Immunological Assays Research Articles

Recent Advances in Mycotoxin Detection: A Review

Mycotoxins, toxic compounds produced by fungi like Aspergillus and Penicillium, pose significant health risks even at low concentrations. Detecting these contaminants in food requires intricate methods due to their low levels and complex sample matrices. Historically, toxic fungi research began with mushrooms but expanded in the mid-1800s to include other fungi, such as Claviceps purpurea, which caused ergotism through contaminated rye. Mycotoxins severely impact both human and animal health, causing issues like cancer, immune suppression, and nervous system damage, while also disrupting global food trade, affecting around 25% of crops worldwide. Traditional detection methods include chromatography (TLC, LC, GC, HPLC) and immunological assays like ELISA and LFI, though these methods have limitations. Recent advances in mycotoxin detection feature innovations like biosensors, fluorescence-based techniques, phage display technology, and smartphone-enabled systems, which offer enhanced sensitivity, specificity, and rapid detection capabilities. These methods represent a promising shift towards more efficient and precise mycotoxin analysis in food safety.

Identification of new antigenic epitopes of porcine reproductive and respiratory syndrome virus nsp12 protein using monoclonal antibodies

Porcine reproductive and respiratory syndrome virus (PRRSV), a member of the arteritis virus family, significantly impacts the swine industry due to its high infectiousness. nsp12, the nonstructural protein encoded by PRRSV, is a membrane-associated protein, with limited knowledge about its antigenic properties and functions. In this study, we used the expressed and purified nsp12 protein as antigen to immunize mice, and successfully screened five positive hybridoma cell lines that stably secrete anti-nsp12 monoclonal antibodies using immunological assays such as indirect ELISA and IFA. The antigenic epitopes recognized by the five monoclonal antibodies were identified using the fusion expression of peptides derived from the overlapping truncators of the nsp12 gene. The results showed that monoclonal antibodies 3G11 and 9C2 recognized the antigenic epitope 93TWGFESDTAY102, 2E3 recognized 115DYNDAFRARQ124, and 10G6 and 2A4 recognized 142PGPVIEPTL150. Furthermore, the three newly identified antigenic epitopes were all immunodominant and located on the surface of nsp12 protein. Notably, the antigenic epitopes 93–102 aa are all highly conserved across PRRSV-2 strains, making them suitable targets for PRRSV-2 detection. In conclusion, our findings advance the understanding of the antigenic properties of the PRRSV nsp12 protein and facilitate the development of assays for PRRSV detection.

Structure analysis and immunomodulatory activity of novel oligosaccharide from Nicotiana tabacum roots

A novel oligosaccharide (NTRP60-W-2) with an average molecular weight of 1377 Da was isolated and purified from Nicotiana tabacum roots. Its structural characteristics and immunomodulatory properties were investigated. Structural analysis revealed that NTRP60-W-2 was composed exclusively of glucose, featuring →1)-α-D-Glcp-(6→ backbone. Immunological assays demonstrated that NTRP60-W-2 significantly enhanced cell viability, nitric oxide production and cytokine secretion (IL-6 and TNF-α) in RAW264.7 cells. These findings provide a foundation for further exploration of Nicotiana tabacum carbohydrates and their potential biological activities.

Unveiling the dynamics of circulating tumor cells in colorectal cancer: from biology to clinical applications

This review delves into the pivotal role of circulating tumor cells (CTCs) in colorectal cancer (CRC) metastasis, focusing on their biological properties, interactions with the immune system, advanced detection techniques, and clinical implications. We explored how metastasis-competent CTCs evade immune surveillance and proliferate, utilizing cutting-edge detection and isolation technologies, such as microfluidic devices and immunological assays, to enhance sensitivity and specificity. The review highlights the significant impact of CTC interactions with immune cells on tumor progression and patient outcomes. It discusses the application of these findings in clinical settings, including non-invasive liquid biopsies for early diagnosis, prognosis, and treatment monitoring. Despite advancements, challenges remain, such as the need for standardized methods to consistently capture and analyze CTCs. Addressing these challenges through further molecular and cellular research on CTCs could lead to improved interventions and outcomes for CRC patients, underscoring the importance of unraveling the complex dynamics of CTCs in cancer progression.

HIV-1 reservoir landscape of post-treatment control.

This review explores the viral reservoir landscape in individuals who control viral replication after treatment interruption (TI), designated as post-treatment controllers (PTCs). Identifying their virologic features is crucial to inform drug-free HIV remission strategies. Traditionally characterized as small, likely due to early treatment, the viral reservoir of PTCs, after TI, exhibits limited transcriptional activity, residual viral replication and subsequent proviral diversity. Intact proviruses are found to be restricted. In nonhuman primate PTCs, this depletion of intact proviruses is already observed in lymph nodes before TI, suggesting that control mechanisms begin during antiretroviral therapy. Furthermore, recent studies suggest immune-driven proviral deep latency associated with repressive epigenetic features and integration sites in PTCs. While molecular mapping of virological features of PTCs is increasingly precise and coupled with in-depth immunologic assays, robust predictive biomarkers of PTCs are still lacking. Despite limited sample sizes and heterogeneous definitions, common virologic features of PTCs include restricted reservoir size and transcriptional activity, fewer intact proviruses and deep proviral latency. Ongoing research using innovative technologies will further elucidate the mechanisms underlying post-treatment control, paving the way for successful HIV cure interventions.

Envelope Protein in Differential Serodiagnosis of Dengue, Zika, and Chikungunya Viruses: A Systematic Review.

This systematic review was conducted to evaluate the applicability of the envelope (E) protein in the diagnosis of arboviruses. This review was performed in accordance with the PRISMA statement. Five databases were explored (PubMed, Web of Science, Scopus, EMBASE, and IEDB). The inclusion and exclusion criteria were applied to study eligibility. After data extraction, the risk of bias and evidence certainty were evaluated according to QUADAS and GRADE assessments, respectively. Eleven studies were included. A total of 11 studies were included in the review. ELISA was the most frequently utilized technique, with two studies employing it for antigen detection and nine for antibodies. The E protein was used as a whole protein, heterologous protein, and peptides. The diagnostic metrics were enhanced by optimizations on techniques, such as antibody capture, competitors, and nanosensors. Monoclonal antibodies showed improved specificity, including in coinfected samples. Seven studies demonstrated a minimal risk of bias, and the evidence certainty was considered moderate for dengue diagnosis. The E protein was successfully employed in different immunological assays with large-scale strategies, enhancing the applicability potential for differential arboviruses' diagnosis. Furthermore, both the antigen design and the implementation of innovative methodologies will have a substantial impact on the quality of the new tests. The PROSPERO protocol related to this work: CRD42021265243.

Intraspecific venom variation in the medically important puff adder (Bitis arietans): Comparative venom gland transcriptomics, in vitro venom activity and immunological recognition by antivenom.

Variation in snake venoms is well documented, both between and within species, with intraspecific venom variation often correlated with geographically distinct populations. The puff adder, Bitis arietans, is widely distributed across sub-Saharan Africa and into the Arabian Peninsula where it is considered a leading cause of the ~310,000 annual snakebites across the region, with its venom capable of causing substantial morbidity and mortality. Despite its medical importance and wide geographic distribution, there is little known about venom variation between different B. arietans populations and the potential implications of this variation on antivenom efficacy. We applied a range of analyses, including venom gland transcriptomics, in vitro enzymatic assays and reverse phase chromatography to comparatively analyse B. arietans venoms originating from Nigeria, Tanzania, and South Africa. Immunological assays and in vitro enzymatic neutralisation assays were then applied to investigate the impact of venom variation on the potential efficacy of three antivenom products; SAIMR Polyvalent, EchiTAb-Plus and Fav-Afrique. Through the first comparison of venom gland transcriptomes of B. arietans from three geographically distinct regions (Nigeria, Tanzania, and South Africa), we identified substantial variation in toxin expression. Findings of venom variation were further supported by chromatographic venom profiling, and the application of enzymatic assays to quantify the activity of three pathologically relevant toxin families. However, the use of western blotting, ELISA, and in vitro enzymatic inhibition assays revealed that variation within B. arietans venom does not appear to substantially impact upon the efficacy of three African polyvalent antivenoms. The large distribution and medical importance of B. arietans makes this species ideal for understanding venom variation and the impact this has on therapeutic efficacy. The findings in this study highlight the likelihood for considerable venom toxin variation across the range of B. arietans, but that this may not dramatically impact upon the utility of treatment available in the region.

Marine macroalgae Chaetomorpha aerea as a dietary supplement: Optimizing immunity and resistance to Edwardsiella tarda in tilapia (Oreochromis mossambicus)

The intensification of aquaculture has led to a rise in fish infections, necessitating the search for alternative antibiotics. In this context, our study investigated the effects of dietary supplementation with Chaetomorpha aerea, a filamentous green algae, on the immune health and resistance to infections in tilapia (Oreochromis mossambicus). Diets containing varying concentrations of C. aerea (0, 1, 2, 5, and 10 g/kg) were prepared and administered to the fish for 30 days, followed by a challenge with Edwardsiella tarda to evaluate survival rates. The results were significant. The diet containing 5 g/kg of C. aerea (group T3) brought about substantial improvements in hematological parameters, including increases in red blood cell count (RBC), hematocrit (Hct), and hemoglobin (Hb). The T3 group exhibited a robust immune response, with higher lysozyme and ceruloplasmin activity in immunological assays. LBP gene expression was significantly elevated in the spleen and thymus of fish in the T3 group, which correlated with higher survival after bacterial challenge compared to the control group. Principal Component Analysis (PCA) and cluster analysis confirmed that the 5 g/kg concentration stood out for maximizing immunological benefits without compromising the overall health of the fish. These findings highlight the robust immune response in the T3 group, a key finding of our study. We conclude that supplementation with C. aerea represents a promising and sustainable alternative in the formulation of diets for tilapia, contributing to improved health and resistance to diseases. Future studies are recommended to explore its application in other species and development stages, in addition to evaluating other health biomarkers.

Diagnostic value of IFN-gamma in tuberculous pleural effusion

BackgroundSimple, rapid, and accurate diagnosis of tuberculous pleural effusion (TPE) remains challenging. This study aimed to determine the accuracy of IFN-γ in diagnosing TPE. MethodsWe quantified the expression of interferon-gamma (IFN-γ) in blood (B), adenosine deaminase (ADA), and IFN-γ in pleural effusions (PE) from 25 TPE patients and 31 non-TPE patients using a combination of immunological assays and flow cytometric analysis. The diagnostic performance of these three biomarkers was evaluated using receiver operating characteristic (ROC) curves. ResultsWe found that IFN-γ levels in blood and pleural fluid were higher in the TPE group than in the non-TPE group. The mean concentration of IFN-γ in pleural fluid of the TPE group was 3140.90 (1817.94, 6611.05) pg/mL, while that of the non-TPE group was 4.91 (0.69, 8.6) pg/mL), and the difference was statistically significant (z = 6.39, P < 0.001). The mean blood IFN-γ was 40.19 (16.45, 59.08) pg/mL in the TPE group and 2.76 (1.96, 6.02) pg/mL in the non-TPE group, which was statistically different (z = 5.12, P < 0.001). The area under the ROC curve (AUC) for pleural fluid IFN-γ, blood IFN-γ, and ADA were 0.999 (95 % CI: 0.994–1.00), 0.901 (95 % CI: 0.798–1.00) and 0.996 (95 % CI: 0.987–1.00), respectively. ConclusionThis study confirms that IFN-γ has high diagnostic validity in patients with TPE and can potentially be an excellent biomarker.

Immunomodulatory and growth promotors effects of rosemary (Rosmarinus officinalis) and/or Spirulina with respect to some gene expression on Nile tilapia (Oreochromis niloticus) in Aswan Governorate

The purpose of this work trial was to determine the true impact of spirulina and rosemary in a combination treatment on Nile tilapia performance before experimental infection. Moreover, the effects of these treatments on the liver and kidney functions, the histology of certain organs, and the expression of certain antioxidant and immune-related genes in the liver tissue both before and after Aeromonas hydrophila (A. hydrophila) infection are also included. To achieve this purpose, four diets were prepared: the first was free of additives as a control group (Con), the second contained (SP) spirulina at 7.5 g/kg of diet, the third contained (RM) rosemary at a rate of 10 g/kg of diet, and the fourth one contained rosemary and spirulina as a SP + RM group. The results of the different parameters indicated the growth-stimulant effects of rosemary and spirulina either alone or in a combination when compared with the control group (P ≤ 0.05), with a superior effect to combination treatment. In addition, improved hematological parameters, immunological assays, phagocytic activity and index, and expression of antioxidant and immune-related genes occurred before and after infection. Moreover, there were positive effects of spirulina and rosemary without pathological lesions in the different organs before infection and fewer lesions after infection.

Extraction and Analysis of Mycotoxins from Whole Wheat Flour - A Methods Efficiency Comparison

Abstract Wheat is considered as staple food source for 40% of the population worldwide. Yet, the yield and quality can be compromised by fungal diseases, which are also responsible for mycotoxins presence at wheat seeds and originating foodstuff. In this context, the tackling of this problem by developing regulatory limits and standards have induced the development of various methods for sampling, extraction, identification and quantification of mycotoxins in food samples. This review addresses the comparison of the technical and cost efficiency of methods for the extraction and qualitative- quantitative analysis of mycotoxins from whole wheat flour. Methods of extraction such as the Solvent Extraction method, the Liquid Liquid Extraction, the Solid Liquid Extraction, the Solid Phase Extraction, the Immuno-Affinity Columns, the QuEChERS, and the use of absorbent nanomaterials such as graphene oxide and multi-walled carbon nanotubes in extraction procedures, are described in principle, technical details are presented, and examples of reported use are given. Methods of mycotoxin analysis such as Immunological Assays (LFIA, ELISA, FPIA), the Sensor-based (Surface Plasmon Resonance Sensor, Piezoelectric Sensors, Electrochemical Sensors, Colorimetric Sensors), and Chromatographic Techniques (TLC, GC, HPLC, HPLC-FLD, LC-MS/MS, UPLC-MS/MS, UHPLC-MS/MS, UFLC-MS/MS) are reviewed. To compare their efficiency, main advantages and disadvantages, the ongoing improvements, as well as the validation parameters (linearity, recovery range, RSDr range, RSDR %, LOQ range, and cut off) are summarized, and pairing of extraction to analysis methods for specific mycotoxins is provided. It was evidenced that none of methods already in use is capable of analyzing all mycotoxin categories at once, because of their chemical characteristics (volatile/non-volatile, co-elution, UV absorption, fluorescence) versus methods restrictions (matrix interferences, cross-reactivity of antibodies, selectivity and reproducibility of data, need for derivatization, etc). Also, depending on the purpose of the analysis (research or screening as part of legal requirements), to date the immunological methods are only suitable for validated matrices, biosensors can be used for routine screening, and that GC-MS and HPLC-based methods fulfill the legal requirements. In conclusion, while the selectivity and accuracy of methods for mycotoxin detection is being improved rapidly (those sensor-based thanks to the use of nanoparticles, nanomaterials, aptasensors, etc., and the chromatographic techniques coupled with mass spectrometry offer a higher selectivity and sensitivity, low detection limits, maintained resolution performance), and the duration of the analysis, the cost, and the need for highly-skilled staff go in favor of rapid methods (immunological and sensors-based), it is the capacity to fulfill legal requirements, which will determine the trend and their future success in the market.

A study on loading multiple epitopes with a single peptide.

Epitopes, the basic functional units of antigens, hold great significance in the field of immunology. However, the structure and composition of epitopes and their interactions with antibodies remain unclear, which limits in-depth studies on epitopes and the development of subunit vaccines. In a previous study on the localization of anti-influenza HA monoclonal antibodies (mAbs), three strains with different characteristics reacted with the same peptide. In this study, by conventional immunological assays, computer homology modeling, and molecular docking simulations, we found that (1) the peptide could bind to three strains of mAbs with different reaction characteristics utilizing different combinations of immunodominant groups. (2) By computer molecular docking and simulation methods, the immunodominant groups on the two peptides could be combined into a multi-epitope peptide bound to six strains of mAbs. We established a method for multi-epitope peptide recombination from these immunodominant groups. (3) The immune effect of the recombinant multi-epitope peptide was better than that of a single peptide. Our findings facilitate the understanding of the composition of antigen epitopes and provide a theoretical and experimental basis for developing polyvalent vaccines and understanding immune responses at the molecular level.

Immunofluorescent detection of protein CoAlation in mammalian cells under oxidative stress.

Previously, we reported the generation and characterisation of highly specific anti-CoA monoclonal antibodies capable of recognizing CoA in various immunological assays. Utilizing these antibodies in conjunction with mass spectrometry, we identified a wide array of cellular proteins modified by CoA in bacteria and mammalian cells. Furthermore, our findings demonstrated that such modifications could be induced by oxidative or metabolic stress. This study advances the utility of anti-CoA monoclonal antibodies in analysing protein CoAlation, highlighting their effectiveness in immunofluorescent assay. Our data corroborates a significant increase in cellular protein CoAlation induced by oxidative agents. Additionally, we observed that hydrogen-peroxide induced protein CoAlation is predominantly associated with mitochondrial proteins.

Antioxidant Activity and Hypoallergenicity of Egg Protein Matrices Containing Polyphenols from Citrus Waste.

This study reports on the interactions of egg proteins, which represent a major health concern in food allergy, with polyphenols obtained from orange and lemon peels. The antioxidant properties of such citrus peel extracts prior to protein binding were evaluated. The resulting edible, and therefore inherently safe, matrices exhibit reduced IgE binding compared to pure proteins in indirect immunological assays (ELISA) using individual sera from patients allergic to ovalbumin and lysozyme. The reduced allergenicity could arise from the interactions with polyphenols, which alter the structure and functionality of the native proteins. It is hypothesized that the anti-inflammatory and antioxidant properties of the polyphenols, described as inhibitors of the allergic response, could add immunomodulatory features to the hypoallergenic complexes. A docking analysis using lysozyme was conducted to scrutinize the nature of the protein-polyphenol interactions. An in silico study unravelled the complexity of binding modes depending on the isoforms considered. Altogether, the presented results validate the antioxidant properties and reduced allergenicity of polyphenol-fortified proteins. Lastly, this study highlights the upgrading of vegetable wastes as a source of natural antioxidants, thus showing the benefits of a circular economy in agri-food science.

Identification of three novel linear B-cell epitopes on VP7 of African horse sickness virus using monoclonal antibodies

African horse sickness (AHS) is an acute and subacute infectious disease of equine species caused by the African horse sickness virus (AHSV). The VP7 of AHSV is a group-specific protein conserved in all serotypes and is an excellent candidate for the serological diagnosis and an AHS vaccine component. However, to date, B-cell epitopes on the AHSV VP7 recognized by humoral immune responses remain unclear. This study expressed the recombinant AHSV VP7 soluble in Escherichia coli and purified it for mouse immunization. Four monoclonal antibodies (mAbs) were screened and identified by hybridoma cell fusion, clonal purification, and immunological assays. The B-cell epitopes, recognized by monoclonal antibodies 4B5, 3G10, 3D7, and 4D6, were identified by a series of truncated overlapping peptides expressed as glutathione S-transferase (GST)-fusion proteins. The results revealed that 4B5 recognized the 124VQTGRYAGA132 motif, 3G10 recognized the 140RYYVPQGRT148 motif, while 3D7 and 4D6 recognized the 292QPINPPIFP300 motif. Amino acid sequence alignment indicated that three novel B-cell epitopes were conserved among various AHSV serotypes but unconserved in other orbiviruses, such as the bluetongue and epidemic hemorrhagic disease viruses. This study informs on the antigenic epitopes of AHSV VP7, facilitating future investigations into the serological diagnosis method and epitope-based vaccines against AHSV.

Precise location of three novel linear epitopes using the generated monoclonal antibodies against the Knob domain of FAdV-4 surface structural protein, fiber1.

Fowl adenovirus serotype 4 (FAdV-4) is the main pathogen of hepatitis-hydropericardium syndrome (HHS), which brings huge economic losses to the poultry industry worldwide. Fiber-1 protein plays an important role in viral infection and pathogenesis by binding directly to cellular receptors of FAdV-4. In particular, the knob domain of fiber-1 protein has been reported to induce the production of neutralizing antibodies and arouse protection against the lethal challenge of chickens with FAdV-4. The fiber-1 knob (F1K) protein was expressed in a prokaryotic expression system and purified using Ni-NTA affinity chromatography. Monoclonal antibodies (mAbs) against FAdV-4 were generated by immunizing BALB/c mice with the purified F1K protein and screened using a series of immunoassays. Potential B cell epitopes on the knob domain of fiber-1 protein were mapped using enzyme-linked immunosorbent assay (ELISA) and dot-blot. Precious location and crucial amino acids of the identified epitopes were determined using peptide array scanning, truncations and alanine-scanning mutagenesis. The epitopes were analyzed and visualized on the knob trimer of FAdV-4 fiber-1 protein using the PyMOL software. Water-soluble recombinant fiber-1 knob (F1K) protein was obtained with the assistance of chaperone. Four monoclonal antibodies (5C10, 6F8, 8D8, and 8E8) against FAdV-4 were generated and characterized using indirect ELISA, Western blot, dot-blot, and immunological fluorescence assay (IFA). The mAbs were demonstrated to be from different hybridoma cell lines based on the sequences of the variable regions. Meanwhile, three distinct novel linear B-cell epitopes (319SDVGYLGLPPH329, 328PHTRDNWYV336, and 407VTTGPIPFSYQ417) on the knob domain of fiber-1 protein were identified and the key amino acid residues in the epitopes were determined. Structural analysis showed that the two adjacent epitopes 319SDVGYLGLPPH329 and 328PHTRDNWYV336 were exposed on the surface of the fiber-1 knob trimer, whereas the epitope 407VTTGPIPFSYQ417 was located inside of the spatial structure. This was the first identification of B-cell epitopes on the knob domain of fiber-1 protein and these findings provided a sound basis for the development of subunit vaccines, therapeutics, and diagnostic methods to control FAdV infections.

Design of TOLERANT: phase I/II safety assessment of intranodal administration of HSP70/mB29a self-peptide antigen-loaded autologous tolerogenic dendritic cells in patients with rheumatoid arthritis

IntroductionIn rheumatoid arthritis (RA), immunosuppressive therapies may achieve symptomatic relief, but do not induce long-term, drug-free remission. Meanwhile, the lifelong use of immunosuppressive drugs confers increased risk for malignancy and...

In Vitro Anti-inflammatory Effects of Larch Turpentine, Turpentine Oil, Eucalyptus Oil, and Their Mixture as Contained in a Marketed Ointment.

An ointment containing larch turpentine, turpentine oil, and eucalyptus oil has been used for almost a century for the symptomatic treatment of mild, localized, purulent inflammations of the skin. Its clinical efficacy in the treatment of skin infections has been shown in clinical trials, but the mode of action of the active ingredients on inflammation is not known. We studied the anti-inflammatory properties of the active ingredients of the ointment and their mixture in a human monocyte cell model, in which the cells were stimulated with lipopolysaccharide and incubated with the test substances. The cytotoxic threshold of each test substance and the mixture was identified using the alamarBlue assay, and their anti-inflammatory activity was assessed by measuring the release of interleukins IL-1β, IL-6, IL-8, monocyte chemoattractant protein-1, prostaglandin E2, and TNF-α. Cell toxicity was observed at a mixture concentration of 10 µg/mL. All immunological assays were carried out at nontoxic concentrations. Larch turpentine decreased IL-1β, monocyte chemoattractant protein-1, and prostaglandin E2 release at a concentration of 3.9 µg/mL and TNF-α at concentrations > 1.95 µg/mL, whereas eucalyptus oil and turpentine oil had no relevant inhibitory effects. The mixture dose-dependently inhibited IL-1β, IL-6, monocyte chemoattractant protein-1, prostaglandin E2, and TNF-α release at concentrations > 1 µg/mL. IL-8 release was only marginally affected. The anti-inflammatory activity of the herbal ingredients and their mixture was confirmed in this model. This effect seems to be mediated mainly by larch turpentine, with turpentine oil and eucalyptus oil exerting an additive or possibly synergistic function.

Improving Reliability of Immunological Assays by Defining Minimal Criteria for Cell Fitness.

Human PBMC-based assays are often used as biomarkers for the diagnosis and prognosis of disease, as well as for the prediction and tracking of response to biological therapeutics. However, the development and use of PBMC-based biomarker assays is often limited by poor reproducibility. Complex immunological assays can be further complicated by variation in cell handling before analysis, especially when using cryopreserved cells. Variation in postthaw viability is further increased if PBMC isolation and cryopreservation are done more than a few hours after collection. There is currently a lack of evidence-based standards for the minimal PBMC viability or "fitness" required to ensure the integrity and reproducibility of immune cell-based assays. In this study, we use an "induced fail" approach to examine the effect of thawed human PBMC fitness on four flow cytometry-based assays. We found that cell permeability-based viability stains at the time of thawing did not accurately quantify cell fitness, whereas a combined measurement of metabolic activity and early apoptosis markers did. Investigation of the impact of different types and levels of damage on PBMC-based assays revealed that only when cells were >60-70% live and apoptosis negative did biomarker values cease to be determined by cell fitness rather than the inherent biology of the cells. These data show that, to reproducibly measure immunological biomarkers using cryopreserved PBMCs, minimal acceptable standards for cell fitness should be incorporated into the assay protocol.

Bilateral vocal cords palsy in adolescent Systemic Lupus Erythematosus patient

Background: Executive functions such as speech are uncommonly involved in systemic lupus erythematosus (SLE) presentation. We report a case of SLE who presented with a seizure followed by an inability to speak. Case description: A 17-year-old girl was presented with a sudden loss of voice for 2 days after a seizure following fever for 4 months. The patient was aware and could understand words but could not execute verbal sounds; the sound she produced was muffled. The generalized seizure occurred without any trigger, lasted 2 minutes, and stopped spontaneously. Fever occurred primarily at night which subsided without treatment. She also complained of severe hair loss. Patient with leucopenia, lymphopenia, anemia hemolytic, thrombocytopenia, proteinuria, hematuria, brain atrophy from magnetic resonance imaging (MRI) examination, and echocardiography revealed mild carditis and mild circumferential pericardial effusion. Immunological assay showed antinuclear antibodies (ANA) &gt;1:1000 with nuclear homogenous, low C3 complement, and positive Anti-dsDNA. Systemic Lupus International Collaborating Clinics (SLICC) 2015 total score was 14, and the European League Against Rheumatism (EULAR/ACR) 2019 total score was 31, confirmed patient for SLE diagnosis. Further exploration from fiberoptic laryngoscopy confirmed bilateral vocal cord palsy due to SLE. Management included high dose methylprednisolone, cyclophosphamide, and speech therapy which results in gradual improvement from producing mumble sounds, then words followed by sentences with nasal sounds. Laryngeal involvement is uncommonly reported as an SLE manifestation. Management includes steroids and immunosuppressive for pharmacological and speech therapy as an exercise for vocal cord palsy. Conclusion: The patient must be continuously monitored for complete resolution of the palsy.