Determination Of Expression Research Articles

Advancing magnetic flow cytometry to quantitative epitope analysis in high hematocrit conditions for point-of-care testing

Quantitative cell function measurements are essential for many clinical decisions but are primarily tied to centralized laboratories. Limited access to these laboratories in low-resource settings or for immobile patients highlights the urgent need for Point-of-Care testing (POCT) infrastructure. Magnetic flow cytometers (MFC) offer a solution, albeit phenotyping is limited, and sample processing steps like cell lysis or washing increase MFC's workflow complexity. Here, we investigate conditions for novel phenotyping and direct cell concentration quantification in a streamlined workflow suitable for POCT in high hematocrit environments. We characterize magnetic nanoparticles (MNP) by their size, magnetic moment, and opportunities for high signal-to-noise ratios. With adapted theoretical models, we provide the framework for quantifying bound MNPs per cell. This reveals labeling quality and gives insight into system requirements for reliable cell detection and rational cell phenotyping. We investigate temporal labeling dynamics, which show suboptimal MNP binding kinetics in whole blood (WB), leading to long incubation periods and only 50% recovery concentrations. With our streamlined workflow favoring small (<50 nm) MNPs, we quantify CD14+ monocytes in WB and achieve coefficients of variation of <11%. By simultaneously assessing quantitative epitope expression, we extend MFC's capabilities to clinical subtyping for POCT.

Comprehensive pan-cancer analysis of FUTs family as prognostic and immunity markers based on multi-omics data

BackgroundThe dysregulation of fucosyltransferases (FUTs) contributes to alterations in fucosylated epitope expression, which serve as distinctive features of cancer cells. Nonetheless, a comprehensive elucidation of the prognostic biological marker and therapeutic target of the FUTs family in pan-cancer remains elusive.MethodsOver 10,000 individuals' profiling information was examined, including information on 750 small molecule drugs, 33 types of cancer, and 24 types of immune cells. We focused on POFUT2's function and applied GSVA (Gene Set Variation Analysis) to calculate the FUT score. Survival and cancer pathways were found to be correlated with this score. After deriving a signature via univariate Cox and LASSO regression, we generated and analyzed the ROC curve and developed a nomogram.ResultsOur comprehensive analysis revealed epigenetic, genomic, and immunogenomic changes in FUTs, particularly POFUT2, resulting in aberrant expression. Elevated frequencies of CNV (Copy number variation), SNV (Single Nucleotide Variant), and hypermethylation were observed in FUTs. Additionally, the survival of patients with various types of cancers may be predicted by FUT expression. Immune response and prognosis in numerous types of cancer were found to be strongly linked to aberrant POFUT2 expression. Pathway analysis unveiled the role of FUTs in apoptosis, epithelial-to-mesenchymal transition (EMT), cell cycle, DNA damage response, RAS/MAPK, TSC/mTOR, PI3K/AKT, AR, ER, and RTK. A prognostic index for patients diagnosed with adrenocortical carcinoma (ACC) was established by applying a risk model incorporating nine FUTs and based on the findings of the GSVA.ConclusionsFUTs, particularly POFUT2, emerge as candidate targets for improving the outcomes of immune therapy. The significance of aberrant MUC12 expression, cancer immune therapy, and patient survival in the context of diverse malignancies is enhanced by the strong correlation observed among these factors. Our five-gene risk signature provides patients with ACC with an independent prognostic indicator, emphasizing the critical function of these genes in inhibiting the immune system's response in ACC.

Genotyping for Weak and Partial D Rh Status to Optimize Usage of Limited Blood Supplies

Abstract Introduction The Rh antigen group, expressed on erythrocytes and encoded by highly polymorphic RHD and RHCE genes on chromosome 1 (1p36-p34), is notable in transfusion medicine due to its high polymorphism, strong immunogenicity and ability to cause hemolytic transfusion reactions in patients and hemolytic disease of the fetus and newborn (HDFN). The D antigen specifically contains multiple epitopes with variations that include decreased expression (weak D) or loss of certain epitopes (partial D) which expose patients to risk of alloimmunization depending on the subtype of the Rh(D) antigen. Hence, serologic weak D testing followed by molecular testing is not performed except for donors testing negative for Rh(D) antigen and newborns typing negative for Rh(D) and born to Rh(D) negative mothers. Given the current shortages of blood supplies, understanding patients’ genotypes in the context of weak or partial D-typing and risk of alloimmunization can be essential in the management of increasingly limited supplies of red blood cell (RBC) units. Objective To further characterize patients that initially type negative for Rh(D) but positive for weak D with molecular testing to investigate risk of alloimmunization, retype the Rh(D) and avoid unnecessary use of Rh(D) negative RBC units during critical shortages of RBC units. Methods We collected the results of ABO/Rh(D) typing for patients with negative Rh(D) testing but positive weak D test over a period of four weeks in December 2023 and had D-variant molecular testing performed by a reference laboratory. Initial serologic ABO/Rh(D) was performed in tube followed by weak D testing by indirect Coombs test in tube. Results The total number of patients who initially tested negative for Rh(D) but positive (weak to +4) for weak-D was three patients. Two patients showed a probable genotype of RHD*01W.1 (RHD* weak type 1, 809T&amp;gt;G (V270G)) homozygous which indicated the presence of weak D+ type 1. Patients with weak D+ type 1 can receive Rh(D) positive red cell units, and they are not at risk of alloimmunization and developing anti-D. The third patient showed a probable genotype RHD*DVI type 2 homozygous (lack of Rh(D) exons 4 through 6) and indicated the presence of partial D antigen. Patients with partial D antigen should receive RH(D) negative red cell units as they are at risk of alloimmunization and developing anti-D. Conclusions Although the vast majority of Transfusion Medicine services do not perform the serologic weak D test with reflex to molecular testing, characterizing patients who test negative with initial serologic Rh(D) testing with serologic and molecular D-tests may aid in retyping the Rh(D) of patients during critical shortages, particularly for patients with ABO blood group O.

Effects of Pressure, Hypoxia, and Hyperoxia on Neutrophil Granulocytes

Background: The application of normo- and hyperbaric O2 is a common therapy option in various disease patterns. Thereby, the applied O2 affects the whole body, including the blood and its components. This study investigates influences of pressure and oxygen fraction on human blood plasma, nutrient media, and the functions of neutrophil granulocytes (PMNs). Methods: Neutrophil migration, reactive oxygen species (ROS) production, and NETosis were examined by live cell imaging. The treatment of various matrices (Roswell Park Memorial Institute 1640 medium, Dulbecco’s Modified Eagle’s Medium, H2O, human plasma, and isolated PMNs) with hyperbaric oxygen (HBO) was performed. In addition, the expression of different neutrophil surface epitopes (CD11b, CD62L, CD66b) and the oxidative burst were investigated by flow cytometry (FACS). The application of cold atmospheric plasma (CAP) to normoxic and normobaric culture media served as a positive control. Soluble reaction products such as H2O2, reactive nitrogen species (RNS: NO2− and NO3−), and ROS-dependent dihydrorhodamine oxidation were quantified by fluoro- and colorimetric assay kits. Results: Under normobaric normoxia, PMNs migrate slower and shorter in comparison with normobaric hyper- or hypoxic conditions and hyperbaric hyperoxia. The pressure component has less effect on the migration behavior of PMNs than the O2 concentration. Individual PMN cells produce prolonged ROS at normoxic conditions. PMNs showed increased expression of CD11b in normobaric normoxia, lower expression of CD62L in normobaric normoxia, and lower expression of CD66b after HBO and CAP treatment. Treatment with CAP increased the amount of ROS and RNS in common culture media. Conclusions: Hyperbaric and normobaric O2 influences neutrophil functionality and surface epitopes in a measurable way, which may have an impact on disorders with neutrophil involvement. In the context of hyperbaric experiments, especially high amounts of H2O2 in RPMI after hyperbaric oxygen should be taken into account. Therefore, our data support a critical indication for the use of normobaric and hyperbaric oxygen and conscientious and careful handling of oxygen in everyday clinical practice.

Characterization of a novel chicken γδ TCR-specific marker

Chickens are a species with a high number of γδ T cells in various tissues. Despite their abundance, γδ T cells are poorly characterized in chickens, partially due to a lack of specific reagents to characterize these cells. Up until now, the TCR1 clone has been the only γδ T cell-specific monoclonal antibody (mAb) in chickens and additional reagents for γδ T cell subsets are needed. In order to address this issue, new mAb were generated in our laboratory by immunizing mice with in vitro cultured γδ T cells. In an initial flow cytometric screen a new mAb, clone “8D2”, displayed an interesting staining pattern that mirrored γδ TCR up- and downregulation in the γδ T cell line D4 over time, prompting us to characterize this antibody further. We compared the expression of the unknown 8D2 epitope in combination with TCR1 staining across various primary cells. In splenocytes, peripheral blood lymphocytes and intestinal epithelial cells, 8D2 consistently labeled a subset of TCR1+ cells. To determine, whether specific γδ T cell receptors were recognized by 8D2, we sorted γδ T cells according to their 8D2 and TCR1 expression and analyzed their TCR V(D)J gene usage by TCR profiling. Strikingly, sorted 8D2+ cells preferentially expressed Vγ3 genes, whereas the TCR Vγ genes used by TCR1+ 8D2- cells were more variable. γδ TCR in 8D2+ cells were most frequently comprised of gamma chain VJ genes TRGV3-8 and TRGJ3, and delta chain VDJ genes TRDV1-2, TRDD2, TRDJ1. To confirm binding of 8D2 to specific γδ TCR, the preferentially utilized combination of TRG and TRD was expressed in HEK293 cells in combination with CD3, demonstrating surface binding of the 8D2 mAb to this Vγ3 γδ TCR-expressing cell line. Conversely, HEK293 cells expressing either Vγ1 or Vγ2 TCR did not react with 8D2. In conclusion, 8D2 is a novel tool for identifying specific Vγ3 bearing γδ T cells.

Three novel Er blood group system alleles and insights from protein modeling.

The Er blood group system was recently shown to be defined by PIEZO1. The system consists of high prevalence antigens Era, Er3, ERSA, and ERAMA; and low prevalence antigen Erb. Era/Erb are antithetical with Er(a-b+) defined by the ER*B allele [c.7180G>A p.(Gly2394Ser)]. A nonsense variant c.5289C>G p.(Tyr1763*) is associated with a predicted Ernull phenotype, and a missense variant c.7174G>A p.(Glu2392Lys) in close proximity to p.2394 causes loss of both Era and Erb expression. We investigated PIEZO1 in four Er(a-) individuals who presented with anti-Era. Whole genome sequencing (WGS) and Sanger sequencing were performed. The location and structural differences of predicted protein changes were visualized using the predicted 3-D structure of Piezo1 created using AlphaFold2. One individual was homozygous for the reported ER*B. A second had a novel heterozygous nonsense variant c.3331C>T p.(Gln1111*), but a second allelic variant was not found. In the remaining two individuals, two different heterozygous novel missense variants, c.7184C>T p.(Ala2395Val) or c.7195G>A p.(Gly2399Ser), were in trans to the reported c.7180G>A variant, ER*B. AlphaFold2 protein modeling showed that each of the missense variants is predicted to encode an altered structural conformation near Era and Erb. Investigation of archived samples resulted in the identification of three novel PIEZO1 alleles including a predicted Ernull and two missense variants. Structural modeling suggests that the missense changes potentially alter Era/Erb epitope expression with p.2399Ser resulting in a small increase in the negative electrostatic potential.

Platelet integrin αIIbβ3 plays a key role in venous thrombogenesis in a mouse model.

Venous thrombosis (VT) is a common vascular disease associated with reduced survival and a high recurrence rate. Previous studies have shown that the accumulation of platelets and neutrophils at sites of endothelial cell activation is a primary event in VT, but a role for platelet αIIbβ3 in the initiation of venous thrombosis has not been established. This task has been complicated by the increased bleeding linked to partial agonism of current αIIbβ3 inhibitory drugs such as tirofiban (Aggrastat ® ). Here, we show that m-tirofiban, an engineered version of tirofiban, is not a partial agonist of αIIbβ3. This is based on its cryo-EM structure in complex with human full-length αIIbβ3 and its inability to increase expression of an activation-sensitive epitope on platelet αIIbβ3. m-tirofiban abolished agonist-induced platelet aggregation ex vivo at concentrations that preserved clot retraction and markedly suppressed the accumulation of platelets, neutrophils, and fibrin on thrombin-activated endothelium in real-time using intravital microscopy in a mouse model of venous thrombogenesis. Unlike tirofiban, however, m-tirofiban did not increase bleeding at the thrombosis-inhibitory dose. These findings establish a key role for αIIbβ3 in the initiation of VT, provide a guiding principle for designing potentially safer inhibitors for other integrins, and suggest that pure antagonists of αIIbβ3 like m-tirofiban merit further consideration as potential thromboprophylaxis agents in patients at high-risk for VT and hemorrhage.

Some determinantal representations of derangement polynomials of types B and D

Chow (2024) recently computed expressions of the types B and D derangement polynomials dnB(q)=∑σ∈DnBqfmaj(σ) and dnD(q)=∑σ∈DnDqmaj(σ) as tridiagonal and lower Hessenberg determinants of order n. Qi, Wang, and Guo (2016), based on a determinantal formula for the nth derivative of a quotient of two functions, derived an expression of the classical derangement number dn=n!∑k=0n(−1)k/k! as a tridiagonal determinant of order n+1. By q-extending the approach of Qi et al., we present in this work yet another determinantal expressions of dnB(q) and dnD(q) as determinants of order n+1.

CRPA1A2: A novel TCR-based T-cell engager targeting MAGEA1-positive solid tumors.

e14589 Background: MAGEA1, identified as a cancer/testis antigen, is highly expression in various solid tumors, including hepatocellular carcinoma, non-small cell lung cancer, but absent in normal tissues except male germ cells and placenta, both of which lacking HLA class molecules. This selective expression of HLA-MAGEA1 epitope makes it a promising target for cancer therapy. However, its intracellular localization poses a challenge for conventional antibodies designed to target surface-expressed antigens. TCR-based T-cell engagers, such as Tebentafusp, overcome this hurdle by targeting intracellular protein-derived peptides presented on cancer cell surfaces via HLA, enabling precise cytotoxic T cell attacks. Notably, Tebentafusp has shown significant clinical benefits by improving survival rates in metastatic uveal melanoma, underscoring the efficacy of TCR-based T-cell engagers in treating MAGEA1-positive tumors. Methods: We have developed an advanced structural format, CR62, consisting of a anti-CD3 single chain fragment, a soluble modified TCR, and an Fc region. Additionally, the use of CorreGene's SMART-TCR system has facilitated the acquisition of TCRs with high affinities in the pico-molar range. By leveraging CR62 and a high-affinity TCR, we have conceptualized the TCR-based T-cell engager, CRPA1A2. Comprehensive preclinical assessments have been conducted to evaluate CRPA1A2's safety and efficacy. Results: CRPA1A2 is designed to specifically target the HLA-A*02:01-restricted MAGEA1 epitope, effectively inhibiting tumor growth. CRPA1A2 is characterized by its exceptional soluble expression in CHO cells, minimal homodimer miss-assembly and simplifying purification. It is strategically engineered with a high-affinity TCR and a low-affinity anti-CD3 scFv to optimize the efficacy and safety. In vitro studies, CRPA1A2 showed strong ability to eliminate tumor cells and induce IFN-γ release, effective even at low MAGEA1 epitope densities, with a therapeutic window that spans over three orders of magnitude, suggesting a 1000-fold greater activity against antigen-positive cancer cells compared to antigen-negative cells. In vivo, using a tumor-bearing mouse model, CRPA1A2 has been shown to recruit T cells into subcutaneous solid tumors, expand CD8+ T cell populations, and significantly reduce tumor mass. Notably, CRPA1A2 has an extended half-life of 120 hours and demonstrates preferential accumulation in tumor tissues, thereby enhancing its therapeutic potential. Moreover, the safety assessments for CRPA1A2 highlight its exceptional specificity, with in vitro studies showing no evidence of off-target toxicity, on-target off-tumor toxicity, or alloreactivity and favorable tolerance in mice during in vivo studies. Conclusions: CRPA1A2 has emerged as a superior TCR-based T-cell engager with exceptional antitumor efficacy and promising safety profile.

Display of porcine epidemic diarrhea virus spike protein B-cell linear epitope on Lactobacillus mucosae G01 S-layer surface induce a robust immunogenicity in mice

The Porcine epidemic diarrhea virus (PEDV) presents a substantial risk to the domestic pig industry, resulting in extensive and fatal viral diarrhea among piglets. Recognizing the mucosal stimulation triggered by PEDV and harnessing the regulatory impact of lactobacilli on intestinal function, we have developed a lactobacillus-based vaccine that is carefully designed to elicit a strong mucosal immune response. Through bioinformatics analysis, we examined PEDV S proteins to identify B-cell linear epitopes that meet the criteria of being non-toxic, soluble, antigenic, and capable of neutralizing the virus. In this study, a genetically modified strain of Lactobacillus mucosae G01 (L.mucosae G01) was created by utilizing the S layer protein (SLP) as a scaffold for surface presentation. Chimeric immunodominant epitopes with neutralizing activity were incorporated at various sites on SLP. The successful expression of SLP chimeric immunodominant epitope 1 on the surface of L.mucosae G01 was confirmed through indirect immunofluorescence and transmission electron microscopy, revealing the formation of a transparent membrane. The findings demonstrate that the oral administration of L.mucosae G01, which expresses the SLP chimeric immunodominant gene epitope1, induces the production of secreted IgA in the intestine and feces of mice. Additionally, there is an elevation in IgG levels in the serum. Moreover, the levels of cytokines IL-2, IL-4, IFN-γ, and IL-17 are significantly increased compared to the negative control group. These results suggest that L. mucosae G01 has the ability to deliver exogenous antigens and elicit a specific mucosal immune response against PEDV. This investigation presents new possibilities for immunoprophylaxis against PEDV-induced diarrhea.

MODELS OF HLA-DPB1 PERMISSIVENESS AND UNRELATED DONOR HEMATOPOIETIC CELL TRANSPLANTATION

In current allo-HCT practice, a fully HLA-matched sibling donor is the best donor associated with improved transplant outcomes. When a matched sibling donor is unavailable, the second best available donor option is a matched unrelated donor (MUD), either HLA 8/8 or HLA 10/10. One notable characteristic in the MUD setting is that HLA-DPB1 mismatches are present in around 80/85% of unrelated donor/recipient pairs. This unique feature has an additional layer of complexity as these HLA-DPB1 incompatibilities may be further divided into permissive and non-permissive mismatches by two biological-driven permissiveness models, namely T-cell epitope (TCE) and DP expression. In the current review article, we described the basics of T-cell allorecognition, the unique HLA-DPB1 immunogenetics, the early conflicting results regarding HLA-DPB1 mismatching in allo-HCT, the development and the clinical impact of T-cell epitope and Expression models, the new indirect allorecognition algorithm of HLA-DPB1 permissiveness (PIRCHE model), the role of HLA-DPB1 in nonmalignant disease setting, and future perspectives on HLA-DPB1 permissiveness. Keywords: HLA-DPB1, Permissive mismatch, Unrelated donor

GENETIC VARIANCE IN HEPARAN SULFATION IS ASSOCIATED WITH SALT-SENSITIVITY: A GENE-ENVIRONMENT ANALYSIS

Objective: The high heritability of salt sensitivity suggests an essential role for genetic variants in the relationship between sodium intake and blood pressure (BP). Candidate gene studies have mainly focused on renal and vascular pathways, but the role of glycosaminoglycan (GAG) genes, which are crucial for salinity tolerance, remains to be elucidated. Design and method: Interactions between 54,126 variants in 130 GAG genes and daily sodium excretion on BP were explored in 20,420 EPIC-Norfolk subjects. UK Biobank (n=414,132) and the multi-ethnic HELIUS study (n=2,239) were used for validation. In HELIUS sodium intake was estimated with Food Frequency Questionnaires (FFQ). Urinary GAG composition in 57 Dutch origin HELIUS participants stratified by genotype were analyzed to assess the association between SNP and GAG difference. Also, urinary GAG composition in 12 healthy volunteers after a sodium intervention (7-day low (50 mmol Na+/day) and 7-day high (200 mmol Na+/day) diet) were studied to assess if differences in genotype are associated with different GAG expression. Results: The intron variant rs2892799, mapped to NDST3, showed the strongest interaction with sodium on mean arterial pressure (MAP) (FDR 0.03), and the intron variant rs9654628 mapped to HS3ST5 with sodium on systolic BP (FDR 0.03). These interactions were multi-ethnically validated (Fig 1A, B). When stratified for rs2892799 genotype, T-allele carriers had a significantly higher urinary expression of N-sulfated heparan sulfate epitope D0S0 (Fig1C). Conversely, upon dietary salt loading, urinary D0S0 expression differed substantially between salt-sensitive and salt-resistant individuals (Fig1D). Sodium-induced effects on this epitope were opposite in salt-sensitive in salt-resistant individuals. Conclusions: Genetic variants associated with NDST3 and HS3ST5 interact with relation between sodium consumption and BP. rs2892799 (NDST3) genotype results in a different expression of the N-sulfated D0S0 epitope. In turn, D0S0 expression is associated with salt-mediated BP increase, indicating that genetic GAG variation and metabolic GAG adaptation to a high salt environment might be important for human BP regulation.

Inequalities for totally nonnegative matrices: Gantmacher–Krein, Karlin, and Laplace

A real linear combination of products of minors which is nonnegative over all totally nonnegative (TN) matrices is called a determinantal inequality for these matrices. It is referred to as multiplicative when it compares two collections of products of minors and additive otherwise. Set theoretic operations preserving the class of TN matrices naturally translate into operations preserving determinantal inequalities in this class. We introduce index-row (and index-column) operations that act directly on all determinantal inequalities for TN matrices, and yield further inequalities for these matrices. These operations assist in revealing novel additive inequalities for TN matrices embedded in the classical identities due to Laplace [Mem. Acad. Sciences Paris 1772] and Karlin (1968). In particular, for any square TN matrix A, these derived inequalities generalize – to every ith row of A and jth column of adjA – the classical Gantmacher–Krein fluctuating inequalities (1941) for i=j=1. Furthermore, our index-row/column operations reveal additional undiscovered fluctuating inequalities for TN matrices.The introduced index-row/column operations naturally birth an algorithm that can detect certain determinantal expressions that do not form an inequality for TN matrices. However, the algorithm completely characterizes the multiplicative inequalities comparing products of pairs of minors. Moreover, the underlying index-row/column operations add that these inequalities are offshoots of certain “complementary/higher” ones. These novel results seem very natural, and in addition thoroughly describe and enrich the classification of these multiplicative inequalities due to Fallat–Gekhtman–Johnson [Adv. Appl. Math. 2003] and later Skandera [J. Algebraic Comb. 2004] (and revisited by Rhoades–Skandera [Ann. Comb. 2005, J. Algebra 2006]).

Risk prediction and function evaluation by T-cell epitope model and expression model of HLA-DPB1 mismatching in unrelated-donor hematopoietic stem cell transplantations

Objective: To evaluate the risk prediction and assessment function of HLA-DPB1 T-cell epitope (TCE) model and expression model in human leukocyte antigen (HLA)-matched unrelated hematopoietic stem cell transplantation (MUD-HSCT) with HLA-DPB1 mismatching. Methods: A total of 364 (182 pairs) potential MUD-HSCT donors and recipients confirmed by HLA high-resolution typing in Shaanxi Blood Center from 2016 to 2019 were analyzed retrospectively. Of the 182 recipients, there were 121 males and 61 females with an average age of (26.3±14.2) years. Of the 182 donors, there were 148 males and 34 females with an average age of (33.7±7.5) years. Polymerase chain reaction-sequence-based typing (PCR-SBT), next-generation sequencing (NGS) and polymerase chain reaction-sequence specific oligonucleotide probe (PCR-SSO) based on LABScan®3D platform were used for high-resolution typing of HLA-A, B, C, DRB1, DQB1, DPB1 gene, and PCR-SBT was used for single nucleotide polymorphism (SNP) typing. TCE model and expression model were used to predict and evaluate the HLA-DPB1 mismatch pattern and acute graft-versus-host-disease (aGVHD) risk. Results: A total of 26 HLA-DPB1 alleles and their 3'-UTR rs9277534 SNP genotypes were detected in this study population, and two new alleles HLA-DPB1*1052∶01 and HLA-DPB1*1119∶01 were found and officially named. The overall mismatch rate of HLA-DPB1 in MUD-HSCT donors and recipients was 90.66% (165/182). In TCE model, the HLA-DPB1 mismatch rates of permissible mismatch (PM) and non-permissible mismatch (non-PM) were 47.80% (87/182) and 42.86% (78/182), respectively. The non-PM in GvH direction was 13.73% (25/182), and which in HvG direction was 29.12% (53/182). A total of 73 pairs of donors and recipients in TCE model met the evaluation criteria of expression model. Among of TCE PM group, recipient DP5 mismatches accounted for 34.25% (25/73) were predicted as aGVHD high risk according to expression model. For the TCE non-PM group, both the recipient DP2 mismatches of 6.85% (5/73) and recipient DP5 mismatches of 10.86% (8/73) were predicted to be at high risk for aGVHD. Risk prediction by TCE model and expression model was 27.27% concordant and 16.97% unconcordant. Conclusions: TCE model and expression model are effective tools to predict aGVHD risk of MUD-HSCT. Comprehensive application of the two models is helpful to the hierarchical assessment of HSCT risk.

Correlations between components of the immune system

Background No evidence of the possibility of altering a constituent of the immune system without directly affecting one of its associated components has yet been shown. Methods A schematic model was developed in which two triggers, fasting and splenectomy, were studied for their ability to affect the expression of cell membrane epitopes and the cytokine secretion of out-of-body autogeneic and syngeneic lymphocytes. Results The effect of fasting and/or splenectomy on promoting correlations between immune systems was studied by determining the alterations in expressions of cell membrane epitopes and in cytokine secretion by out-of-body autogeneic and syngeneic lymphocytes. The effect of fasting as a trigger decreased expression of CD8 and CD25 and increased TNFα levels. The effect of splenectomy as a trigger was investigated in non-fasting mice by comparing splenectomized and non-splenectomized mice. An increase in the CD8 expression and in TNFα, IFNg, and IL10 secretion was noted. The effect of splenectomy as a trigger in fasting mice was determined by comparing splenectomized and non-splenectomized mice. Splenectomy significantly affected the expression of CD25 and CD4 CD25 and on secretion of TNFα, IFNg, and IL10. To determine the effect of keeping the cells in an out-of-body location on the expression of lymphocyte epitopes, tubes kept on top of the cages of the fasting mice were compared with tubes kept on top of empty cages. The results showed a significant change in the CD8 expression was noted. To determine the effect of keeping cells in an out-of-body location on cytokine secretion, tubes kept on cages were tested for cytokine levels significant decrease was noted in the secretion of TNFα and IFNg. Conclusions The study showed that a mouse could affect cells at a distance and alter the expression of surface markers and cytokine secretion following two types of triggers: fasting and/or splenectomy. The data characterized a system for the induction of correlations between two’s immune system components without a transfer of mediators. It suggests that an out-of-body correlation can be induced between two components of the immune system.

Identification of novel blood-based extracellular vesicles biomarker candidates with potential specificity for traumatic brain injury in polytrauma patients.

The goal of this study was to identify changes in extracellular vesicles (EV) surface proteins specific to traumatic brain injury (TBI), which could be used as a diagnostic and prognostic tool in polytrauma patients. Known serum TBI-specific biomarkers (S100B, NSE, and GFAP), which can predict the severity and outcome of isolated TBI, lose their predictive value in the presence of additional extracranial injuries. Extracellular vesicles (EVs) are released from cells in response to various stimuli and carry specific cargo/surface molecules that could be used for tracking injury-responding cells. EVs were isolated using size exclusion chromatography (SEC) from the plasma of two groups of patients (with isolated TBI, ISS≥16, AIShead≥4, n=10; and polytraumatized patients without TBI ISS≥16, AIShead=0, n=10) collected in the emergency room and 48h after trauma. EVs' surface epitope expression was investigated using a neurospecific multiplex flow cytometry assay and compared with healthy controls (n=10). Three enrichments of EV epitopes found to be specific to TBI were validated by western blot. The expression of 10 EV epitopes differed significantly among the patient and control groups, and five of these epitopes (CD13, CD196, MOG, CD133, and MBP) were TBI-specific. The increased expression of CD196, CD13, and MOG-positive EVs was validated by western blot. Our data showed that TBI is characterized by a significant increase of CD13, CD196, MOG, CD133, and MBP-positive EVs in patients' plasma. A high level of MOG-positive EVs negatively correlated with the Glasgow Coma Scale score at admission and could be an indicator of poor neurological status.

Chagasin from Trypanosoma cruzi as a molecular scaffold to express epitopes of TSA-1 as soluble recombinant chimeras

Trypanosoma cruzi is the causative agent of Chagas disease, a global public health problem. New therapeutic drugs and biologics are needed. The TSA-1 recombinant protein of T. cruzi is one such promising antigen for developing a therapeutic vaccine. However, it is overexpressed in E. coli as inclusion bodies, requiring an additional refolding step. As an alternative, in this study, we propose the endogenous cysteine protease inhibitor chagasin as a molecular scaffold to generate chimeric proteins. These proteins will contain combinations of two of the five conserved epitopes (E1 to E5) of TSA-1 in the L4 and L6 chagasin loops. Twenty chimeras (Q1-Q20) were designed, and their solubility was predicted using bioinformatics tools. Nine chimeras with different degrees of solubility were selected and expressed in E. coli BL21 (DE3). Western blot assays with anti-6x-His and anti-chagasin antibodies confirmed the expression of soluble recombinant chimeras. Both theoretically and experimentally, the Q12 (E5-E3) chimera was the most soluble, and the Q20 (E4-E5) the most insoluble protein. Q4 (E5-E1) and Q8 (E5-E2) chimeras were classified as proteins with medium solubility that exhibited the highest yield in the soluble fraction. Notably, Q4 has a yield of 239 mg/L, well above the yield of recombinant chagasin (16.5 mg/L) expressed in a soluble form. The expression of the Q4 chimera was scaled up to a 7 L fermenter obtaining a yield of 490 mg/L. These data show that chagasin can serve as a molecular scaffold for the expression of TSA-1 epitopes in the form of soluble chimeras.

A tumor-restricted glycoform of podocalyxin is a highly selective marker of immunologically cold high-grade serous ovarian carcinoma.

Targeted-immunotherapies such as antibody-drug conjugates (ADC), chimeric antigen receptor (CAR) T cells or bispecific T-cell engagers (eg, BiTE®) all aim to improve cancer treatment by directly targeting cancer cells while sparing healthy tissues. Success of these therapies requires tumor antigens that are abundantly expressed and, ideally, tumor specific. The CD34-related stem cell sialomucin, podocalyxin (PODXL), is a promising target as it is overexpressed on a variety of tumor types and its expression is consistently linked to poor prognosis. However, PODXL is also expressed in healthy tissues including kidney podocytes and endothelia. To circumvent this potential pitfall, we developed an antibody, named PODO447, that selectively targets a tumor-associated glycoform of PODXL. This tumor glycoepitope is expressed by 65% of high-grade serous ovarian carcinoma (HGSOC) tumors. In this study we characterize these PODO447-expressing tumors as a distinct subset of HGSOC using four different patient cohorts that include pre-chemotherapy, post-neoadjuvant chemotherapy (NACT) and relapsing tumors as well as tumors from various peritoneal locations. We find that the PODO447 epitope expression is similar across tumor locations and negligibly impacted by chemotherapy. Invariably, tumors with high levels of the PODO447 epitope lack infiltrating CD8+ T cells and CD20+ B cells/plasma cells, an immune phenotype consistently associated with poor outcome. We conclude that the PODO447 glycoepitope is an excellent biomarker of immune "cold" tumors and a candidate for the development of targeted-therapies for these hard-to-treat cancers.

Tau maturation in clinicopathological spectrum of Lewy body and Alzheimer’s disease

AbstractBackgroundIn Alzheimer’s disease neuropathologic change (ADNC), maturation of neurofibrillary tangles and tau pathology are characterized by posttranslational modifications of tau. ANDC and Lewy body disease (LBD) commonly co‐exist in aging brains and there are overlapping clinical syndromes and heterogeneity in clinical features among cases with mixed ADNC and LBD pathology. Given this clinical and pathological overlap, we examined the postmortem expression of tau conformational epitopes that mark various stages of tangle maturation in clinicopathologic spectrum of Alzheimer’s disease (AD) and LBD.MethodAutopsy‐confirmed cases with diffuse neocortical LBD with/without ADNC (n = 59) were selected (Table1). We used digital histology methods to measure percent area occupied (%AO) with pathology in three regions (middle frontal, anterior cingulate, and superior temporal cortices) immunohistochemically stained with MJF‐R13 (α‐synuclein), NAB228 (beta‐amyloid), and tau monoclonal antibodies marking phosphorylated (AT8), early conformational (MC1, preferentially reactive in pre‐tangles), and C‐terminally truncated tau epitopes (TauC3, preferentially reactive in mature intracellular and ghost tangles). Linear models tested the group comparisons, and correlation between α‐synuclein or beta‐amyloid with outcome of average neocortical tau. In models, %AO were log‐transformed, and age and sex were included as covariates.Resultα‐synuclein was strongly correlated with early tau epitopes labelled with AT8 (β = 1.37, p&lt;0.001) and MC1 (β = 1.20, p&lt;0.001), but not with mature tau epitopes detected by TauC3 (p = 0.38). There were significant positive correlations between all tau epitopes and beta‐amyloid, including TauC3 immunoreactive tau (p≤0.013) (Figure1). In the mixed pathology group, those with clinical LBD had a positive association between α‐synuclein and MC1 (β = 1.61, p = 0.014) and AT8 (β = 1.67, p = 0.005) positive tau, while those with clinical AD had no correlation between α‐synuclein and tau epitopes. There was greater MC1 (β = 3.40, p&lt;0.001), AT8 (β = 3.59, p&lt;0.001), and TauC3 (β = 1.43, p = 0.005) pathology in patients with clinical AD compared to clinical LBD.ConclusionIn LBD, higher α‐synuclein is associated with greater phosphorylated and early tau conformations, but not later conformation of mature tau, which was selectively associated with beta‐amyloid pathology. Our findings suggest that tau pathology may modify the clinical expression of synucleinopathies and C‐terminal truncation of tau may be more closely related to AD pathophysiology than synucleinopathy.

Differential CD38 Expression on Daratumumab-Naïve Vs. Resistant Multiple Myeloma - a Direct Stochastic Optical Reconstruction Microscopy ( dSTORM) Analysis in 53 Patients

Background: The anti-CD38 antibody Daratumumab (Dara) has become the new standard of care for the treatment of newly-diagnosed and relapsed/ refractory multiple myeloma (MM). At the same time, Dara resistance remains poorly understood. Potential resistance mechanisms have been proposed and range from intrinsic downregulation in CD38 dim MM cells to genetic alterations and splicing variants which may affect CD38 epitope expression (Adamia, ASH 2022). Here, we used super-resolution microscopy for high-precision quantification of CD38 in MM and piloted this approach in a large cohort of 53 MM patients with vs. without Dara resistance. Methods: Direct stochastic optical reconstruction microscopy ( dSTORM) is a novel imaging modality based on photoswitching of certain fluorophores coupled to antibodies for molecular representation of surface molecules at unprecedented levels of resolution (&amp;lt;20 nM). To correlate CD38 expression data with clinical outcome, we divided our cohort based on treatment status into two groups: Dara-naïve (n = 31) vs. Dara-resistant (n = 22) patients. Imaging data was complemented by phenotypic assessment using a well-established multi-epitope anti-CD38 antibody (Cytognos), as per standard-of-care. Results: Mean CD38 receptor density as measured with Dara via dSTORM was significantly higher in Dara-naïve (of 48.81 ± 29.65 receptors/µm²) vs. Dara-resistant patients (15.52 ± 3.9 receptors/µm², p = 0.016). This finding was corroborated by multi-epitope phenotypic analysis which revealed mean CD38 receptor expression levels of 32.09 ± 13.14 receptors/µm² in Dara-naïve vs. 21.65 ± 17.73 receptors/µm² in Dara-resistant patients ( p = 0.05). Lower CD38 expression via dSTORM vs. multi-epitope anti-CD38 staining was consistently observed in our cohort and may reflect ongoing blockage of the receptor binding site by Dara in cases with shorter time to last Dara application (median 53 days, range 1-189 days). As there was no correlation with the interval between Dara application and sampling however, splice-site mutations affecting CD38 receptor accessibility as proposed by Adamia et al. may depict a more relevant mechanism of resistance in our Dara-resistant cohort. In a next step, we therefore investigated alternative epitope binding in Dara-resistant patients. To this end, we investigated antibody binding by dSTORM imaging for isatuximab (Isa), an alternative CD38-directed monoclonal antibody that binds to a location in the C-terminal portion of the CD38 extracellular domain which is entirely different from the Dara epitope. Interestingly, there was a trend for increased detection rate with Isa for mean CD38 receptor expression in Dara-resistant patients (22.2 ± 4.6 receptors/µm²) vs. Dara (15.5 ± 3.9 receptors/µm², p = 0.1) and on par with CD38 receptor expression. No data was available to assess Dara binding in Isa pre-exposed patients. Conclusion: Innovative microscopy using dSTORM allows for high-resolution epitope quantification at unprecedented granularity. While this finding enables visual proof of differential epitope binding of Dara vs. Isa on CD38 in a clinically feasible setting, correlation studies to determine the clinical relevance of this observation remain ongoing.