Blood Samples Of Healthy Donors Research Articles

Comparison of results obtained using clot-fibrinolysis waveform analysis and global fibrinolysis capacity assay with rotational thromboelastography

Global fibrinolysis assays detect the fibrinolysis time of clot dissolution using tissue-type plasminogen activator (tPA). Two such assays, clot-fibrinolysis waveform analysis (CFWA) and global fibrinolysis capacity (GFC) assay, were recently developed. These were compared with rotational thromboelastography (ROTEM). Healthy donor blood samples were divided into four groups based on tPA-spiked concentrations: 0, 100, 500, and 1000 ng/mL. CFWA and GFC fibrinolysis times, including 4.1 µg/mL and 100 ng/mL tPA in the assays, were determined, denoted as CFWA-Lys and GFC-Lys, respectively. Statistical differences were recognized between tPA concentrations of 0 and 500/1000 ng/mL for CFWA-Lys, and 0 and 100/500/1000 ng/mL for GFC-Lys. The correlation coefficients with lysis onset time (LOT) of extrinsic pathway evaluation and intrinsic pathway evaluation in ROTEM were statistically significant at 0.610 and 0.590 for CFWA-Lys, and 0.939 and 0.928 for GFC-Lys, respectively (p-values < 0.0001 for all correlations). Both assays showed significant correlations with ROTEM; however, the GFC assay proved to have better agreement with ROTEM compared with the CFWA assay. These assays have the potential to reflect a hyperfibrinolysis status with high tPA concentrations.

Ultrasensitive detection of circulating tumor cells in clinical blood samples by a three-dimensional network nanovehicle-based aptasensor platform

A burgeoning topic in the early diagnosis and treatment of cancer is the sensitive and precise quantification of circulating tumor cells (CTCs). However, due to the extremely low CTC concentrations in the blood, traditional enrichment and identification methods for CTCs are prone to false negative results. Here, to specifically identify and improve the sensitivity of CTC detection in the blood, we designed a novel fluorometric turn-on aptasensor platform based on a three-dimensional network nanovehicle (TDNN) modified with a PEG derivative and AS1411 aptamer specifically targeting the nucleolin protein. The limit of detection (LOD) of the aptasensor platform was as low as 2 cells per mL, which was remarkably better than that of the single nanoparticle ASS1. Notably, the novel aptasensor platform showed excellent specificity and sensitivity as well as negligible fluorescent background for quantitative and visualization detection of CTCs in peripheral blood samples without enrichment, centrifugation and washing steps. More importantly, the number of CTCs identified by TDNN was significantly higher in metastatic patients than in nonmetastatic patients, while CTCs were not detected in the blood samples of healthy donors. In conclusion, the aptasensor platform provides an available and promising tool for CTC identification and cancer metastasis prediction in clinical blood samples.

A Pan-Cancer Single-Cell Transcriptomic Atlas of Natural Killer (NK) Cells Reveals Intrinsic and Extrinsic Mediators of NK Cell Anti-Tumor Functions

Background: Natural killer (NK) cells are critical mediators of anti-tumor immunity, engaging in direct cytotoxic responses and promoting pro-inflammatory immune microenvironments. The molecular mechanisms underlying these functions, however, have yet to be fully elucidated. This is due in part to a lack of understanding of the phenotypic and functional diversity of NK cells across cancer types. Here, we present a single-cell transcriptomic atlas of &amp;gt;250,000 NK cells spanning 340 patients across 17 cancer types to advance our understanding of tumor-infiltrating NK cells. Methods: We re-analyzed publicly available single-cell RNA-sequencing (scRNA-seq) datasets of primary, therapy-naïve tumors, as well as matching peripheral blood samples and healthy donor blood samples. We employed systematic computational analyses to deconvolute the phenotypic and functional diversity of tumor-infiltrating NK cells and define key regulators of NK cell functions in both a cancer type-specific and pan-cancer manner. Feature expression module scoring was used to infer phenotypic and functional characteristics of NK cell clusters in each cancer type. Transcription factor regulon and cell-cell communication prediction algorithms were applied to understand the mechanistic drivers of NK cell populations within tumors. Results: Tumor-infiltrating NK cells display distinct patterns of phenotypic and functional features across cancer types. Nonetheless, we identify consensus NK cell populations across cancers that demonstrate the utility and performance of our gene module-based phenotypic and functional annotations. Interestingly, we observe the enrichment of adaptive-like and CD56 bright NK cell populations in tumors that exhibit features of tissue residency, including the upregulation of ITGA1, ITGAE, and CD69 alongside the downregulation of FCG3RA. Notably, these tissue-resident populations display enhanced chemokine and cytokine activity compared to their recruited counterparts, suggesting the significance of tissue-resident NK cell populations in immune recruitment to tumors. We find the phenotypic composition of intra-tumoral NK cells is vastly different than that of circulating NK cells which harbor predominantly CD56 dim populations, as well as a significant proportion of adaptive-like NK cells in several cancer types. Leveraging the paired nature of several tumor and peripheral blood samples, we performed temporal comparative analyses that revealed the robust expression of KLF2 and S1PR5 in circulation which is then lost in paired tumor tissues. While KLF2 and S1PR5 have been previously implicated in the homing and migration of immune cells, their roles in the trafficking of NK cells to tumors have yet to be defined. In modeling transitions from canonical to stressed NK cell states, we not only identify well-established NK cell checkpoints ( TIM-3, LAG3, and CISH) but also identify novel putative checkpoints yet to be investigated in NK cells, such as the AP-1 family of transcription factors. Additionally, we examine extrinsic factors contributing to intra-tumoral NK cell phenotypes, detecting significant crosstalk between NK cells and immunomodulatory macrophages and CD4+ T cells mediated by galectin-9 and TGFβ interactions. Conclusions: We define the phenotypic and functional pan-cancer landscape of NK cells and uncover both intrinsic and extrinsic factors influencing their anti-tumor functions. We envision our NK cell atlas will facilitate the development of strategies to enhance NK cell-based immunotherapies against cancer.

Effect of streptococcal arginine deiminase on the function of CD4&lt;sup&gt;+&lt;/sup&gt; and CD8&lt;sup&gt;+&lt;/sup&gt;T lymphocytes

Arginine metabolism plays an important role in regulating the functions of immune cells in mammals. Pathogenic microbes use the mechanism of arginine depletion to suppress the immune response during infection. Arginine deiminase is a microbial arginine-hydrolyzing enzyme important for survival at low pH in the focus of infection, or in phagolysosomes, as well as under low-glucose conditions. The effect of bacterial arginine deiminase on the functions of adaptive immune cells remains poorly understood. The aim of our study was to evaluate the effect of streptococcal arginine deiminase on the proliferation and autophagy of CD4+ and CD8+ human peripheral blood T lymphocytes.The enzyme effects were studied with supernates of ultrasonic lysates from parental Streptococcus pyogenes M49-16, and its isogenic mutant with inactivated arcA gene (Streptococcus pyogenes M49-16delarcA). The study was performed with blood samples of healthy donors. The fraction of mononuclear leukocytes was isolated by centrifugation in a Ficoll density gradient. To evaluate proliferation levels, a method based on the staining of intracellular proteins with vital fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) was used. The level of autophagy was studied using the fluorescent Lysotracker Green DND-26 dye. To analyze the proliferation and autophagy of T helper cells (CD3+CD4+) and cytotoxic T lymphocytes (CD3+CD4-), the obtained cell suspensions were stained with antibodies against CD4, CD45RA, and CD3. The proportion of necrotic cells was determined by staining with a fluorescent DNA-binding DAPI dye. The normality of the distribution was assessed by Shapiro–Wilk test. The data were analyzed using Kruskal–Wallis criterion, followed by Mann–Whitney criterion for pairwise comparisons and expressed as median and interquartile ranges (Q0.25-Q0.75).When comparing the effects of supernatants from the parental and mutant streptococcal strains, which differed in expression of arginine deiminase gene, we have shown that the bacterial enzyme had no effect on the functions of inactive lymphocytes. However, streptococcal arginine deiminase completely suppressed proliferation of CD4+ and CD8+T lymphocytes stimulated with anti-CD2/CD3/CD28 antibodies. These effects were accompanied by a decrease in the autophagy levels. At the same time, arginine deiminase did not exert cytotoxic effects upon lymphocytes. L-arginine applied at the doses exceeding physiological levels caused restoration of the cellular functions. There were no differences between the studied parameters of CD4+ and CD8+T lymphocyte subsets.The obtained data show that the antiproliferative effect of arginine demimnase may be associated with ability of the enzyme to inhibit autophagy and confirm an opportunity of the bacterial enzyme to suppress host adaptive immune responses.

A-384 Laboratory Evaluation of the SeptiCyte Rapid Host Response Assay In Differentiating Infection-Positive Versus Infection-Negative Systemic Inflammation Patients Suspected of Sepsis

Abstract Background Early diagnosis of sepsis is critical for timely treatment but remains challenging. A rapid host response assay, SeptiCyte, was recently approved by the FDA for aiding early differentiation of infection-positive from infection-negative systemic inflammation in patients suspected of sepsis. This test is a fully automated reverse transcription polymerase chain reaction (RT-qPCR) assay that quantifies changes in the expression of two host immune response genes and results are available in about 1 hour. The present study aimed to evaluate the SeptiCyte assay performance in a clinical setting at a large medical center. Methods Eighty-two patients admitted to the University Hospitals Cleveland Medical Center with suspected sepsis from July to September 2022 were included in the study. SeptiCyte samples were obtained at the time of initial evaluation. In addition, healthy donor blood samples and extracted RNA samples were also utilized for assay validation. The SeptiCyte assay was performed according to the manufacturer's instructions using multi-chambered fluidic cartridges and the Idylla Console (Immunexpress, Seattle, WA). The assay result was reported as the SeptiScore (range 0–15), a calculated ratio of the expression of two host immune response genes, and the SeptiCyte band (ranging from 1 to 4, with 1 indicating the lowest risk of sepsis and 4 indicating the highest risk). The assay cutoff values are 5.0, 6.2 and 7.4 respectively for the band 2, 3 and 4. Results Intra- and inter-assay precision was 0.2–6.2% and 0–3.2%, respectively. % Recovery at the cutoff for band 3 (SeptiScore 6.0) was 102.4% with a CV of 2.4%. Method comparison with the Immunexpress lab demonstrated equivalent performance, as evidenced by a slope of 0.99 and an intercept of 0.27 by Deming regression. SeptiScore showed a weak positive correlation with CRP (Correlation Coefficient 0.389, P &amp;lt; 0.01) but no association with procalcitonin (n = 76), lactate (n = 78), international normalized ratio (INR), platelet, neutrophil, lymphocyte, or white blood cell (WBC) count (n = 82). CRP (n = 71) median concentration in patients with band 4 (16.8) was significantly higher than in patients who were band 1 (3.6). The SeptiScore of blood culture positive cases (n = 22) was 7.2 ± 2.0 (mean ± SD) and that of pathogen negative non-sepsis (by sepsis retrospective review of event only, n = 10) cases was 5.6 ± 1.6 (P &amp;lt; 0.05). In addition, the median SeptiScore was notably higher in patients with viral infection compared to those without infection (9.2 vs 6.2, P &amp;lt; 0.05). Further analysis showed 92% (11/12) of patients with hematologic cancer were categorized as band 4 including one non-sepsis case per the retrospective review. SeptiScore was similar between patients with non-hematologic cancer and those without malignancy (P &amp;gt; 0.05). Conclusion The SeptiCyte RAPID assay showed good analytical performance. SeptiScore represents an independent factor to quantify host response in patients suspected of sepsis. It may add value in aiding differentiation between infection-positive from infection-negative systemic inflammation with promising results in patients with viral infections or those with positive blood cultures. The negative predictive value of low SeptiScore within band 1 or 2 may need further investigation.

Cell-free plasma miRNAs analysis for low invasive lung cancer diagnostics

Introduction. The high mortality rate in patients with lung cancer (LC) is due to the lack of highly sensitive diagnostic markers of this disease. Genetic and epigenetic alterations in tumor cells, for example, aberrant microRNA expression, can be proposed. It is known that extracellular/circulating microRNA of biological fluids, in complexes with proteins, or packaged in extracellular vesicles is of interest for the diagnosis of tumor diseases.Aim. To perform a comparative analysis of miRNA expression in plasma and plasma extracellular vesicles of LC patients and healthy donors. Based on the obtained results, to propose a diagnostic panel to identify patients with LC.Materials and methods. Blood plasma was obtained from blood samples of healthy donors and LC patients by sequential centrifugation. Then, a fraction of extracellular vesicles (40–150 nm in size) was isolated from a part of the obtained plasma supernatant by the method of aggregation-precipitation with polyethylene glycol/blue dextran. MicroRNAs were isolated from both blood plasma fractions of patients and healthy donors using guanidine isothiocyanate and octanoic acid. Expression of 17 miRNAs most characteristic for the development of LC according to our and literature data in the above-mentioned blood plasma fractions was analyzed by stem-loop reverse transcription polymerase chain reaction.Results. 29 and 10 miRNA pairs were differentially expressed in plasma extracellular vesicles and plasma of lung cancer patients and donors. Thus, plasma extracellular vesicles are characterized by greater potential as a source for miRNA based lung cancer diagnostic panels in comparison with blood plasma. Diagnostic algorithm based on aberrant miRNA expression of 8 different miRNAs (miRNA-30e, -1, -125b, -133, -222, -374, -425, -660) composed in 6 pairs was designed. This algorithm allows to diagnose 100 % of patients with lung cancer stages II–IV.Conclusion. Extracellular plasma vesicles represent a promising source of diagnostically significant microRNAs compared to plasma microRNAs. For the diagnosis of patients with non-small cell lung cancer with 100 % sensitivity and specificity, a panel of 8 microRNAs (6 miRNA pairs) was proposed.

#3764 SINGLE-CELL RNA-SEQ REVEALS IMMUNE AND KIDNEY CELL TYPE–SPECIFIC MOLECULAR ASSOCIATIONS TO IGA NEPHROPATHY

Abstract Background and Aims IgA nephropathy is the most common primary glomerulonephritis worldwide. There is growing evidence of host immunity is closely related to the pathogenesis of IgAN, but the detailed mechanism in the early stages of IgAN remain largely unknown. In this study, we applied single-cell RNA-seq technology to investigate the transcriptomic changes in PBMCs and kidney tissues of early stage IgAN patients at the single-cell resolution. This study aims to establish a immune and kidney cell landscape of IgAN, and offers new insights for the diagnosis and treatment of IgAN. Method In strict accordance to the admission criteria, collect peripheral venous blood and kidney samples of healthy donors and newly diagnosed IgAN patients, prepare single cell solution, verify cell viability by flow cytometry, and perform single-cell RNA-seq using a BD Rhapsody platform. Meanwhile, the clinical data of healthy donors and IgAN patients were collected and analysed. Finally, the correlation and association of differential expressed genes (DEGs) and cell clusters-identified by single-cell RNA-seq with the clinical data of IgAN were investigated. Results First, we established immune cells and kidney cells landscape of IgAN. The differentially expressed genes (DEGs) between the control group and IgAN were mainly concentrated in the NK cell-mediated cytotoxicity and cell killing pathways. Interestingly, we found significant decreases in NK cell numbers and cytotoxicity, and NK cell numbers and marker genes were negatively correlated with clinical parameters, including urinary protein creatinine ratio (UPCR) and serum galactose-deficient IgA1 and IgA. In contrast, B cell DEGs were enriched in different viral infection pathways, and one specific B cell subgroup showed inhibition of NFκB signaling, which was positively correlated with clinical parameters. In addition, a subpopulation of monocytes expressing interferon-inducing genes was positively associated with clinical severity of IgAN. In case of the kidney tissue, compared with the control group, mesangial cells of IgAN patients up-regulated extracellular matrix, transcription factors, kidney development and genes related to the regulation of Wnt signaling pathway. The expression of structural protein ITGA8 on mesangial cells was significantly decreased, indicating the mesangial cells were damaged in early IgAN. Pseudo-time analysis indicated that extracellular matrix-related genes were involved in mesangial cell lesion. We also found that the patie'ts' glomerular endothelial cells expressed high level of angiogenesis and VEGF- related genes. Moreover, podocytes of IgAN patients expressed genes of cytoskeletal recombination and FGFR activation. Conclusion We successfully established the single-cell landscapes of peripheral blood immune cells and kidney cells in early IgAN patients, which revealed changes in the number and status of immune cells and kidney cells closely related to clinical manifestations and phenotypic transformation. Our study offers new insights for the diagnosis and treatment of IgAN.

NF-κB fingerprinting reveals heterogeneous NF-κB composition in diffuse large B-cell lymphoma.

Improving treatments for Diffuse Large B-Cell Lymphoma (DLBCL) is challenged by the vast heterogeneity of the disease. Nuclear factor-κB (NF-κB) is frequently aberrantly activated in DLBCL. Transcriptionally active NF-κB is a dimer containing either RelA, RelB or cRel, but the variability in the composition of NF-κB between and within DLBCL cell populations is not known. Here we describe a new flow cytometry-based analysis technique termed "NF-κB fingerprinting" and demonstrate its applicability to DLBCL cell lines, DLBCL core-needle biopsy samples, and healthy donor blood samples. We find each of these cell populations has a unique NF-κB fingerprint and that widely used cell-of-origin classifications are inadequate to capture NF-κB heterogeneity in DLBCL. Computational modeling predicts that RelA is a key determinant of response to microenvironmental stimuli, and we experimentally identify substantial variability in RelA between and within ABC-DLBCL cell lines. We find that when we incorporate NF-κB fingerprints and mutational information into computational models we can predict how heterogeneous DLBCL cell populations respond to microenvironmental stimuli, and we validate these predictions experimentally. Our results show that the composition of NF-κB is highly heterogeneous in DLBCL and predictive of how DLBCL cells will respond to microenvironmental stimuli. We find that commonly occurring mutations in the NF-κB signaling pathway reduce DLBCL's response to microenvironmental stimuli. NF-κB fingerprinting is a widely applicable analysis technique to quantify NF-κB heterogeneity in B cell malignancies that reveals functionally significant differences in NF-κB composition within and between cell populations.

Validation of a single-cell sequencing assay for the detection of circulating tumor cells in metastatic breast cancer.

1053 Background: Blood Biopsy and the evaluation of Circulating Tumor Cells (CTC) has provided valuable clinical information for patients with metastatic breast cancer, specifically where a tissue biopsy is not feasible. A critical problem in the field is that most assays cannot distinguish true CTCs from other types of rare cells. By combining established protein biomarkers associated with circulating epithelial cells with single-cell whole-genome sequencing to assess copy number variations (CNVs, a signature for genomic instability - GI), we differentiate rare cells from true CTCs. Additionally, we can determine the presence or absence of regions that contain gene amplifications (e.g., ERBB2/HER2). Here we report the validation of a clinical blood biopsy CTC assay that can enumerate CTCs, determine if those CTCs have GI and if selected CTCs are expressing HER2 or ER antigens. Methods: Relevant cell line controls of epithelial origin and known expression levels of ER or HER2 were spiked into healthy donor whole blood at varying concentrations. The nucleated cells were deposited on microscope glass slides at high density. Immunofluorescence staining of slides was performed to identify the presence and localization of nuclei (via DAPI), pan-CK, HER2, ER, CD45 and CD31. Nucleated cells were classified as CTC candidates if they were pan-cytokeratin(+) and CD45/CD31(-), and were further characterized for expression of ER and HER2 protein. Defined CTC candidates were imaged and located by specific coordinates allowing single-CTC isolation for whole genome amplification. Single-cell sequencing data was then analyzed with the Epic CTC DNA copy number analysis pipeline to detect GI and amplification at the ERBB2 locus. Results: The limit of detection for CTCs was determined to be 1 CTC in 6 million white blood cells with a linear range of 1- 300 CTC per slide. The sensitivity and specificity for HER2 detection was determined to be 94% and 97%, respectively. ER assay sensitivity and specificity were 91% and 100%, respectively. Overall accuracy was 100% for HER2 and 94% for ER detection. Overall precision across all relevant variables produced %CVs &lt;10% for HER2 and ER. A genome possessed GI if more than 12 large-scale transitions (LSTs) were detected. For detection of 2-fold amplification at the ERBB2 locus, gene amplification was detected with 85% sensitivity and 94% specificity. Tested healthy donor blood samples were absent of detected CTCs and lacked LST/GI or ERBB2 amplification in all (46) isolated WBCs. Conclusions: These results reflect the validation of a novel clinical assay combining CTC detection with single-cell GI and amplification of the ERBB2 locus. This assay better discriminates CTC detection with the addition of an assessment of GI. Additionally, along with protein expression measures of ER and HER2, it provides the ability to assess if HER2 expression is driven by ERBB2 amplification.

Activation effects on the physical characteristics of T lymphocytes.

The deformability of leukocytes is relevant to a wide array of physiological and pathophysiological behaviors. The goal of this study is to provide a detailed, quantitative characterization of the mechanical properties of T cells and how those properties change with activation. We tested T cells and CD8+ cells isolated from peripheral blood samples of healthy donors either immediately (naïve population) or after 7days of activation in vitro. Single-cell micropipette aspiration was used to test the mechanical properties. T cells exhibit the general characteristics of a highly viscous liquid drop with a cortical "surface" tension, T cort. The time course of each cell entry into the micropipette was measured at two different aspiration pressures to test for shear thinning behavior. The data were analyzed in the framework of an approximate mechanical model of the cell deformation to determine the cortical tension, the cell volume, the magnitude of the initial cell entry, the characteristic viscosity μ o, and the shear thinning coefficient, b. Activation generally caused increases in cellular resistance to deformation and a broadening of the distribution of cell properties. The cell volume increased substantially upon cell activation from ∼200μm3 to ∼650μm3. Naive and activated T cells had similar mean cortical tension (∼150pN/μm). However, compared to naïve CD8+ cells, the cortical tension of activated CD8+ cells increased significantly to ∼250pN/μm. Dynamic resistance of naive CD8+ T cells, as reflected in their characteristic viscosity, was ∼870Pa and significantly increased to 1,180Pa after in vitro activation. The magnitude of the instantaneous projection length as the cell enters the pipette (L init) was more than doubled for activated vs. naive cells. All cell types exhibited shear thinning behavior with coefficients b in the range 0.5-0.65. Increased cell size, cortical tension, and characteristic viscosity all point to increased resistance of activated T cells to passage through the microvasculature, likely contributing to cell trapping. The increased initial elastic response of cells after activation was unexpected and could point to instability in the cell that might contribute to spontaneous cell motility.

Detection of Circulating Tumor Cells Using the Attune NxT.

Circulating tumor cells (CTCs) have been detected in many patients with different solid malignancies. It has been reported that presence of CTCs correlates with worse survival in patients with multiple types of cancer. Several techniques have been developed to detect CTCs in liquid biopsies. Currently, the only method for CTC detection that is approved by the Food and Drug Administration is CellSearch. Due to low abundance of CTCs in certain cancer types and in early stages of disease, its clinical application is currently limited to metastatic colorectal cancer, breast cancer and prostate cancer. Therefore, we aimed to develop a new method for the detection of CTCs using the Attune NxT-a flow cytometry-based application that was specifically developed to detect rare events in biological samples without the need for enrichment. When healthy donor blood samples were spiked with variable amounts of different EpCAM+EGFR+ tumor cell lines, recovery yield was on average 75%. The detection range was between 1000 and 10 cells per sample. Cell morphology was confirmed with the Attune CytPix. Analysis of blood samples from metastatic colorectal cancer patients, as well as lung cancer patients, demonstrated that increased EpCAM+EGFR+ events were detected in more than half of the patient samples. However, most of these cells showed no (tumor) cell-like morphology. Notably, CellSearch analysis of blood samples from a subset of colorectal cancer patients did not detect CTCs either, suggesting that these blood samples were negative for CTCs. Therefore, we anticipate that the Attune NxT is not superior to CellSearch in detection of low amounts of CTCs, although handling and analysis of samples is easier. Moreover, morphological confirmation is essential to distinguish between CTCs and false positive events.

Low level of dengue infection and transmission risk in Hong Kong: an integrated analysis of temporal seroprevalence results and corresponding meteorological data

ABSTRACT Hong Kong is an Asia-Pacific City with low incidence but periodic local outbreaks of dengue. A mixed-method assessment of the risk of expansion of dengue endemicity in such setting was conducted. Archived blood samples of healthy adult blood donors were tested for anti-dengue virus IgG at 2 time-points of 2014 and 2018/2019. Data on the monthly notified dengue cases, meteorological and vector (ovitrap index) variables were collected. The dengue virus (DENV) IgG seroprevalence of healthy adults in 2014 was 2.2% (95%C.I. = 1.8–2.8%, n = 3827) whereas that in 2018/2019 was 1.7% (95%C.I. = 1.2–2.3%, n = 2320). Serotyping on 42 sera in 2018/2019 showed that 22 (52.4%) were DENV-2. In 2002–2019, importation accounted for 95.3% of all reported cases. By wavelet analysis, local cases were in weak or no association with meteorological and vector variables. Without strong association between local cases and meteorological/vector variables, there was no evidence of increasing level of dengue infection in Hong Kong.

End-point detection of loop-mediated isothermal amplification (LAMP) on malaria by direct observation with colorimetric dyes

In order to ascertain the results of the LAMP technique, different end-point detection methods can be employed. However, these methods require sophisticated equipment. To simplify current end-point detection methods for the diagnosis of malaria, we propose the incorporation of colorimetric dyes: malachite green (MG), phenol red (PR), and xylenol orange (XO) in the LAMP assay. To evaluate the optimum concentration of dyes, 5 different concentrations (50 μM, 75 μM, 100 μM, 125 μM, and 150 μM) were used with buffer pH 8.5 and pH 8.8, respectively. The results showed that 125 μM of MG at pH 8.8 produced the most obvious colour change. A total of 71 clinical blood samples of Plasmodium knowlesi, Plasmodium malariae, Plasmodium vivax, Plasmodium falciparum, and healthy donors were tested using MG-LAMP. It showed 100% sensitivity and specificity. The simplicity and affordability of this method make it ideal to be used as an end-point detection method for malaria diagnosis in resource limited settings.

Combined treatment with anti-PSMA CAR NK-92 cell and anti-PD-L1 monoclonal antibody enhances the antitumour efficacy against castration-resistant prostate cancer.

BackgroundThe chimeric antigen receptor NK‐92 (CAR NK‐92) cell targeting the prostate‐specific membrane antigen (PSMA) has shown antitumour effects in castration‐resistant prostate cancer (CRPC). However, the expression changes of programmed death ligand 1 (PD‐L1) and its mechanisms on CAR NK‐92 and CRPC cells and the effect of the anti‐PD‐L1 monoclonal antibody (mAb) on PD‐L1 expressed on CAR NK‐92 cells remain unknown.MethodsHuman dendritic cells and CD8+ T cells were acquired from blood samples of healthy donors and cocultured with C4‐2 cells. Changes in PD‐L1 expression were detected by flow cytometry. Differential gene expressions were investigated by RNA sequence analysis, while the regulation of PD‐L1 molecular signaling was explored using western blotting. In vitro cytotoxicity was evaluated using the Cell Counting Kit‐8 assay and the bioluminescent intensity (BLI) of green fluorescent protein‐labelled C4‐2 cells. CRPC growth in vivo was monitored using callipers and BLI in male NOD/SCID mice subcutaneously injected with C4‐2 cells and treated intravenously with anti‐PD‐L1/PD‐1 mAb, CAR NK‐92 or cocultured CD8+ T cells.ResultsSignificantly upregulated expression of PD‐L1k was observed in cocultured C4‐2 and CAR NK‐92 cells. In addition, upregulation of PD‐L1 expression was dependent on interferon‐γ in C4‐2 cells, while it was dependent on direct cell‐to‐cell interaction via the NK group 2 member D/ phosphatidylinositol 3‐kinase/AKT pathway in CAR NK‐92 cells. The anti‐PD‐L1 mAb directly acted on PD‐L1 expressed on CAR NK‐92 cells and augmented the cytotoxicity of CAR NK‐92 cells against C4‐2 and CRPC cells from one patient in vitro. Anti‐PD‐L1 mAb significantly enhanced the antitumour effect of CAR NK‐92 cells against CRPC cells in vivo when compared to treatment with CAR NK‐92 cells or combined with anti‐PD‐1 mAb in the absence or presence of cocultured CD8+ T cells.ConclusionCombined treatment with CAR NK‐92 and anti‐PD‐L1 mAb improved the antitumour efficacy against CRPC, which is of extraordinary translational value in the clinical treatment of CRPC.

AK4P1 is a cancer-promoting pseudogene in pancreatic adenocarcinoma cells whose transcripts can be transmitted by exosomes.

Adenylate kinase 4 pseudogene 1 (AK4P1) is a processed pseudogene whose function in cancer biology remains largely underexplored. Bioinformatics analysis suggested an association between the expression levels of adenylate kinase 4 (AK4) gene and AK4P1, as well as a clinical significance in relation to the increased transcription levels of AK4P1 in pancreatic adenocarcinoma (PAAD). In the present study, the expression levels of AK4P1 and AK4 were compared by RT-qPCR and western blotting between PAAD tissue and paired adjacent tissue. The level of AK4P1 transcript was compared between the circulating exosomes derived from patients with PAAD and those derived from healthy donors. Overall survival of the patients with PAAD with high or low expression levels of AK4P1 or AK4 was compared. AK4 gene expression level, in vitro cell viability and gemcitabine-induced apoptosis in PAAD cells with or without AK4P1 overexpression were also assessed using Cell Counting Kit-8 and TUNEL assays. It was identified that the transcription level of AK4P1 and the expression level of AK4 in PAAD tissue were significantly higher compared with those in paired non-cancerous tissue specimens. Transcription levels of AK4P1 and AK4 showed a significant relationship in PAAD. Circulating exosomes derived from patients with PAAD showed significantly higher level of AK4P1 transcript compared with that from circulating exosomes derived from blood samples of healthy donors. Patients with high expression of AK4P1 or AK4 exhibited significantly reduced overall survival compared with those with low expression. AK4P1 overexpression significantly upregulated the expression levels of AK4 in PAAD cells and rescued the viability and survival under gemcitabine challenge decreased by AK4 knockdown but not that by AK4 knockout. Treatment with exosomes secreted by AK4P1-overexpressing PAAD cells but not with those secreted by wild-type PAAD cells significantly increased the viability and survival under gemcitabine challenge of the recipient cells. These results suggested that AK4P1 affects the cellular biological functions of PAAD cells in vitro by upregulating the expression level of AK4. AK4P1 transcripts with elevated expression levels can be transmitted between PAAD cells through exosomes and exert pro-oncogenic effects in recipient cells.

Epigallocatechin gallate (EGCG) restores 25-hydroxy vitamin D levels in rheumatoid arthritis patients by attenuating ROS-mediated activation of NF-κB

Purpose: To investigate the mechanism by which epigallocatechin gallate (EGCG) attenuates ROS-induced pathogenesis in a rheumatoid arthritis (RA) patients.&#x0D; Methods: Fifty patients (33 male and 17 female) aged between 25 and 60 years who satisfied ACR 1987 criteria for active RA, and thirty healthy individuals were recruited. Total peripheral blood mononuclear cells (PBMCs), neutrophils and monocytes were isolated from the blood samples of RA patients and healthy donors using commercial extraction kits. Levels of reactive oxygen species (ROS) and superoxide radical (O2-) in isolated cells were measured using fluorescence spectroscopy while the levels of the associated inflammatory cytokines (TNF-α and IL-6) were determined in PBMCs isolated from RA patients using enzyme linked immunosorbent assay (ELISA). These parameters were monitored in EGCG-treated and untreated cells. Moreover, the status of 25(OH) vitamin D and NK-κB were investigated in lipopolysaccharide (LPS)-challenged PBMCs in the presence and absence of EGCG.&#x0D; Results: Elevated levels of superoxide radical and ROS were observed in neutrophils from RA patients in the absence of ECGC, but in the presence of EGCG, the levels of the two parameters were significantly reduced (p &lt; 0.05). Similarly, EGCG treatment downregulated the ROS-mediated increases in TNF-α and IL-6 in the PBMCs from RA patients (p &lt; 0.05). Treatment of PBMCs with the ROS-inducing agent, LPS, resulted in the activation of NK-κB via phosphorylation, and also depletion of 25(OH) vitamin D levels (p &lt; 0.05). However, pre-treatment of the LPS-challenged cells with EGCG inhibited NK-κB activation and 25(OH)-vitamin D depletion (p &lt; 0.05). Moreover, 25(OH)vitamin D levels were restored in the presence of NK-κB inhibitor, flavopiridol, thereby confirming the direct regulatory role of NK-κB in 25(OH) vitamin D levels.&#x0D; Conclusion: EGCG restores 25(OH) vitamin D levels by attenuating ROS-mediated NK-κB activation. Thus, EGCG may be a promising drug candidate for RA.

High CD169 Monocyte/Lymphocyte Ratio Reflects Immunophenotype Disruption and Oxygen Need in COVID-19 Patients.

Background: Sialoadhesin (CD169) has been found to be overexpressed in the blood of COVID-19 patients and identified as a biomarker in early disease. We analyzed CD169 in the blood cells of COVID-19 patients to assess its role as a predictive marker of disease progression and clinical outcomes. Methods: The ratio of the median fluorescence intensity of CD169 between monocytes and lymphocytes (CD169 RMFI) was analyzed by flow cytometry in blood samples of COVID-19 patients (COV) and healthy donors (HDs) and correlated with immunophenotyping, inflammatory markers, cytokine mRNA expression, pulmonary involvement, and disease progression. Results: CD169 RMFI was high in COV but not in HDs, and it correlated with CD8 T-cell senescence and exhaustion markers, as well as with B-cell maturation and differentiation in COV. CD169 RMFI correlated with blood cytokine mRNA levels, inflammatory markers, and pneumonia severity in patients who were untreated at sampling, and was associated with the respiratory outcome throughout hospitalization. Finally, we also report the first evidence of the specific ability of the spike protein of SARS-CoV-2 to trigger CD169 RMFI in a dose-dependent manner in parallel with IL-6 and IL-10 gene transcription in HD PBMCs stimulated in vitro. Conclusion: CD169 is induced by the spike protein and should be considered as an early biomarker for evaluating immune dysfunction and respiratory outcomes in COVID-19 patients.

Plasmacytoid Dendritic Cells in Patients with MGUS and Multiple Myeloma.

Background: Plasmacytoid dendritic cells (pDCs) play prominent roles in mediating innate and adaptive immune responses. However, it is unclear how pDCs contribute to the immunosuppressive tumor microenvironment described in multiple myeloma (MM). Methods: Newly diagnosed myeloma patients (MM, n = 37) were analyzed to determine the pDC counts in comparison to peripheral blood (PB, n = 53) and bone marrow (BM, n = 10) samples of age-matched healthy donors (HD) using flow cytometry. Second, proliferation of myeloma tumor cells in the presence of freshly isolated pDCs was examined. Third, production of IFNα by pDCs co-cultured with MM cells was determined by intracellular staining. Results: We found a highly significant reduction of circulating pDCs (p < 0.0001) and in bone marrow (p < 0.0001) of MM patients compared to HD. We also observed a significant decrease of pDCs (p = 0.004) in BM in patients with monoclonal gammopathy of undetermined significance (MGUS, n = 12). Importantly, we determined that pDCs promote proliferation specifically of MM cells and not the stromal cells and that pDCs secrete IFNα upon co-culture with MM tumor cells. Conclusions: Our results show altered pDC frequencies in the BM microenvironment in MGUS and MM patients at diagnosis. We showed the tumor-promoting function of pDCs that may mediate immune deficiencies affecting long-term disease control and treatment outcome.

Abstract 1170: MEN1112/OBT357, a first-in-class humanized de-fucosylated monoclonal antibody targeting CD157 positive cells in acute myeloid leukemia and myelodysplastic syndrome

Abstract Bst1/CD157 is a GPI-anchored transmembrane protein encoded by a member of the CD38 NADase/ADP-ribosyl cyclase gene family, which is expressed on some blood cells of the myeloid lineage, such as monocytes and neutrophils. The antigen is found to be highly expressed, at diagnosis and relapse, in primary samples derived from Acute Myeloid Leukemia (AML) patients, showing the brightest expression in monocyte-oriented blasts. Moreover, CD157 is expressed in high and low-risk Myelodysplastic Syndrome (MDS) patients. MEN1112/OBT357, is a humanized de-fucosylated monoclonal antibody, in clinical development (clinicaltrials.gov identifier: NCT02353143), that is characterized by high affinity and specificity against CD157, inducing potent in vitro Antibody-Dependent Cell-mediated Cytotoxicity (ADCC). The de-fucosylation improved the binding of MEN1112/OBT357 to both high and low-affinity alleles of FcγRIIIa/CD16a, allowing stronger recognition by NK cells. Since no Complement-Dependent Cytotoxicity (CDC) activity was detected, the MEN1112/OBT357 mechanism of action should mainly rely on immune effector cells. Indeed, in an in vitro reporter assay, MEN1112/OBT357 significantly triggered ADCC activity against AML target cell lines with different expression of CD157. Since the ex vivo sensitivity assay is considered a pharmacologically and clinically relevant model to evaluate the preclinical efficacy of therapeutic antibodies in AML (1), MEN1112/OBT357 activity was tested in 38 AML primary samples, demonstrating promising efficacy in an autologous setting, both in peripheral blood and bone marrow samples, in comparison to healthy donor blood samples and independently from FcγRIIIa polymorphisms. A similar activity was also observed in CD157 positive MDS primary samples. Since Myeloid-Derived Suppressor Cells (MDSCs) have been described to be increased in the bone marrow of both AML and MDS patients and their presence to be associated with resistance to chemotherapy (2, 3), we investigated CD157 expression in MDSCs, demonstrating a high expression of the antigen in the bone marrow and peripheral blood of AML patients. Importantly, we confirmed in ex vivo experiments, the depletion of these immunosuppressive cells by MEN1112/OBT357. Overall, our data demonstrate that MEN1112/OBT357 is a novel antibody with potent ex vivo activity against blast cells in AML and MDS patients, suggesting a potential contribution of MEN1112/OBT357 in altering the immunosuppressive environment in the bone marrow niche.

Evidence of the pathogenic HERV-W envelope expression in T lymphocytes in association with the respiratory outcome of COVID-19 patients

BackgroundDespite an impressive effort in clinical research, no standard therapeutic approach for coronavirus disease 2019 (COVID-19) patients has been established, highlighting the need to identify early biomarkers for predicting disease progression and new therapeutic interventions for patient management. The present study aimed to evaluate the involvement of the human endogenous retrovirus -W envelope (HERV-W ENV) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection considering recent findings that HERVs are activated in response to infectious agents and lead to various immunopathological effects. We analysed HERV-W ENV expression in blood cells of COVID-19 patients in correlation with clinical characteristics and have discussed its potential role in the outcome of the disease. MethodsWe analysed HERV-W ENV expression in blood samples of COVID-19 patients and healthy donors by flow cytometry and quantitative reverse transcriptase PCR analysis, and evaluated its correlation with clinical signs, inflammatory markers, cytokine expression, and disease progression. FindingsHERV-W ENV was highly expressed in the leukocytes of COVID-19 patients but not in those of healthy donors. Its expression correlated with the markers of T-cell differentiation and exhaustion and blood cytokine levels. The percentage of HERV-W ENV-positive lymphocytes correlated with inflammatory markers and pneumonia severity in COVID-19 patients. Notably, HERV-W ENV expression reflects the respiratory outcome of patients during hospitalization. InterpretationGiven the known immuno- and neuro-pathogenicity of HERV-W ENV protein, it could promote certain pathogenic features of COVID-19 and therefore serve as a biomarker to predict clinical progression of disease and open to further studies for therapeutic intervention. FundingInformation available at the end of the manuscript.