Median Fluorescence Intensity Research Articles

Safety, acceptability, and feasibility of acquiring endothelial cells from guidewires in patients having major surgery or with acute critical illness

Abstract Background Endothelial cells (EC) play a key role in vascular homeostasis, and endothelial dysfunction has been linked to the development of cardiovascular disease. While most EC research relies on indirect assessment of EC function in humans, in vitro systems and in vivo animal models, the development of a safe protocol to isolate EC from patients would provide a new window into EC function. Purpose To assess (a) the feasibility of isolating EC from discarded guidewires used as part of the standard of care (SOC) in surgical patients and patients admitted to intensive care (ICU) and (b) to compare EC phenotypes in the patient cohorts. Methods Discarded guidewires were collected from arterial lines and central venous catheters (CVC) from 15 patients before abdominal surgery and 16 patients admitted to ICU requiring organ support. Additionally, J-wires were inserted into forearm veins using an proven technique for EC biopsy (ECBx) in all surgical patients. ECs were dissociated from guidewires, stained for CD31, CD144, CD202b, and nuclear integrity then analysed using flow-cytometry. EC were characterised based on surface marker expression and granularity. Blood biomarkers and clinical information was collected. Organ function was assessed using sequential organ failure assessment (SOFA). Tolerability and adverse event (AE) data was also collected using structured interviews to support the analysis. Results From the surgical cohort 6 arterial lines, 14 CVC and 15 ECBx were collected. From the ICU patients, 8 arterial lines and 9 CVC were collected. EC were identified as live and triple positive for CD31, CD144, CD202b. A median of 536 (IQR 160-796 ) EC were obtained from arterial lines, 622 (IQR 329-1045) from CVCs and 768 (IQR 210-1001) from ECBx with no significant difference between method of collection (Kruskal-Wallis p>0.05). Median granularity showed a significant increase in granularity in EC collected from ICU patients compared to the surgical patients. This could be attributed to internalisation of cell surface proteins or apoptosis caused by the hyperinflammatory conditions in ICU patients (SOFA 7, IQR 5-8.25, CRP 118, IQR 66.2-298) when compared to the surgical patients (SOFA 1, IQR 0-2, CRP <4mg/l). Median fluorescent intensity analysis of all three markers did not show a significant difference between patient groups. AEs to ECBx reported only 4 patients experienced light bruising. High tolerability was reported for ECBx by structured interview, with patients agreeing that they theoretically would tolerate up to 3 ECBx. Conclusion(s) It is feasible to isolate ECs from patients during SOC using discarded guidewires. As an isolated procedure, ECBx from forearm veins is well tolerated and carries low risk. This enables the characterisation of EC phenotypes and obtain unique insight in their roles in health and disease.

CD64 Expression on Neutrophils (nCD64) as a Biomarker in Adult Patients With Sepsis: A Cross-Sectional Study.

Introduction Neutrophils that are at rest exhibit very low expression of CD64, the high-affinity immunoglobulin fragment crystallizable γ receptor I, which is also found in monocytes. Neutrophils that have been exposed to endotoxins or are infected express more CD64. Objectives The aim of this study was to assess the CD64 biomarker expression on neutrophils using the flow cytometry method and its comparison with total leukocyte count, C-reactive protein (CRP) level, and blood culture sensitivity test in the diagnosis of sepsis in adults. Materials and methods Using the quick Sequential Organ Failure Assessment (qSOFA) scoring criteria, 94 blood samples from individuals with clinical indications of sepsis were included in this investigation. Samples were collected in K2 EDTA (ethylenediaminetetraacetic acid) vacutainers and analyzed for neutrophil CD64 (nCD64) levels using BD FACSLyricTM flow cytometer (BD, Franklin Lakes, NJ) within 24 hours of collection. Total leukocyte count, CRP levels, and blood culture sensitivity tests were also assessed simultaneously. Results Majority of the patients, i.e., 29 (30.9%), belonged to the age group of 19 to 35 years, with a female preponderance. The mean total leukocyte count was 19.49 ± 8.12 x 103/µL, mean CRP level was 81.19 ± 56.33 mg/dL, and the mean nCD64 expression - median fluorescence intensity was 197.26 ± 79.56. A positive blood culture was found in 59 (62.8%) cases. Neutrophils showed bright expression of nCD64 in 39 (41.5%) patients, dim expression in 35 (37.2%) patients, and moderate expression in 20 (21.3%) patients in the present study. The correlation between nCD64 expression with CRP, total leukocyte count, and blood culture sensitivity test was statistically significant (p=0.001). nCD64 expression showed a sensitivity of 93.2% and specificity of 91.4% for diagnosing sepsis, with an area under the curve (AUC) of 0.973. This proves nCD64 to be a highly effective biomarker compared to conventional methods. Conclusion nCD64 has a higher sensitivity and specificity as compared to total leukocyte count, CRP levels, and blood culture sensitivity test in the diagnosis of sepsis. It is an effective and rapid test that can enhance sepsis diagnostic accuracy, when combined with other biomarkers. Multicentric research needs to be conducted to validate these findings.

Causal role of 731 immune cells in diabetic nephropathy: a bi-directional two-sample Mendelian randomization study.

The primary cause of end-stage renal disease (ESRD) is diabetic nephropathy (DN), and a growing body of research indicates that immunology plays a part in how DN develops into ESRD. Our objective is to identify causal relationships between various immune invading cells and DN to identify possible targets for immunotherapy. This study used a complete Mendelian randomization (MR) analysis with two samples to identify the underlying mechanism linking immune cell characteristics with DN. Using publicly available genetic data, we investigated the causal link between 731 immune cell profiles and DN risk. Included were four different types of immune systems: morphological parameters (MP), absolute cell (AC), relative cell (RC), and median fluorescence intensities (MFI). The results' robustness, heterogeneity, and horizontal pleiotropy were confirmed through extensive sensitivity analysis. Following FDR (False Discovery Rate correction method) correction, no statistically significant differences were observed; however, six immunophenotypes were shown to be significantly associated with DN risk at the 0.25 level. Only CD28+ CD4- CD8- T cells were identified as the protective immunophenotype (OR = 0.588, 95% CI 0.437-0.792, P = 4.71 × 10-4). Moreover, DN had no discernible impact on immunophenotyping after FDR correction. Surprisingly, three unadjusted phenotypes with low P values were discovered to be positively correlated with the risk of DN: CD20 on IgD- CD27- B cells (OR = 1.263, 95% CI 1.076-1.482, P = 4.22 × 10-3), CD8 on naive CD8 + T cells with Effector Memory (OR = 1.107, 95% CI 1.013-1.209, P = 2.40 × 10-2), and CD8 on Effector Memory CD8 + T cells (OR = 1.126, 95% CI 1.024-1.239, P = 1.46 × 10-2). Our findings provide a genetic basis for the association between immune cells and DN and should inform future clinical research.

B-094 Assessment of a Live Cell-based Flow Cytometry Assay for MuSK-IgG4 Testing in Myasthenia Gravis

Abstract Background Myasthenia gravis (MG) is caused by disrupting the function of postsynaptic acetylcholine receptors (AChRs) at the neuromuscular junction. 85% of MG patients have autoantibodies targeting AChRs, while 6% of MG patients have autoantibodies targeting the muscle-specific receptor tyrosine kinase (MuSK). MuSK is located on the muscle membrane and is activated by the low-density lipoprotein receptor-related protein 4 (LRP4) upon the binding with ligand Agrin. Activated MuSK undergoes autophosphorylation and stimulates downstream signaling to cluster AChRs, which is important for the formation and maintenance of neuromuscular synapses. IgG4 antibodies that bind MuSK are thought to directly disrupt the ability of MuSK to cluster AChR. The gold standard methodology for MuSK antibody testing is radioimmunoprecipitation assay (RIA) using anti-human IgG secondary antibodies. Performing RIA methods in the clinical laboratory is a burden due to radioactive biohazard concerns and the analytical limitations of these assays. This study is to assess the analytical and clinical performance of a live cell-based flow cytometry assay as a potential replacement for the MuSK RIA. Methods HEK293T cells were transiently transfected with a vector co-expressing human MuSK protein and GFP. Patient sera were incubated with a mix of HEK293 cells (GFP-positive cells that express MuSK protein on the cell surface and GFP-negative cells that do no express MuSK) followed by incubation with anti-human IgG4 secondary antibody conjugated with alexa fluor 647 (AF647). An IgG binding index (IBI) was calculated for each sample as the ratio of the median AF647 fluorescence intensity (MFI) of the MuSK-expressing cells (GFP+) to the MFI of the non-MuSK expressing cells (GFP-). An arbitrary positive IBI cut-off of 2.0 was selected. A preliminary assessment of assay imprecision was performed for the flow cytometry assay, as well as a method comparison study to compare this assay to RIA utilizing a cohort of 130 (34 positive and 96 negative) residual samples previously tested by the gold-standard assay. 133 disease control samples were also tested to assess the clinical specificity of the flow cytometry-based assay. Results To assess the imprecision, eight samples with varying IBIs were tested with 5 replicates in each assay over five days. The coefficient of variation (CV) for intra-assay replicates ranged from 1% to 7% for all samples, while CV for inter-assay replicates ranged from 2% to 15%. Based on the 130 samples previously tested by RIA, the Positive Percentage Agreement (PPA) between these two assays was 76.5% and the negative Percentage Agreement (NPA) was 100%. Of the discordant samples (n=8), clinical information was available for only 1 sample (positive by RIA, negative by flow). This patient had a final non-MG diagnosis (ALS). All control samples were negative on the flow cytometry-based assay. Conclusions The live cell-based flow cytometry MuSK IgG4 assay demonstrated encouraging preliminary analytical performance that warrants further development and validation.

Converting an HLA-B27 flow assay from the BD FACSCanto to the BD FACSLyric.

HLA-B27 is a major histocompatibility complex (MHC) class I antigen which exhibits strong association (90%) with ankylosing spondylitis. HLA-B27 detection in patients by flow cytometry is a widely used clinical test, performed on many different flow cytometer models. We sought to develop and validate a test conversion protocol for the HLA-B27 test performed on the BD FACSCanto to BD's newer FACSLyric flow cytometers. The development and validation experiments were performed using anti-HLA-B27*FITC/CD3*PE antibody-stained whole blood patient specimens. The anti-HLA-B27*FITC logarithmic median fluorescence (LMF) results on the BD FACSCanto were converted to median fluorescence intensity (MFI) values on the BD FACSLyric. Clustering of the HLA-B27 positive and negative values, using a 3rd order polynomial equation, resulted in a conversion of the BD FACSCanto cutoff values, negative (<150 LMF) and positive (≥160 LMF), to negative (<4530 MFI) and positive (≥6950 MFI) on the BD FACSLyric. Accuracy was assessed by comparing the flow results obtained on the BD FACSCanto and BD FACSLyric to a molecular PCR based assay. Additional validation parameters (compensation verification, intra- and inter-assay precision, and instrument comparison) were performed per the recommendations outlined in the Clinical and Laboratory Standards Institute (CLSI) H62 guidelines for validation of flow cytometry assays.

Genetically defined causal effects of natural killer cells related traits in risk of infection: a Mendelian randomization study

BackgroundThe intricate interplay between genetics and immunology often dictates the host’s susceptibility to various diseases. This study explored the genetic causal relationship between natural killer (NK) cell-related traits and the risk of infection.MethodsSingle-nucleotide polymorphisms (SNPs) significantly associated with NK cell-related traits were selected as instrumental variables to estimate their genetic causal effects on infection. SNPs from a genome-wide association study (GWAS) on NK cell-related traits, including absolute cell counts, median fluorescence intensities reflecting surface antigen levels, and relative cell counts, were used as exposure instruments. Summary-level GWAS statistics of four phenotypes of infection were used as the outcome data. The exposure and outcome data were analyzed via the two-sample Mendelian randomization method.ResultsEach one standard deviation increase in the expression level of human leukocyte antigen (HLA)-DR on HLA-DR+ NK cells was associated with a lower risk of pneumonia (P < 0.05). An increased HLA-DR+ NK/CD3− lymphocyte ratio was related to a lower of risk of pneumonia (P < 0.05). Each one standard deviation increase in the absolute count of HLA-DR+ NK cells was associated with a lower risk of both bacterial pneumonia and pneumonia (P < 0.05). An increased HLA-DR+ NK/NK ratio was associated with a decreased risk of both pneumonia and bacterial pneumonia (P < 0.05). The results were robust under all sensitivity analyses. No evidence for heterogeneity, pleiotropy, or potential reverse causality was detected. Notably, our analysis did not reveal any significant associations between NK cell-related traits and other phenotypes of infection, including cellulitis, cystitis, and intestinal infection.ConclusionsHLA-DR+ NK cells could be a novel immune cell trait associated with a lower risk of bacterial pneumonia or pneumonia.

Can median fluorescence intensity of HLA antibody detected by LABScreen Single Antigen Bead assay absolutely predict C1q activity?

Can median fluorescence intensity of HLA antibody detected by LABScreen Single Antigen Bead assay absolutely predict C1q activity?

Flow cytometric expression of Bcl-2, Mcl-1, and their ratios correlates with primary and secondary cytogenetic changes and their combinations in multiple myeloma.

Response to BH3 mimetics in multiple myeloma (MM) correlates with CCND1-rearrangement or expression of anti-apoptotic molecules, particularly Bcl-2 and Mcl-1. Our study investigates the relationship between cytogenetic abnormalities (CGAs) and intracellular Bcl-2 and Mcl-1 expression in myeloma plasma cells(MPCs) using flow cytometry (FCM). We measured median fluorescence intensity (MFI) of Bcl-2 and Mcl-1 in 163 bone marrow samples (143 MM, 20 controls) across various cell types. Both Bcl-2MFI and Mcl-1MFI were significantly higher in MPCs compared to other cells, with Bcl-2 MFI exceeding Mcl-1 MFI in MPCs. Bcl-2 expression peaked in CCND1-rearranged cases, while Mcl-1 expression was highest in cases with 1q21 gain/amplification. Notably, 65-74% of cases with other CGAs exhibited moderate to strong Bcl-2 or Mcl-1 expression, indicating potential utility of BH3 mimetics in this group, while 25% showed dim to absent expression of one or both markers, suggesting potential futility in these patients. Our study highlights FCM's potential for rapid Bcl-2 and Mcl-1 quantification, surpassing traditional methods. We propose that direct measurement of Bcl-2 and Mcl-1 expression in PCs by FCM, combined with cytogenetic characterization, could improve therapeutic decision-making regarding the use of BH3 mimetics in MM, potentially enhancing outcomes and overcoming resistance.

Causal role of immune cells in aplastic anemia: Mendelian randomization (MR) study

Prior research has identified associations between immune cells and aplastic anaemia (AA); however, the causal relationships between them have not been conclusively established. A two-sample Mendelian randomisation analysis was conducted to investigate the causal link between 731 immune cell signatures and AA risk using publicly available genetic data. Four types of immune signatures, including relative cell, absolute cell (AC), median fluorescence intensities and morphological parameters, were considered sensitivity analyses were also performed to verify the robustness of the results and assess potential issues such as heterogeneity and horizontal pleiotropy. Following multiple test adjustments using the False Discovery Rate (FDR) method, no statistically significant impact of any immunophenotype on AA was observed. However, twelve immunophenotypes exhibited a significant correlation with AA without FDR correction (p of IVW < 0.01), of which eight were harmful to AA: CD127- CD8br %T cell (Treg panel), CD25 on IgD + CD38dim (B cell panel), CD38 on naive-mature B cell (B cell panel), CD39 + resting Treg % CD4 Treg (Treg panel), CD39 + secreting Treg AC (Treg panel), CD8 on CD28 + CD45RA- CD8br (Treg panel), HLA DR + NK AC (TBNK panel), Naive DN (CD4−CD8−) AC (Maturation stages of T cell panel); and four were protective to AA: CD86 on CD62L + myeloid DC (cDC panel), DC AC (cDC panel), DN (CD4−CD8−) NKT %T cell (TBNK panel), and TD CD4 + AC (Maturation stages of T cell panel). The results of this study demonstrate a close link between immune cells and AA by genetic means, thereby improving the current understanding of the interaction between immune cells and AA risk and providing guidance for future clinical research.

Quantification of the median fluorescence intensity of CD3 and CD4 in mycosis fungoides/Sezary syndrome versus non-neoplastic control cases in peripheral blood.

Peripheral blood involvement by MF/SS has significant implications for prognosis and treatment. Flow cytometry is commonly used to assess MF/SS by analyzing the ratio of CD26- and/or CD7-CD4 + T cells and assessment of immunophenotypic abnormalities. However, distinguishing normal from abnormal cells is not always easy. In this study, we aimed to establish quantitative thresholds to better distinguish normal CD4 + T cells from neoplastic CD4 + T cells. A retrospective analysis of flow cytometry data was performed on 30 MF/SS patients with a detectable abnormal T cell population (positive), 63 patients with suspected or confirmed cutaneous involvement without a detectable abnormal T cell population (negative), and 60 healthy controls (control). CD3 and CD4 median fluorescence intensity (MFI) was normalized to internal control subsets. Among the positive cases, 50% had CD3 expression outside ± 2 SD from the mean of the negative and control group in the CD4 + CD26- subset. The corresponding specificity of this threshold was 94%. The ± 2 SD threshold showed a sensitivity of 57% and a specificity of 94% for the CD3 intensity among the CD7-negative subset. For CD4 intensity, the ± 2 SD threshold had a sensitivity of 33.3% and specificity of 95% for the CD26-negative subset and a sensitivity of 37% and specificity of 95% for the CD7-negative subset. In our study, although changes in CD3 and CD4 intensity greater than ± 2 SD were specific for MF/SS, more subtle differences in the intensity of CD3 and CD4 should not be used as the sole abnormality to make a diagnosis of circulating MF/SS.

Genome editing in K562 cells suggests a functional role for the XmnI Gg polymorphism: a widely used genetic marker in β-thalassemia and sickle cell disease patients.

The XmnI Gg -158 C/T polymorphism has been widely associated with fetal hemoglobin (HbF) levels, the severity of disease, and the response to the drug hydroxyurea (HU) in both β-thalassemia (β-thal) and sickle cell disease (SCD) patients. However, the functional significance of this single nucleotide polymorphism (SNP) remains unclear. To gain insight, green fluorescence protein (GFP) cassettes harboring the XmnI C or T alleles in their left homology arms (i.e. Gg promoters) were knocked into the Gg gene(s) of K562 cells via CRISPR/Cas9. Subsequently, the GFP fluorescence levels were compared in the ensuing cell populations and isolated clones. In both instances, median fluorescence intensities (MFI) of the knockin cells having the inserted XmnI T allele were higher than those having the XmnI C allele. Our results suggest that the XmnI T allele can increase Gg expression in K562 cells. The possible functional significance of the XmnI Gg -158 C/T polymorphism provides a rationale for the aforementioned associations. Furthermore, the XmnI polymorphism as a functional SNP substantiates its importance as a prognostic marker.

Causal association of circulating immune cells and lymphoma: A Mendelian randomization study.

Malignant lymphoma (ML) is a group of malignant tumors originating from the lymphatic hematopoietic system. Previous studies have found a correlation between circulating immune cells and ML. Nonetheless, the precise influence of circulating immune cells on ML remains uncertain. Based on publicly available genetic data, we explored causal associations between 731 immune cell signatures and ML risk. A total of four types of immune signatures, median fluorescence intensities, relative cell, absolute cell, and morphological parameters were included. Primary analysis was performed using inverse variance weighting (IVW) to assess the causal relationship between circulating immune cells and the risk of ML. Sensitivity analysis was conducted using Cochran's Q test, the Mendelian randomization Egger regression intercept test, and leave-one-out analysis. ML had a statistically significant effect on immunophenotypes. Twenty-three immunophenotypes were identified to be significantly associated with Hodgkin lymphoma risk through the IVW approach, and the odds ratio values of CD64 on CD14- CD16+ monocyte [2.31, 95% confidence interval (CI) = 1.41-3.79, P1 = 0.001], IgD+ CD24+ B-cell %lymphocyte (2.06, 95% CI = 1.13-3.79, P1 = 0.018), B-cell %lymphocyte (1.94, 95% CI = 1.08-3.50, P1 = 0.027), CD24+ CD27+ B-cell %lymphocyte (1.68, 95% CI = 1.03-2.74, P1 = 0.039), and CD14+ CD16- monocyte %monocyte (1.60, 95% CI = 1.15-2.24, P1 = 0.006) ranked in the top five. Eleven immunophenotypes were identified to be significantly associated with non-Hodgkin lymphoma risk, CD86 on granulocyte (2.35, 95% CI = 1.18-4.69, P1 = 0.015), CD28-CD8+ T-cell absolute count (1.76, 95% CI = 1.03-2.99, P1 = 0.036), CCR2 on myeloid dendritic cell (CD24+ CD27+ B cell, 95% CI = 1.02-1.66, P1 = 0.034), CD3 on effector memory CD8+ T cell (1.29, 95% CI = 1.02-1.64, P1 = 0.012), and natural killer T %lymphocyte (1.28, 95% CI = 1.01-1.62, P1 = 0.046) were ranked in the top five. This study presents compelling evidence indicating the correlation between circulating immune cells and lymphoma, thus providing guidance for future clinical research.

Functionalized magnetic nanoparticles remove donor-specific antibodies (DSA) from patient blood in a first ex vivo proof of principle study

The presence of donor-specific antibodies (DSA) such as antibodies directed against donor class I human leucocyte antigen (e.g., HLA-A) is a major barrier to kidney transplant success. As a proof of concept, functionalized magnetic nanoparticles have been designed to eliminate DSA from saline, blood and plasma of healthy donors and sensitized patients. Specific HLA-A1 protein was covalently bound to functionalized cobalt nanoparticles (fNP), human serum albumin (HSA) as control. fNP were added to anti-HLA class I-spiked saline, spiked volunteers’ whole blood, and to whole blood and plasma of sensitized patients ex vivo. Anti-HLA-A1 antibody levels were determined with Luminex technology. Antibodies' median fluorescent intensity (MFI) was defined as the primary outcome. Furthermore, the impact of fNP treatment on blood coagulation and cellular uptake was determined. Treatment with fNP reduced MFI by 97 ± 2% and by 94 ± 4% (p < 0.001 and p = 0.001) in spiked saline and whole blood, respectively. In six known sensitized anti-HLA-A1 positive patients, a reduction of 65 ± 26% (p = 0.002) in plasma and 65 ± 33% (p = 0.012) in whole blood was achieved. No impact on coagulation was observed. A minimal number of nanoparticles was detected in peripheral mononuclear blood cells. The study demonstrates—in a first step—the feasibility of anti-HLA antibody removal using fNP. These pilot data might pave the way for a new personalized DSA removal technology in the future.

Causal role of immune cells in uveitis: Mendelian randomization study.

Uveitis, characterized by inflammation of the iris, ciliary body, and choroid, presents a significant global clinical challenge, contributing substantially to visual impairment. Risk factors include autoimmune diseases and immune cell dysfunctions, yet many remain unidentified. Immune cells, notably T cells, B cells, and monocytes, play pivotal roles in uveitis pathogenesis. While biologic agents show promise, comprehensive studies on immune cell types in ocular diseases are lacking. Genome-wide association studies (GWAS) and Mendelian randomization (MR) present promising avenues to elucidate genetic susceptibilities and causal relationships between immune cell traits and uveitis risk. Two-sample MR analysis was used to evaluate the causal relationship between 731 immune cells and uveitis, and genome-wide significance analysis was performed for genetic variation in 731 immune cells traits (P < 5 × 10-8). Immune characteristics include median fluorescence intensity (MFI), relative cell counts (RC), absolute cell counts (AC), and morphological parameters (MP), which were determined by published GWAS, and public data from the IEU Open GWAS database. The main analysis method of MR is inverse variance weighting (IVW). Heterogeneity and horizontal pleiotropy were also assessed. 5 immunophenotypes, including CD62L-DC %DC, IgD+ CD38dim %B cell, CD3 on CM CD4+T cell, CD3 on CD45RA-CD4 +T cell, and CD3 on CD39+ CD4+ Treg may increase the risk of uveitis. 5 immunophenotypes, including CD11b on CD33dim HLA DR-Myeloid cell, HLA DR on CD33dim HLA DR+ CD11b-myeloid cell, CD14-CD16 + %monocyte, HLA DR on CD14-CD16 + monocyte and PDL-1 on CD14-CD16 + monocyte was negatively associated with the risk of uveitis. Among them, HLA DR on CD14-CD16 + monocyte (OR=0.921, 95%CI =0.875-0.970, P=0.001) and HLA DR on CD33dim HLA DR+ CD11b- (OR=0.879, 95%CI = 0.833-0.927, P=0.00) were negatively associated with the risk of uveitis in bi-direction. These results indicate that 10 immune cells traits are significantly associated with the risk of developing uveitis and 2 of them were strongly associated with uveitis bi-directionally, after excluding the effects of confounding factors such as some immune diseases, which provided new ideas and therapeutic targets for the study of immune mechanism of uveitis.

Neutrophil CD64 – A Reliable Predictive Marker of Sepsis in Adult Intensive Care Unit

Abstract BACKGROUND: Sepsis poses a significant threat to adult patients admitted to intensive care units (ICUs), contributing to substantial morbidity and mortality. Neutrophil CD64 (nCD64) expression has been linked to inflammatory responses during infection or tissue injury, suggesting its potential as a predictive marker for sepsis. MATERIALS AND METHODS: We conducted a prospective analytical study over 2 years at a tertiary care hospital, enrolling 91 sepsis cases from the adult ICU. Alongside routine laboratory parameters, including complete blood count, prothrombin time, activated partial thromboplastin time, fibrinogen, C-reactive protein, and procalcitonin, the expression of nCD64 was analyzed using a Beckman Coulter Navios flow cytometer. Median fluorescence intensity (MFI) was calculated for these cases. Statistical analysis was performed using SPSS version 25.0 software, with a significance threshold of P &lt; 0.05. RESULTS: MFI scores demonstrated a notable increase in diagnosed sepsis cases, as determined by clinical and biochemical parameters. Moreover, changes in MFI scores on Day 4 exhibited a correlation with other clinical and biochemical parameters, strengthening the association between nCD64 expression and sepsis severity. CONCLUSION: Our findings suggest that nCD64 serves as an independent prognostic factor in adult ICU sepsis patients. It offers a promising alternative to traditional sepsis markers for predicting patient outcomes, emphasizing its potential clinical utility in guiding therapeutic interventions and improving patient care in ICU settings.

The relationship of microvascular inflammation with antibody-mediated rejection in kidney transplantation

Microvascular inflammation (MVI) is a key diagnostic feature of antibody-mediated rejection (AMR); however, recipients without donor-specific antibodies (DSA) defy etiologic classification using C4d staining of peritubular capillaries (C4dptc) and conventional DSA assignment. We evaluated MVI ≥ 2 (Banff g + ptc ≥ 2) using Banff 2019 AMR (independent of MVI ≥ 2 but including C4dptc) with unconventional endothelial C4d staining of glomerular capillaries (C4dglom) and - arterial endothelium and/or intima (C4dart) using tissue immunoperoxidase, shared-eplet and subthreshold DSA (median fluorescence intensity, [MFI] 100-499), and capillary ultrastructure from 3398 kidney transplant samples for evidence of AMR. MVI ≥ 2 (n = 202 biopsies) from 149 kidneys (12.4% prevalence) correlated with DSA+, C4dptc+, C4dglom+, Banff cg, i, t, ti scores, serum creatinine, proteinuria, and graft failure compared with 202 propensity score matched normal controls. The laboratory reported DSA− MVI ≥ 2 (MFI ≥500) occurred in 34.7%; however, subthreshold (28.6%), eplet-directed (51.4%), and/or misclassified anti-Human leukocyte antigen (HLA) DSA (12.9%) were identified in 67.1% by forensic reanalysis, with vascular C4d+ staining in 67.1%, and endothelial abnormalities in 57.1%, totaling 87.1%. Etiologic analysis attributed 62.9% to AMR (77.8% for MVI with negative reported DSA [DSA− MVI ≥2] with glomerulitis) and pure T cellular rejection in 37.1%. C4dptc−DSA− MVI ≥ 2 was unrecognized AMR in 48.0%. Functional outcomes and graft survival were comparable to normal controls. We concluded that DSA− MVI ≥ 2 frequently signified a mild “borderline” phenotype of AMR which was recognizable using novel serologic and pathological techniques.

Causal relationships between immune cell phenotypes and lung adenocarcinoma: A bidirectional two-sample Mendelian randomization study.

Lung adenocarcinoma (LUAD) is the most common type of lung cancer and closely associated with the immune system. Emerging evidence suggests that blood immune cell phenotypes in patients with LUAD may undergo alterations. Nevertheless, the limited amount of relevant research makes it difficult to understand the causal links between LUAD and changes in the immune cells. This study aimed to reveal the potential causal relationships between 731 immune cell phenotypes and LUAD. A bidirectional two-sample Mendelian randomization (MR) analysis was used to clarify causal relationships. Four types of immune phenotypes, absolute cell counts, relative cell counts, median fluorescence intensities (MFIs) of surface antigens, and morphological parameters, were investigated in this study. Heterogeneity tests, horizontal pleiotropy tests, and leave-one-out analyses were performed to validate the reliability of our study. A total of 26 immune cell characteristics were identified as contributing to the occurrence of LUAD. Memory B cells, IgD-CD38br cells, CD4+ regulatory T cells (Tregs), and plasmacytoid dendritic cells (DCs) may play a role in the development of LUAD. Through reverse MR, our study discovered that the presence of LUAD also induced changes in the expression levels of 16 immune cell traits involving specific surface markers and various types of immune cells, some of which pertain to antigen presentation and immune activation processes. Our study demonstrated causal links between several immune cell phenotypes and LUAD, thereby providing indications of the potentially oncogenic physiological state and early screening biomarkers for future research.

Immune cell signatures and causal association with irritable bowel syndrome: A mendelian randomization study

BACKGROUND The mucosal barrier's immune-brain interactions, pivotal for neural development and function, are increasingly recognized for their potential causal and therapeutic relevance to irritable bowel syndrome (IBS). Prior studies linking immune inflammation with IBS have been inconsistent. To further elucidate this relationship, we conducted a Mendelian randomization (MR) analysis of 731 immune cell markers to dissect the influence of various immune phenotypes on IBS. Our goal was to deepen our understanding of the disrupted brain-gut axis in IBS and to identify novel therapeutic targets. AIM To leverage publicly available data to perform MR analysis on 731 immune cell markers and explore their impact on IBS. We aimed to uncover immunophenotypic associations with IBS that could inform future drug development and therapeutic strategies. METHODS We performed a comprehensive two-sample MR analysis to evaluate the causal relationship between immune cell markers and IBS. By utilizing genetic data from public databases, we examined the causal associations between 731 immune cell markers, encompassing median fluorescence intensity, relative cell abundance, absolute cell count, and morphological parameters, with IBS susceptibility. Sensitivity analyses were conducted to validate our findings and address potential heterogeneity and pleiotropy. RESULTS Bidirectional false discovery rate correction indicated no significant influence of IBS on immunophenotypes. However, our analysis revealed a causal impact of IBS on 30 out of 731 immune phenotypes (P &lt; 0.05). Nine immune phenotypes demonstrated a protective effect against IBS [inverse variance weighting (IVW) &lt; 0.05, odd ratio (OR) &lt; 1], while 21 others were associated with an increased risk of IBS onset (IVW ≥ 0.05, OR ≥ 1). CONCLUSION Our findings underscore a substantial genetic correlation between immune cell phenotypes and IBS, providing valuable insights into the pathophysiology of the condition. These results pave the way for the development of more precise biomarkers and targeted therapies for IBS. Furthermore, this research enriches our comprehension of immune cell roles in IBS pathogenesis, offering a foundation for more effective, personalized treatment approaches. These advancements hold promise for improving IBS patient quality of life and reducing the disease burden on individuals and their families.

The immune factors have complex causal regulation effects on kidney stone disease: a mendelian randomization study

PurposePrevious studies have reported the potential impact of immune cells on kidney stone disease (KSD), but definitive causal relationships have yet to be established. The purpose of this paper is to elucidate the potential causal association between immune cells and KSD by Mendelian randomization (MR) analysis.MethodsIn our study, a thorough two-sample Mendelian randomization (MR) analysis was performed by us to determine the potential causal relationship between immune cell traits and kidney stone disease. We included a total of four immune traits (median fluorescence intensity (MFI), relative cellular (RC), absolute cellular (AC), and morphological parameters (MP)), which are publicly available data. GWAS summary data related to KSD (9713 cases and 366,693 controls) were obtained from the FinnGen consortium. The primary MR analysis method was Inverse variance weighted. Cochran’s Q test, MR Egger, and MR-Pleiotropy RESidual Sum and Outlier (MR-PRESSO) were used to assess the stability of the results.ResultsAfter FDR correction, the CD8 on HLA DR + CD8br (OR = 0.95, 95% CI = 0.93–0.98, p-value = 7.20 × 10− 4, q-value = 0.088) was determined to be distinctly associated with KSD, and we also found other 25 suggestive associations between immune cells and KSD, of which 13 associations were suggested as protective factors and 12 associations were suggested as risk factors. There was no horizontal pleiotropy or significant heterogeneity in our MR analysis, as determined by the p-value results of our Cochrane Q-test, MR Egger’s intercept test, and MR-PRESSO, which were all > 0.05.ConclusionsOur study has explored the potential causal connection between immune cells and KSD by Mendelian randomization analysis, thus providing some insights for future clinical studies.

Exoscopic versus Microscopic Surgery in 5-ALA-Guided Resection of High-Grade Gliomas.

Background: Glioma surgery has been remarkably enhanced in the past 2 decades, with improved safety and limited but improved life expectations. The fluorescence-guided resection of high-grade gliomas (HGGs) plays a central role in this sense, allowing a greater extent of resection (EOR). The introduction of exoscopic-guided surgery may be considered in implementing fluorescence techniques over traditional microscopes. We present the application and the advantages of exoscopic-guided surgery compared to microscopic surgery in tumor resection guided by 5-ALA fluorescence in patients with HGGs. Methods: Ten consecutive patients underwent surgery for HGG resection. The surgery was performed via an exoscopic-guided procedure (Olympus ORBEYE) and after the oral administration of Gliolan 5 h before the procedure. During surgery, the procedure shifted to using a microscopic (Kinevo 900, Zeiss) view. The intensity of the fluorescence under the two different procedures was subjectively measured in different picture samples during the surgery on a 1 to 5 (from minimum to maximum) scale. The brightness of the surgical field and the detailing of the anatomy were also analyzed comparatively. Results: Among the ten patients, the histopathological diagnosis was an high-grade glioma in all cases. In nine cases, it was possible to achieve gross total resection. There was no perioperative mortality. The median fluorescence intensity, on a scale of 1-5, was 4.5 in the exoscope group and 3.5 in the microscope group (p < 0.01). Conclusions: The exoscopic-guided surgery adds advantages to traditional fluorescence-guided surgery with 5-aminolevulinic acid. Beyond the important advantage of low cost and the possibility to perform collaborative surgeries, it adds a plain and continuous visualization of the tumor and offers advantages in the surgical field of fluorescence-guided glioma surgery compared to the microscopic-guided one.