF, GRO-a, and HGF. RESULTS: The correlation between measurements of the same samples analyzed on different dates was reasonable. However, samples run on different dates could exhibit different absolute values. The 75th percentile of the fold differences for samples run on different dates was 2.2. No significant difference was found between one and four freeze-thaw cycles (except for HGF), and the correlation was high. We found significant differences in mean concentrations of the majority of analytes in different media and with different anticoagulants. Only the following analytes did not show difference in mean concentrations: EDTA plasma vs. serum: leptin and VEGFR-2, LH plasma vs. serum: IL-2, IL-13, and VEGF, LH plasma levels vs. EDTA plasma: IL-2 and IL-4. CONCLUSION: Stored serum, LH plasma, and EDTA plasma from clinical trials can be used for analysis of circulating cytokines and proteins. Variations in measurements occur, but are within reasonable ranges. The optimal type of media depends on the analytes, as different analytes have low number of measurements below the lower limit of quantification and higher dynamic ranges in different media.