Immunoglobulin Receptor Research Articles

Abstract IA017: Spontaneous humoral responses in endometrial cancer provide a rationale for novel tumor-penetrating therapeutic antibodies

Abstract The role of humoral responses in endometrial cancer remains insufficiently investigated. Using a cohort of 107 patients with different histological subtypes of endometrial carcinoma, we found that concomitant accumulation of T, B, and plasma cells at tumor beds predicted better survival. However, only B cell markers corresponded with prolonged survival specifically in high-grade endometrioid type and serous tumors. Immune protection was associated with class-switched IgA and, to a lesser extent, IgG. Expression of polymeric immunoglobulin receptor (PIGR) by tumor cells allow dimeric IgA transcytosis. Taking advantage of tumor cell penetration, we demonstrated that mutation-specific dIgA, but not IgG, neutralized KRASG12D inside carcinoma cells and expelled this oncodriver (but not wild-type KRAS) outside tumor cells, abrogating tumor cell proliferation in an antigen-specific manner. Accordingly, KRASG12D-specific dIgA1, but not the same antibody on an IgG backbone, abrogated KRASG12D carcinoma growth in vivo, more effectively than small molecule KRASG12D inhibitors, including in syngeneic tumor-bearing immunocompetent mice. Because >6% of endometrial cancers exhibit KRAS mutations, our results provide a rationale for developing PIGR-targeting tumor cell-penetrating antibodies to effectively target intracellular oncogenes, which could be applicable to many aggressive and frequent human cancers. Citation Format: Jose R. Conejo-Garcia. Spontaneous humoral responses in endometrial cancer provide a rationale for novel tumor-penetrating therapeutic antibodies [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr IA017.

Sperm traits and seminal plasma proteome of locally adapted hairy rams subjected to intermittent scrotal insulation

The present study evaluated the effects of heat stress on reproductive parameters of hairy rams. Six animals were subjected to scrotal insulation during four consecutive nights (6 PM – 6 AM). Day (D) 0 was the first day of insulation. Scrotal circumference increased from 30.5 ± 0.3 cm (at pre-insulation) to 31.8 ± 0.4 cm on D4, decreased 3.9 cm on D28, returning to 30.6 ± 0.6 cm on D57. Sperm concentration decreased from 3.7 ± 0.12 ×109 sperm/mL before insulation to 2.6 ± 0.1 ×109 on D23, returning to normal on D57. Sperm motility averaged 75 ± 2.9% before insulation, was undetectable on D23, and became normal on D77. Sperm with normal morphology reached 5.9 ± 2.6% on D35 but recovered (86.8 ± 2.1%) on D91. Sperm DNA integrity decreased from 86.5 ± 4.7% before insulation to 11.1 ± 3.7% on D63, returning to pre-insulation values on D120. Sperm BSP immunostaining was reduced after scrotal insulation. Variations in seminal protein abundances coincided with changes in sperm parameters. Seminal plasma superoxide dismutase, carboxypeptidase Q-precursor and NPC intracellular cholesterol transporter 2 decreased on D18, returning to normal after D28. Albumin, inhibitor of carbonic anhydrase precursor, EGF-like repeat and discoid I-like domain-containing protein 3 and polymeric immunoglobulin receptor increased after insulation. In summary, intermittent scrotal insulation drastically altered ram sperm attributes and seminal proteins, especially those associated with oxidative stress. Knowledge of animal´s response to thermal stress is vital in the scenario of climate changes.

Targeted Single Particle Tracking with Upconverting Nanoparticles.

Single particle tracking (SPT) is a powerful technique for real-time microscopic visualization of the movement of individual biomolecules within or on the surface of living cells. However, SPT often suffers from the suboptimal performance of the photon-emitting labels used to tag the biomolecules of interest. For example, fluorescent dyes have poor photostability, while quantum dots suffer from blinking that hampers track acquisition and interpretation. Upconverting nanoparticles (UCNPs) have recently emerged as a promising anti-Stokes luminescent label for SPT. In this work, we demonstrated targeted SPT using UCNPs. For this, we synthesized 30 nm diameter doped UCNPs and coated them with amphiphilic polymers decorated with polyethylene glycol chains to make them water-dispersible and minimize their nonspecific interactions with cells. Coated UCNPs highly homogeneous in brightness (as confirmed by a single particle investigation) were functionalized by immunoglobulin E (IgE) using a biotin-streptavidin strategy. Using these IgE-UCNP SPT labels, we tracked high-affinity IgE receptors (FcεRI) on the membrane of living RBL-2H3 mast cells at 37 °C in the presence and absence of antigen and obtained good agreement with the literature. Moreover, we used the FcεRI-IgE receptor-antibody system to directly compare the performance of UCNP-based SPT labels to organic dyes (AlexaFluor647) and quantum dots (QD655). Due to their photostability as well as their backgroundless and continuous luminescence, SPT trajectories obtained with UCNP labels are no longer limited by the photophysics of the label but only by the dynamics of the system and, in particular, the movement of the label out of the field of view and/or focal plane.

LCN2 and ELANE overexpression induces sepsis.

Sepsis is a syndrome characterized by a systemic inflammatory response due to the invasion of pathogenic microorganisms. The relationship between Lipocalin-2 (LCN2), elastase, neutrophil expressed (ELANE) and sepsis remains unclear. The sepsis datasets GSE137340 and GSE154918 profiles were downloaded from gene expression omnibus generated from GPL10558. Batch normalization, differentially expressed Genes (DEGs) screening, weighted gene co-expression network analysis (WGCNA), functional enrichment analysis, Gene Set Enrichment Analysis (GSEA), immune infiltration analysis, construction and analysis of protein-protein interaction (PPI) networks, Comparative Toxicogenomics Database (CTD) analysis were performed. Gene expression heatmaps were generated. TargetScan was used to screen miRNAs of DEGs. 328 DEGs were identified. According to Gene Ontology (GO), in the Biological Process analysis, they were mainly enriched in immune response, apoptosis, inflammatory response, and immune response regulation signaling pathways. In cellular component analysis, they were mainly enriched in vesicles, cytoplasmic vesicles, and secretory granules. In Molecular Function analysis, they were mainly concentrated in hemoglobin binding, Toll-like receptor binding, immunoglobulin binding, and RAGE receptor binding. In Kyoto Encyclopedia of Genes and Genomes (KEGG), they were mainly enriched in NOD-like receptor signaling pathway, Toll-like receptor signaling pathway, TNF signaling pathway, P53 signaling pathway, and legionellosis. Seventeen modules were generated. The PPI network identified 4 core genes (MPO, ELANE, CTSG, LCN2). Gene expression heatmaps revealed that core genes (MPO, ELANE, CTSG, LCN2) were highly expressed in sepsis samples. CTD analysis found that MPO, ELANE, CTSG and LCN2 were associated with sepsis, peritonitis, meningitis, pneumonia, infection, and inflammation. LCN2 and ELANE are highly expressed in sepsis and may serve as molecular targets.

STAT3-Dependent Effects of Polymeric Immunoglobulin Receptor in Regulating Interleukin-17 Signaling and Preventing Autoimmune Hepatitis

One-third of patients with autoimmune hepatitis (AIH) have cirrhosis at the time of diagnosis. The relevance of these variables, although unknown, is believed to be critical in AIH because of suspected interactions between the gut microbiome and genetic factors. Dysbiosis of the gut flora and elevated polymeric immunoglobulin receptor (pIgR) levels have been observed in both patients and mouse models. Moreover, there is a direct relationship between pIgR expression and transaminase levels in patients with AIH. In this study, we aimed to explore how pIgR influences the secretion of regenerating islet-derived 3 beta (Reg3b) and the flora composition in AIH using in vivo experiments involving patients with AIH and a concanavalin A-induced mouse model of AIH. Reg3b expression was reduced in pIgR gene (Pigr)-knockout mice compared to that in wild-type mice, leading to increased microbiota disruption. Conversely, exogenous pIgR supplementation increased Reg3b expression and maintained microbiota homeostasis. RNA sequencing revealed the participation of the interleukin (IL)-17 signaling pathway in the regulation of Reg3b through pIgR. Furthermore, the introduction of external pIgR could not restore the imbalance in gut microbiota in AIH, and the decrease in Reg3b expression was not apparent following the inhibition of signal transducer and activator of transcription 3 (STAT3). In this study, pIgR facilitated the upregulation of Reg3b via the STAT3 pathway, which plays a crucial role in preserving the balance of the intestinal microbiota in AIH. Through this research, we discovered new molecular targets that can be used for the diagnosis and treatment of AIH.

Functional studies with IgM and IgA immunoglobulins: binding to pIgR, FcαμR, FcμR, and CDC activities.

IgMs are the first antibodies produced by the immune system upon encounter of a possible pathogen and are one of five antibody subclasses in humans. For IgG, the most intensively studied antibody class, the N-linked glycosylation site located in the Fc-domain is directly involved in high affinity binding to the respective receptors and initiation ofcorresponding immune response. IgM molecules have five N-glycosylation sites and one N-glycosylation site in the J-chain, which can be incorporated in IgM or IgA molecules. There is only limited knowledge available concerning the function of these N-glycosylations in IgMs. To address this question, we produced IgM molecules lacking a particular N-glycosylation site and tested these variants as well as IgA molecules for binding to the known receptors: the polymeric immunoglobulin receptor (pIgR), the dual receptor for IgA and IgM, FcαμR, and the specific receptor for IgM, FcμR. The single glycosylation sites did not show an impact on expression and multimerization, except for variant N402Q, which could not be expressed. In SPR measurements, no major impact on the binding to the receptors by particular glycosylation sites could be detected. In cellular assays, deglycosylated variants showed some alterations in induction of CDC activity. Most strikingly, we observed also binding of IgA to the FcμR in the same affinity range as IgM, suggesting that this might have a physiological role. To further substantiate the binding of IgA to FcμR we used IgA from different origins and were able to confirm binding of IgA preparations to the FcμR.

Simple methods for measuring milk exosomes using fluorescent compound GIF-2250/2276

Exosomes are small extracellular vesicles (EVs) found in culture supernatants, blood, and breast milk. The size of these nanocomplexes limits the methods of EV analyses. In this study, nitrobenzoxadiazole (NBD), a fluorophore, conjugated endosome-lysosome imager, GIF-2250 and its derivative, GIF-2276, were evaluated for exosome analyses. A correlation was established between GIF-2250 intensity and protein maker levels in bovine milk exosomes. We found that high-temperature sterilization milk may not contain intact exosomes. For precise analysis, we synthesized GIF-2276, which allows for the covalent attachment of NBD to the Lys residue of exosome proteins, and labeled milk exosomes were separated using a gel filtration system. GIF-2276 showed chromatographic peaks of milk exosomes containing >3 ng protein. The area (quantity) and retention time (size) of the exosome peaks were correlated to biological activity (NO synthesis suppression in RAW264.7 murine macrophages). Heat denaturation of purified milk-derived exosomes disrupted these indicators. Proteome analyses revealed GIF-2276-labeled immunomodulators, such as butyrophilin subfamily 1 member A1 and polymeric immunoglobulin receptor. The immunogenicity and quantity of these factors decreased by heat denaturation. When milk exosomes were purified from market-sourced milk we found that raw and low-temperature sterilization milk samples, contained exosomes (none in high-temperature sterilization milk). These results were also supported by transmission electron microscopy analyses. We also found that GIF-2276 could monitor exosome transportation into HEK293 cells. These results suggested that GIF-2250/2276 may be helpful to evaluate milk exosomes.

B-cell receptor immunoglobulin stereotypy in chronic lymphocytic leukemia: Key to understanding disease biology and stratifying patients

Sequence convergence, otherwise stereotypy, of B-cell receptor immunoglobulin (BcR IG) from unrelated patients is a distinctive feature of the IG gene repertoire in chronic lymphocytic leukemia (CLL) whereby patients expressing a particular BcR IG archetype are classified into groups termed stereotyped subsets. From a biological perspective, the fact that a considerable fraction (∼41%) of patients with CLL express (quasi)identical or stereotyped BcR IG underscores the key role of antigen selection in the natural history of CLL. From a clinical perspective, at odds with the pronounced heterogeneity of CLL at large, patients belonging to the same stereotyped subset display consistent clinical presentation and outcome, including response to treatment, likely as a reflection of consistent biological background. Many major stereotyped subsets were recently shown to have satellites, that is, smaller subsets that are immunogenetically similar. Preliminary evidence supports that this similarity extends to shared biological and even clinical features, with important implications for patient stratification. Consequently, BcR IG stereotypy emerges as a powerful tool for dissecting the heterogeneity of CLL toward refined risk stratification and, eventually, more precise therapeutic interventions.

Comparative Serum Proteome Profiling of Canine Benign Prostatic Hyperplasia before and after Castration.

BPH is the most prevalent prostatic condition in aging dogs. Nevertheless, clinical diagnosis and management remain inconsistent. This study employed in-solution digestion coupled with nano-liquid chromatography tandem mass spectrometry to assess serum proteome profiling of dogs with BPH and those dogs after castration. Male dogs were divided into two groups; control and BPH groups. In the BPH group, each dog was evaluated at two time points: Day 0 (BF subgroup) and Day 30 after castration (AT subgroup). In the BF subgroup, three proteins were significantly upregulated and associated with dihydrotestosterone: solute carrier family 5 member 5, tyrosine-protein kinase, and FRAT regulator of WNT signaling pathway 1. Additionally, the overexpression of polymeric immunoglobulin receptors in the BF subgroup hints at its potential as a novel protein linked to the BPH development process. Conversely, alpha-1-B glycoprotein (A1BG) displayed significant downregulation in the BF subgroup, suggesting A1BG's potential as a predictive protein for canine BPH. Finasteride was associated with increased proteins in the AT subgroup, including apolipoprotein C-I, apolipoprotein E, apolipoprotein A-II, TAO kinase 1, DnaJ homolog subfamily C member 16, PH domain and leucine-rich repeat protein phosphatase 1, neuregulin 1, and pseudopodium enriched atypical kinase 1. In conclusion, this pilot study highlighted alterations in various serum proteins in canine BPH, reflecting different pathological changes occurring in this condition. These proteins could be a source of potential non-invasive biomarkers for diagnosing this disease.

MYSM1 attenuates DNA damage signals triggered by physiologic and genotoxic DNA breaks

Patients with deleterious variants in MYSM1 have an immune deficiency characterized by B-cell lymphopenia, hypogammaglobulinemia, and increased radiosensitivity. MYSM1 is a histone deubiquitinase with established activity in regulating gene expression. MYSM1 also localizes to sites of DNA injury but its function in cellular responses to DNA breaks has not been elucidated. This study sought to determine the activity of MYSM1 in regulating DNA damage responses (DDRs) to DNA double-stranded breaks (DSBs) generated during immunoglobulin receptor gene (Ig) recombination and by ionizing radiation. MYSM1-deficient pre- and non-B cells were used to determine the role of MYSM1 in DSB generation, DSB repair, and termination of DDRs. Genetic testing in a newborn with abnormal screen for severe combined immune deficiency, T-cell lymphopenia, and near absence of B cells identified a novel splice variant in MYSM1 that results in nearly absent protein expression. Radiosensitivity testing in patient's peripheral blood lymphocytes showed constitutive γH2AX, a marker of DNA damage, in B cells in the absence of irradiation, suggesting a role for MYSM1 in response to DSBs generated during Ig recombination. Suppression of MYSM1 in pre-B cells did not alter generation or repair of Ig DSBs. Rather, loss of MYSM1 resulted in persistent DNA damage foci and prolonged DDR signaling. Loss of MYSM1 also led to protracted DDRs in U2OS cells with irradiation induced DSBs. MYSM1 regulates termination of DNA damage responses but does not function in DNA break generation and repair.

CD5L is a canonical component of circulatory IgM.

Immunoglobulin M (IgM) is an evolutionary conserved key component of humoral immunity, and the first antibody isotype to emerge during an immune response. IgM is a large (1 MDa), multimeric protein, for which both hexameric and pentameric structures have been described, the latter additionally containing a joining (J) chain. Using a combination of single-particle mass spectrometry and mass photometry, proteomics, and immunochemical assays, we here demonstrate that circulatory (serum) IgM exclusively exists as a complex of J-chain-containing pentamers covalently bound to the small (36 kDa) protein CD5 antigen-like (CD5L, also called apoptosis inhibitor of macrophage). In sharp contrast, secretory IgM in saliva and milk is principally devoid of CD5L. Unlike IgM itself, CD5L is not produced by B cells, implying that it associates with IgM in the extracellular space. We demonstrate that CD5L integration has functional implications, i.e., it diminishes IgM binding to two of its receptors, the FcαµR and the polymeric Immunoglobulin receptor. On the other hand, binding to FcµR as well as complement activation via C1q seem unaffected by CD5L integration. Taken together, we redefine the composition of circulatory IgM as a J-chain containing pentamer, always in complex with CD5L.

Leaves and pseudostems extract of Curcuma longa attenuates immunoglobulin E/bovine serum albumin-stimulated bone marrow-derived cultured mast cell activation and passive cutaneous anaphylaxis in BALB/c mice

Ethnopharmacological relevanceCurcuma longa, known as turmeric, is an herbaceous perennial plant belonging to the genus Curcuma. It is dispersed throughout tropical and subtropical regions worldwide. Since ancient times, turmeric has been used as an ethnomedicinal plant in the Ayurvedic system, particularly in Asian countries. Rhizomes of turmeric possess several pharmacological properties that give high value as a medicinal remedy for treating a range of conditions, including inflammation, pain, allergies, and digestive issues. Moreover, turmeric leaves and pseudostems also contain a variety of health-enhancing secondary metabolites, such as curcumin, flavonoids, and other phenolic compounds, which exhibit anti-inflammatory, antitumor, antibacterial, and antioxidant properties. Aim of the studyAllergic diseases are a group of immune-mediated disorders mainly caused by an immunoglobulin E (IgE)-dependent immunological response to an innocuous allergen. Therefore, this study aimed to investigate the effect of leaves and pseudostems extract of turmeric (TLSWE-8510) on IgE/bovine serum albumin (BSA)-stimulated allergic responses in mouse bone marrow-derived cultured mast cells (BMCMCs) and passive cutaneous anaphylaxis (PCA) in BALB/c mice. Materials and methodsThe effect of TLSWE-8510 on mast cell degranulation has been evaluated by investigating the release of β-hexosaminidase and histamine in IgE/BSA-stimulated BMCMCs. Additionally, anti-allergic properties of TLSWE-8510 on IgE/BSA-stimulated BMCMCs were investigated using suppression of nuclear factor-kappa B (NF-κB), and spleen tyrosine kinase (Syk)-linker for T-cell activation (LAT)-extracellular-signal-regulated kinase (ERK)-GRB2 associated binding protein 2 (Gab2) signaling pathway and downregulation of allergy-related cytokines and chemokines expression. Furthermore, in vivo, studies were conducted using IgE-mediated PCA in BALB/c mice. ResultsTLSWE-8510 treatment significantly inhibited the degranulation of IgE/BSA-stimulated BMCMCs by inhibiting the release of β-hexosaminidase and histamine dose-dependently. Additionally, TLSWE-8510 reduced the expression of high-affinity IgE receptors (Fc epsilon receptor I-FcεRI) on the surface of BMCMCs and the binding of IgE to FcεRI. Besides, the expression of cytokines and chemokines is triggered by IgE/BSA stimulation via activating the allergy-related signaling pathways. TLSWE-8510 dose-dependently downregulated the mRNA expression and the production of allergy-related cytokines (interleukin (IL)-1β, IL-3, IL-4, IL-5, IL-6, IL-13, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ), and chemokines (thymus and activation-regulated chemokine (TARC), and regulated upon activation, normal T cell expressed and secreted (RANTES)) by regulating the phosphorylation of downstream signaling molecules, NF-κB, and Syk, LAT, ERK and Gab2 in IgE/BSA-stimulated BMCMCs. Moreover, PCA reaction in IgE/BSA-stimulated BALB/c mice ears was effectively decreased by TLSWE-8510 treatment in a dose-dependent manner. ConclusionsThese results collectively demonstrated that TLSWE-8510 suppressed mast cell degranulation by inhibiting the release of chemical mediators related to allergies. TLSWE-8510 downregulated the allergy-related cytokines and chemokines expression and phosphorylation of downstream signaling molecules in IgE/BSA-stimulated BMCMCs. Furthermore, in vivo studies with IgE-mediated PCA reaction in the BALB/c mice ears were attenuated by TLSWE-8510 treatment. These findings revealed that TLSWE-8510 has the potential as a therapeutic agent for the treatment of allergic diseases.

Research progress of microglial surface receptors in perioperative neurocognitive disorders

AbstractPerioperative neurocognitive disorder (PND) is a common complication in the perioperative period, which not only prolongs the hospitalization of patients, increases the cost of treatment, but even increases the postoperative mortality of patients, bringing a heavy burden to families and society. Mechanism exploration involves anesthesia and surgery that lead to microglial activation, promote the synthesis and secretion of inflammatory factors, cause an inflammatory cascade, aggravate nerve cell damage, and lead to cognitive dysfunction. It is believed that microglia‐mediated neuroinflammatory responses play a vital role in the formation of PND. Microglia surface receptors are essential mediators for microglia to receive external stimuli, regulate microglial functional status, and carry out intercellular signal transmission. Various microglial surface receptors trigger neuroinflammation, damage neurons, and participate in the development and progression of PND by activating microglia. In this study, the roles of immunoglobulin receptors, chemokine receptors, purinergic receptors, and pattern recognition receptors in microglia surface receptors in PND were reviewed, to provide a reference for the mechanism research, prevention, and treatment of PND.

Routine Whole Genome Sequencing Offers a More Comprehensive Assessment of IG/TCR Gene Rearrangements for MRD Targets in Acute Lymphoblastic Leukemia - Data from the First 62 Cases from the NHS England Genomic Medicine Service

Introduction Minimal residual disease (MRD) monitoring is fundamental for risk stratification and treatment in acute lymphoblastic leukemia (ALL). Sensitive molecular assay requires presentation samples to be screened for immunoglobulin and T-cell receptor (IG/TCR) gene rearrangements. Previously samples were screened by PCR, heteroduplex analysis and Sanger sequencing to identify clone defining IG/TCR gene rearrangements. Subsequently these sequences were used to develop qPCR assays for MRD follow-up monitoring. More recently, targeted IG/TCR high-throughput sequencing (HTS) has been used with similar panels to identify gene rearrangements. Both these methods have limitations, in that only rearrangements covered by the panels are detected, and the techniques are labour intensive and expensive. Whole genome sequencing (WGS) has potential to discover all IG/TCR rearrangements in a single assay giving greater potential to detect markers for follow up MRD monitoring. In England all patients with ALL are eligible for WGS at diagnosis via the National Health Service, Genomic Medicine Service (NHS GMS). Methods Sixty-two patients, presenting with ALL at either Great Ormond Street Hospital or Bristol Children's Hospital, had short-read WGS via the NHS GMS performed concurrently with standard of care (SOC) targeted HTS to identify IG/TCR rearrangements. A bespoke bioinformatic pipeline was developed to identify IG/TCR rearrangements in the WGS samples. SOC targeted HTS analysis was performed using the open source Vidjil bioinformatic software. A cut-off of 5% (clone proportion of total clonotypes) was used to define an acceptable disease-specific clone for monitoring. Results All 62 samples had a greater number of rearrangements detected by WGS than targeted HTS. The mean number of clones >5% detected was 2.7 by targeted HTS and 3.1 by WGS. For rearrangements identified by targeted HTS at a 5% cut-off, 97% (185 out of 190) were identified by WGS. All targets were identified by WGS using a 10% cut-off (Table 1).Despite the lower depth of coverage of the WGS assay, it retrieved 89% of sequences seen in SOC down to a level of 1%, as well as additional re-arrangements not seen by the SOC assay (Figure 1). As well as additional rearrangements being discovered by WGS, we observed novel rearrangements (dd3/ja rearrangements) not previously identified using a targeted approach. To establish if the additional sequences identified by WGS were clinically applicable as MRD targets, we designed allele specific oligonucleotides for a representative 32 rearrangements for testing by qPCR. Over half (n=16) were able to be used to track MRD in follow-up samples. New primer/probe sets were designed for the novel dd3/ja rearrangements identified by WGS, these were also tested by qPCR and performed well enough to be clinically applicable for MRD monitoring. Conclusions WGS approach outperformed targeted HTS, identifying more IG/TCR rearrangements suitable for molecular MRD monitoring in all patients. This is of particularly benefit in cases where targeted HTS only identifies 1 or 2 potential MRD targets. WGS also has the advantage of identifying other key gene rearrangements (BCR-ABL, KMT2A, SIL-TAL, IKZF1) that make excellent targets for MRD monitoring. Beyond this, WGS delivers all diagnostic information, germline and additional somatic findings that aid diagnosis. For ALL, in one test WGS can now replace all molecular diagnostics. In England all patients with leukemia are eligible for centralised WGS. With continued improvements in turnaround times, and reducing costs, we propose that WGS should be the new diagnostic standard for pediatric ALL

Venetoclax and Blinatumomab for Adult Patients with Relapsed/Refractory or MRD Positive Ph-Negative B-Precursor ALL: First Results of the GMALL-Bliven Trial

Introduction Prognosis of adult patients with relapsed/refractory (R/R) B-precursor acute lymphoblastic leukemia (BCP-ALL) remains poor. Monotherapy with Blinatumomab in ALL induced a complete remission (CR) in 44% of patients and measurable residual disease (MRD) negative molecular remission (MOL-CR) in 33%. Median overall survival of 7.7 months remains unsatisfactory. In the MRD setting, median survival was 36 months in the context of subsequent allogeneic stem cell transplantation (SCT). The combination of Blinatumomab with Venetoclax may be an effective option in ALL. Objectives In this multicenter phase 1 clinical trial the GMALL study group evaluates the safety of Blinatumomab in combination with Venetoclax in R/R or MRD positive BCP-ALL (NCT05182385). Using RNA sequencing, functional intracellular BH3 profiling, T cell immunomonitoring and effector cell cytotoxicity assays we aim to further elucidate synergy of this combination. Methods The BLIVEN trial includes patients with CD19-positive Ph-negative BCP-ALL aged 18 years and older, with no upper age limit. Eligible patients had either R/R ALL (bone marrow blasts >5%) or molecular failure/relapse (MRD >1xE-04). To assess safety, we determined maximum tolerated dose (MTD) as primary endpoint of the phase I part of the trial as designated recommended phase 2 dose (RP2D). Key secondary endpoint was the MOL-CR rate. Treatment consisted of oral Venetoclax in continuous dosing on day -7 to day 42 of target doses ranging between 200-800 mg once daily, and Blinatumomab as continuous intravenous infusion of 28 µg/d 4 weeks on and 2 weeks off, starting on day 1. For patients with R/R ALL, a cautionary dose step of 9 µg/d Blinatumomab was implemented for one week. MRD was analyzed in the reference laboratory using real-time quantitative PCR (RQ-PCR) of immunoglobulin and T-cell receptor (IG/TR) rearrangements. Results Between February 2022 and May 2023 a total of 7 patients were enrolled in the phase I part at four German centers (Figure 1). Venetoclax target doses were 400 mg (n=3), 600 mg (n=3), and 800 mg (n=1). Median age was 48.5 years (range 22 - 72 years). MRD assessment was available in 7/7 patients. Molecular BCP-ALL subtypes were identified by RNA sequencing in 5/5 analyzed patients, including ZNF384-rearranged ALL (n=2), BCR::ABL1-like (n=2; IGH::CRLF2 rearranged), and KMT2A-rearranged ALL (n=1). Three of seven patients were treated for hematological relapse, while 4/7 patients had molecular failure/relapse at time of enrolment. Among patients with R/R ALL, 2/3 were refractory to front-line therapy with intensive GMALL induction/consolidation therapy, while 1/3 suffered relapse shortly after failure of second allogeneic SCT. CTCAE grade IV neutropenia was identified as adverse reaction related to Venetoclax in one patient. Neutropenia resolved before dose limiting toxicity (DLT) evaluation time (i.e. 42 days after Venetoclax initiation). Overall, no DLT was identified throughout the trial and MTD was not reached. No treatment interruption was noted during cycle 1. Two of seven patients received one treatment cycle of BLIVEN only due to treatment failure, 5/7 patients received two treatment cycles. Response assessment was available for 7/7 patients. Treatment with Blinatumomab/Venetoclax of R/R ALL resulted in CR in 1/3 patients, with the responding patient showing a MOL-CR. The MOL-CR rate in molecular failure/relapse patients was 3/4. Conclusion Salvage therapy with Blinatumomab and Venetoclax exhibited good tolerability without treatment-related mortality. No treatment withdrawals were attributed to safety concerns. Two patients did not respond to BLIVEN combination therapy. Interestingly, both patients exhibited IGH::CRLF2 fusion transcripts.The remaining 5 patients obtained MRD responses, including complete MRD responses, of which 2/2 occurred in ZNF384 rearranged BCP-ALL. Further follow-up and larger patient numbers are needed to establish the potential benefit of this regimen in molecular driver subtypes of BCP-ALL. Enrollment to complete phase I of the BLIVEN trial is currently ongoing. Updated results including RP2D will be presented at the meeting. Acknowledgments This is an independent academic early phase I clinical trial conducted under the sponsor role of the Goethe University, Frankfurt, Germany. It received financial support and supply of Venetoclax by AbbVie Inc..

Antigen Receptor Gene Repertoire Profiling in Kidney Transplant Recipients Post-Sars-Cov-2 Vaccination

Kidney transplant recipients (KTRs) are highly susceptible to viral infections, while also exhibiting a higher risk of developing severe COVID-19. Recent studies have demonstrated a suboptimal humoral response to current COVID-19 vaccines in this cohort, necessitating multiple vaccine doses. Nevertheless, low rates of seroconversion have been observed even after booster vaccinations, highlighting the need for the in-depth characterization of both humoral and cellular immune responses towards the development of innovative and effective immunoprophylactic strategies tailored for KTRs. Here, we investigated the immunogenetic features of the T cell receptor (TR) and B cell receptor immunoglobulin (BcR IG) gene repertoires in KTRs using targeted Next Generation Sequencing (NGS). In more detail, we studied 19 KTRs at two consecutive timepoints, after the 2 nd (T1) and multiple vaccination doses (T2), respectively. We included as a control group 19 healthy individuals (HIs) sampled either after two mRNA vaccination doses or after a documented SARS-CoV-2 infection. Seroconversion (>1300 AU/ml of anti-RBD IgG) was measured by the Abbott SARS-CoV-2 IgG assay: all HIs tested positive, in sharp contrast to KTRs who failed to exhibit humoral responses after the 2 nd dose (T1). Immunogenetic characterization of TR/BcR IG gene repertoires was performed by paired-end NGS; resultant data were processed through the TRIP software. Only productive V(D)J gene rearrangements were considered for the computation of clonotypes (i.e. TR/BcR IG gene rearrangements with identical V gene usage and complementarity-determining region 3 amino acid sequence-CDR3 aa). Starting with the TR gene repertoire, diversity significantly increased at T2 (average Shannon diversity: KTR-T1=638 vs KTR-T2=3212, p<0.0005| KTR-T2=3212 vs HI=2146, p<0.05). TR clonality (mean cumulative frequency of the 10 dominant TR clonotypes/sample) was significantly increased at T1 in KTRs vs HIs (KTR-T1=37% vs HI=25%, p<0.05), eventually decreasing at T2, reaching comparable levels to those measured in HIs (KTR-T2=27%). At T1, a median of 7.6% of TR clonotypes/sample in KTRs were SARS-specific (high-affinity TR clonotypes to SARS-specific peptide-MHC complexes, >0.9 binding score determined by the ERGO-II algorithm), almost identical to what we found for HIs (7.7%). Although the TR gene repertoire was renewed in T2, the percentage of SARS-specific clonotypes remained essentially identical (median of 7.8%). Turning to the BcR IG gene repertoire, despite the absence of a serological response at T1, the percentage of the predicted SARS-specific BcR IG clonotypes (high-homology with SARS-specific CDR3 aa sequences published at CoV-AbDab, >0.9 identity score) did not differ between KTRs (0.72% at T1 and 0.65% at T2) vs HIs (0.63%), raising the intriguing possibility of B cell anergy in the former. Overtime assessment revealed that BcR IG clonotype diversity significantly increased at T2 vs T1 in KTRs (average Shannon diversity: KTR-T1=12006 vs KTR-T2=38576 p<0.05) and strongly correlated with the presence of SARS-specific BcR IG clonotypes (r=0.65, p<0.001). Nevertheless, the cumulative fraction of SARS-specific BcR IG clonotypes at KTR-T2 decreased (median cumulative frequency of SARS-specific clonotypes: KTR-T2=0.45% vs HI=0.67%, p<0.05). Of note, multiple immunizations resulted to SARS-specific BcR IG clonotypes with a significantly increased number of somatic hypermutations (SHM) compared to either HIs (p<0.0005) or T1 (p<0.05). In conclusion, our findings suggest that the lack of seroconversion at T1 in KTRs is potentially counteracted by cellular sensitization, likely mediated by SARS-specific T cells, that is sustained by booster vaccination. The latter also drives the accumulation of SHM in BcR IG clonotypes, suggesting distinct differentiation trajectories (germinal center-independent at T1 vs germinal center-dependent at T2) that could give rise to new B cell clones with increased neutralizing potency.

Synergistic effect of nickel and temperature on gene expression, multiple stress markers, and depuration: an acute toxicity in fish.

Aquatic animals are prone to extinction due to metal pollution and global climate change. Even though the fish and their products are also unsafe for human consumption, their exports have been rejected due to inorganic and organic contaminants. Nickel (Ni) is a metal that induces toxicity and accumulates in the aquatic ecosystem, posing health threats to humans, animals, and fish. In light of the above, our present investigation aimed to determine the median lethal concentration (96h-LC50) of nickel alone and concurrent with high temperature (34°C) (Ni + T) using static non-renewable bioassay toxicity test in Pangasianodon hypophthalmus. The groups treated under exposure to Ni reared under control condition (25-28.9°C) and Ni + T exposure group reread under 34°C. In this study, chose the definitive dose of Ni and Ni + T as 17, 18, 19, and 20mg L-1 after the range finding test. The median lethal concentration of Ni and Ni + T was determined as 19.38 and 18.75mg L-1, respectively at 96h. Oxidative stress viz. catalase (CAT), superoxide dismutase (SOD), glutathione-s-transferase (GST), and glutathione peroxidase (GPx) in the liver, gill, and kidney were noticeably elevated with Ni and Ni + T during 96h. Whereas, the CAT, GPx, and SOD gene expressions were significantly upregulated with Ni and Ni + T. Trilox equivalent anti-oxidant capacity (TEAC), cupric reducing anti-oxidant capacity (CUPRIC), ferric reducing ability of plasma (FRAP), ethoxy resorufin-O-deethylase (EROD), and acetylcholine esterase (AChE) were reduced due to exposure to Ni and Ni + T. Cellular metabolic stress and lipid peroxidation were highly affected due to Ni and Ni + T exposure. The immunological status, as indicated by total protein, albumin, globulin, A:G ratio, and nitro blue tetrazolium chloride (NBT), was severely affected by the toxicity of Ni and Ni + T. Moreover, the gene expression of interleukin (IL), tumor necrosis factor (TNFα), toll-like receptor (TLR), and total immunoglobulin (Ig) was remarkably downregulated following exposure to Ni and Ni + T. HSP 70, iNOS expression, ATPase, Na + /K + -ATPase, cortisol, and blood glucose was significantly elevated with Ni and Ni + T in P. hypophthalmus. The bioaccumulation of Ni in fish tissues and experimental water was determined. The kidney and liver tissues were highly accumulated with Ni, whereas DNA damage was reported in gill tissue. Interestingly, depuration study revealed that at the 28th day, the Ni bioaccumulation was below the maximum residue limit (MRL) level. Therefore, the present study revealed that Ni and Ni + T led to dysfunctional gene and metabolic regulation affecting physiology and genotoxicity. The bioaccumulation and depuration results also indicate higher residual occurrence of Ni in water and aquatic organisms for longer periods.

SRSF7 is a promising prognostic biomarker in hepatocellular carcinoma and is associated with immune infiltration.

Previous studies indicate that the splicing process, regulated by the cellular machinery of tumors (spliceosome), undergoes alterations, leading to oncogenic splicing events associated with the progression of tumors towards aggressiveness. However, the role of serine/arginine-rich splicing factor 7 (SRSF7) in hepatocellular carcinoma (HCC) and the tumor microenvironment (TME) remains unclear. This study was aimed to explore the role and clinical significance of SRSF7 in HCC. By conducting functional analysis and gene set enrichment analysis, it was discovered that SRSF7 contributes to multiple pathways associated with immune response and tumor advancement. Further experiments verified that silencing of SRSF7 obviously inhibits progression of HCC. Aberrant expression of SRSF7, which were referred as an independent prognostic risk factor, effectively predicts the prognosis of patients with HCC. Functional and gene enrichment analyses revealed that SRSF7 is linked with multiple immune and tumor progression-related pathways, including the B cell receptor signaling pathway, positive regulation of leukocyte and immunoglobulin receptor binding cell activation, nuclear division, membrane invagination, cell cycle, as well as mTOR signaling pathway. Furthermore, increased SRSF7 expression was associated with tumor-infiltrating inflammatory cells (CD4+, monocytes/macrophages, CD8 + and endothelial). Additionally, multiple immune checkpoint genes were markedly positively related to SRSF7. The efficiency of SRSF7 in predicting immunomodulator and chemokine responses were also assessed in microenvironment. Moreover, in vitro analyses demonstrated that knockdown of SRSF7 suppressed the malignant evolution of HCC possibly by deactivating the PI3K/AKT/mTOR signaling. The role of SRSF7 in the tumor microenvironment has been successfully assessed. It may be a valid bio-index for predicting the HCC prognosis, thereby guiding individualized immunotherapy for cancer.

Parasitic infections related to anti-type 2 immunity monoclonal antibodies: a disproportionality analysis in the food and drug administration's adverse event reporting system (FAERS).

Introduction: Monoclonal antibodies (mAbs) targeting immunoglobulin E (IgE) [omalizumab], type 2 (T2) cytokine interleukin (IL) 5 [mepolizumab, reslizumab], IL-4 Receptor (R) α [dupilumab], and IL-5R [benralizumab]), improve quality of life in patients with T2-driven inflammatory diseases. However, there is a concern for an increased risk of helminth infections. The aim was to explore safety signals of parasitic infections for omalizumab, mepolizumab, reslizumab, dupilumab, and benralizumab. Methods: Spontaneous reports were used from the Food and Drug Administration's Adverse Event Reporting System (FAERS) database from 2004 to 2021. Parasitic infections were defined as any type of parasitic infection term obtained from the Standardised Medical Dictionary for Regulatory Activities® (MedDRA®). Safety signal strength was assessed by the Reporting Odds Ratio (ROR). Results: 15,502,908 reports were eligible for analysis. Amongst 175,888 reports for omalizumab, mepolizumab, reslizumab, dupilumab, and benralizumab, there were 79 reports on parasitic infections. Median age was55 years (interquartile range 24-63years) and 59.5% were female. Indications were known in 26 (32.9%) reports; 14 (53.8%) biologicals were reportedly prescribed for asthma, 8 (30.7%) for various types of dermatitis, and 2 (7.6%) for urticaria. A safety signal was observed for each biological, except for reslizumab (due to lack of power), with the strongest signal attributed to benralizumab (ROR = 15.7, 95% Confidence Interval: 8.4-29.3). Conclusion: Parasitic infections were disproportionately reported for mAbs targeting IgE, T2 cytokines, or T2 cytokine receptors. While the number of adverse event reports on parasitic infections in the database was relatively low, resulting safety signals were disproportionate and warrant further investigation.

A novel test for type-I allergy based on crosslink formation of immunoglobulin-E receptors by allergen-specific immunoglobulin-E antibodies and an allergen

Detection of allergen-specific immunoglobulin E (IgE) antibodies (Abs) in serum would allow for screening of the causative allergen in patients with type-I allergy. In this study, we developed a new assay method to detect allergen-specific IgE Abs, which involved crosslinking the plural FcεRIα molecules with an allergen and detection using an amplified luminescence proximity homogeneous assay (AlphaCL). First, the allergen concentration, bead concentrations, and incubation time were optimized for the detection of anti-2,4-dinitrophenyl (DNP) IgE Abs in buffer. Under optimal conditions, AlphaCL was able to detect DNP-specific IgE Abs in simulated human serum at levels comparable to those in serum from type-I allergic patients. When AlphaCL was used to detect anti-DNP IgE Abs, no signal counts were obtained with the monovalent allergen 2,4-dinitrophenylated poly-γ-glutamic acid, whereas high signal counts were obtained with the multivalent allergen DNP-BSA. This confirmed that AlphaCL could specifically detect allergen-specific IgE Abs with the ability to crosslink a multivalent allergen. In summary, we have established a new assay model using AlphaCL to detect allergen-specific IgE Abs with FcεRIα crosslinking ability in human serum. This simple and practical assay model may be applied as a new diagnostic tool for patients with type-I allergy.