Immunoglobulin Heavy Chain Research Articles

Increased c-MYC Expression Associated with Active IGH Locus Rearrangement: An Emerging Role for c-MYC in Chronic Lymphocytic Leukemia

This review examines the pivotal role of c-MYC in Chronic Lymphocytic Leukemia (CLL), focusing on how its overexpression leads to increased genetic instability, thereby accelerating disease progression. MYC, a major oncogene, encodes a transcription factor that regulates essential cellular processes, including cell cycle control, proliferation, and apoptosis. In CLL cases enriched with unmutated immunoglobulin heavy chain variable (IGHV) genes, MYC is significantly overexpressed and associated with active rearrangements in the IGH immunoglobulin heavy chain locus. This overexpression results in substantial DNA damage, including double-strand breaks, chromosomal translocations, and an increase in abnormal repair events. Consequently, c-MYC plays a dual role in CLL: it promotes aggressive cell proliferation while concurrently driving genomic instability through its involvement in genetic recombination. This dynamic contributes not only to CLL progression but also to the overall aggressiveness of the disease. Additionally, the review suggests that c-MYC’s influence on genetic rearrangements makes it an attractive target for therapeutic strategies aimed at mitigating CLL malignancy. These findings underscore c-MYC’s critical importance in advancing CLL progression, highlighting the need for further research to explore its potential as a target in future treatment approaches.

CD38 as theranostic target in oncology.

CD38 is a multifunctional transmembrane glycoprotein found in multiple tissues and overexpressed in many cancer cells, notably in hematological malignancies such as leukemia and multiple myeloma (MM). Therefore, targeting CD38 remains an attractive strategy for cancer treatment in hematological malignancies as well as in solid tumors. It plays a critical role in the progression of these diseases through its ADP-ribosyl cyclase and cADPR-hydrolase activities. Its importance has led to the development of various anti-CD38 monoclonal antibodies (mAbs), including daratumumab and isatuximab, approved for MM treatment. These mAbs exert their anti-tumor effects through Fc-dependent immune mechanisms and immunomodulation, enhancing T-cell and NK-cell-mediated responses. However, resistance mechanisms arise during the treatment with daratumumab, creating the necessity for new therapies. This review explains current knowledge about the role of CD38 as a target in oncology and aims to delineate the use of single domain antibodies (sdAbs) as innovative theranostic tools in nuclear medicine. For diagnostic purposes, PET radionuclides like 68Ga, 64Cu, and SPECT radionuclides like 99mTc and 111In, are commonly used. Significant progress has been made in anti-CD38 radioligand therapy (RLT), with anti-CD38 antibodies providing insights into tumor biology and treatment efficacy. In terms of therapy, RLT is a promising approach that offers precise targeting of malignant cells while minimizing exposure to healthy tissue. This involves the use of radionuclides emitting α particles, like 225Ac, 212Pb or 211At, and β--particles like 90Y, 131I, or 177Lu, to exert cytotoxic effects. Derived from Camelidae heavy chain antibodies, sdAbs offer advantages over conventional mAbs such as small size, high stability, specificity, and ability to recognize hidden epitopes. CD38-specific sdAbs, such as sdAb 2F8, characterized by our laboratory, showing excellent tumor targeting and their engineered constructs, such as biparatopic antibodies and chimeric antibodies, represent a new generation of theranostic agents for diagnosis and treatment CD38-expressing malignancies.

Engineering immunogens that select for specific mutations in HIV broadly neutralizing antibodies

Vaccine development targeting rapidly evolving pathogens such as HIV-1 requires induction of broadly neutralizing antibodies (bnAbs) with conserved paratopes and mutations, and in some cases, the same Ig-heavy chains. The current trial-and-error search for immunogen modifications that improve selection for specific bnAb mutations is imprecise. Here, to precisely engineer bnAb boosting immunogens, we use molecular dynamics simulations to examine encounter states that form when antibodies collide with the HIV-1 Envelope (Env). By mapping how bnAbs use encounter states to find their bound states, we identify Env mutations predicted to select for specific antibody mutations in two HIV-1 bnAb B cell lineages. The Env mutations encode antibody affinity gains and select for desired antibody mutations in vivo. These results demonstrate proof-of-concept that Env immunogens can be designed to directly select for specific antibody mutations at residue-level precision by vaccination, thus demonstrating the feasibility of sequential bnAb-inducing HIV-1 vaccine design.

The identification and correction of pseudohypercalcemia.

We found that a subset of patients with monoclonal gammopathy exhibited hypercalcemia without apparent causes or clinical manifestations In a cohort of 289 monoclonal gammopathy patients, 2.4% presented with such atypical hypercalcemia, with one notable case exhibiting normal ionized calcium levels, suggesting the presence of pseudohypercalcemia. The aim of this study is to elucidate the factors contributing to pseudohypercalcemia in monoclonal gammopathy and to develop a novel globulin-corrected calcium formula for clinical application. This observational study enrolled 110 monoclonal gammopathy patients from our center. An additional 33 patients were recruited to validate the newly proposed formula. Univariate analysis identified potential risk factors. And multivariate logistic regression identified definitive influential factors. The determined influential factors were utilized to develop a formula by multiple linear regression, which was validated by a paired t-test and the Youden index. This study found that globulin was a risk factor for pseudohypercalcemia. It revealed that pseudohypercalcemia should be considered in patients with globulin levels ≥61 g/L (P=0.014). Both albumin and globulin were confirmed as independent factors associated with bound calcium. Given that, we developed a formula to correct ionized calcium levels, which was consistent with ionized calcium tested by blood gas analysis. The diagnostic accuracy of the new formula (Youden index is 0.906) is better than the traditional formula (Youden index is 0.906). Interestingly, all monoclonal immunoglobulin types, except for light chains, showed an equal propensity to develop pseudohypercalcemia (P=0.306). It also showed a linear correlation between IgA, IgG, and IgM and bound calcium. This study confirmed that elevated globulin affects serum total calcium and offered the threshold of globulin ≥ 61 g/L in the differential diagnosis of peudohypercalcemia from hypercalcemia. The new formula based on albumin and globulin was developed, which was verified to be better than the traditional formula for correctly diagnosing hypercalcemia. In addition, we found that neither light chains nor heavy chains of monoclonal immunoglobulin alone can result in pseudohypercalcemia.

Preparation of divalent camelid single-domain antibody and its application in immunoassays for Salmonella detection in food.

Salmonella-related foodborne infections are commonly caused by the serovars of S. Typhimurium, which can be detected using antibody-based immunoassays. The monovalent variable domain of thecamelid heavy chain antibody (VHH) performs excellently in constructing multivalent VHH variants, which generally exhibit higher affinities with antigens and consequently enhance the assay sensitivity. In this study, the divalent variants of VHHs (diVHHs) targeting S. Typhimurium were generated by encoding the monovalent VHH genes assembled in tandem with a flexible linker peptide (G4S)2. Soluble diVHHs were produced in a prokaryotic expression system and purified with a yield of 4.22 mg/L. Benefiting from their stability and antigen-binding abilities towards tested Salmonella serovars, diVHH-based immunoassays were developed. The diVHH-based sandwich immunoassay, using diVHH as capture antibody, exhibited a detection limit of 1.04×102 CFU/mL and enabled as low as 10 CFU/mL S. Typhimurium to be detectedafter 6 h of enrichment in lettuce. Furthermore, this assay can be applied to spiked lettuce, chicken, and pork samples, showing acceptable recoveries ranging from 83 to 106%. This study presented feasible strategies for VHH multivalence and established a superior sensitivity VHH-based immunoassay for monitoring and analyzing Salmonella contamination in food.

Ultrasensitive allele inference from immune repertoire sequencing data with MiXCR.

Allelic variability in the adaptive immune receptor loci, which harbor the gene segments that encode B cell and T cell receptors (BCR/TCR), is of critical importance for immune responses to pathogens and vaccines. Adaptive immune receptor repertoire sequencing (AIRR-seq) has become widespread in immunology research making it the most readily available source of information about allelic diversity in immunoglobulin (IG) and T cell receptor (TR) loci. Here we present a novel algorithm for extra-sensitive and specific variable (V) and joining (J) gene allele inference, allowing reconstruction of individual high-quality gene segment libraries. The approach can be applied for inferring allelic variants from peripheral blood lymphocyte BCR and TCR repertoire sequencing data, including hypermutated isotype-switched BCR sequences, thus allowing high-throughput novel allele discovery from a wide variety of existing datasets. The developed algorithm is a part of the MiXCR software. We demonstrate the accuracy of this approach using AIRR-seq paired with long-read genomic sequencing data, comparing it to a widely used algorithm, TIgGER. We applied the algorithm to a large set of IG heavy chain (IGH) AIRR-seq data from 450 donors of ancestrally diverse population groups, and to the largest reported full-length TCR alpha and beta chain (TRA; TRB) AIRR-seq dataset, representing 134 individuals. This allowed us to assess the genetic diversity within the IGH, TRA and TRB loci in different populations and to establish a database of alleles of V and J genes inferred from AIRR-seq data and their population frequencies with free public access through an online database.

PCR GeneScan Analysis of Rearranged Immunoglobulin or T-Cell Receptor Genes for Clonality Diagnostics in Suspect Lymphoproliferations.

Assessment of the presence of clonal lymphoproliferations via polymerase chain reaction (PCR)-based analysis of rearranged immunoglobulin (IG) or T-cell receptor (TR) genes is a valuable method in the diagnosis of suspect lymphoproliferative disorders. Additionally, this methodology can be used for evaluating dissemination of lymphoma cells and for studying the clonal relationship between multiple (different locations) and consecutive (over time) lymphomas. Here we describe an integrated approach to assess clonality via analysis of Ig heavy chain (IGH), Ig kappa (IGK), TCR beta (TRB), and TCR gamma (TRG) gene rearrangements, based on the standardized multiplex PCRs as originally developed by the European BIOMED-2 consortium (currently named EuroClonality). The described protocol covers the pre-analytical phase of DNA isolation (from formalin-fixed paraffin-embedded and fresh tissues, body fluids, peripheral blood, and bone marrow), the analytical phase of PCR GeneScan analysis, and the post-analytical interpretation of the obtained profiles, following established guidelines.

MRD Detection in B-Cell Non-Hodgkin Lymphomas Using Ig Gene Rearrangements and Chromosomal Translocations as Targets for Real-Time Quantitative PCR and ddPCR.

Minimal residual disease (MRD) diagnostics is of high clinical relevance in patients with indolent B-cell non-Hodgkin lymphomas (B-NHL), B-cell chronic lymphocytic leukemia (CLL), and multiple myeloma and serves as a surrogate parameter to evaluate treatment effectiveness and long-term prognosis. Real-time quantitative PCR (RQ-PCR) targeting circulating lymphoma cells is still the gold standard for MRD detection in indolent B-NHL and currently the most sensitive and the most broadly applied method in follicular lymphoma (FL) and mantle cell lymphoma (MCL). Alternatively, droplet digital PCR (ddPCR) can be used for MRD monitoring in multiple myeloma, mantle cell lymphoma, CLL, and FL with comparable sensitivity, accuracy, and reproducibility.The most broadly applicable MRD target in B-NHL is the junctional regions of the rearranged immunoglobulin heavy (IGH) and light chain genes. Complete and incomplete IGH and additionally IG kappa light chain rearrangements can be used as targets for MRD. Next-generation sequencing (NGS) of IG-rearrangements (IG-NGS) as new sequencing-based technology can overcome the limitation of PCR-based approaches and has a potential for higher sensitivity. Chromosomal translocations like the t(14;18)(q32;q21) translocation associated with IGH::BCL2 fusion in FL and t(11;14)(q13;q32) translocation in MCL leading to the IGH::CCND1 fusion can be used as MRD target in selected lymphoma subtypes. In patients with CLL, both flow-cytometry and RQ-PCR are equally suited for MRD assessment as long as a sensitivity of 10-4 is achieved.MRD diagnostics targeting the IG loci is complex and requires extensive knowledge and experience because the junctional regions of each clonal rearranged gene have to be identified before the patient-specific PCR assays can be designed for MRD monitoring. In addition, the presence and load of somatic hypermutation within the rearranged IGH gene occurring during B-cell development of germinal center and post-germinal center B-cell lymphomas may hamper appropriate primer binding leading to false-negative results. The translocations mentioned above have the advantage that consensus forward primers and probes, both placed in the breakpoint regions of chromosome 18 in FL and chromosome 11 in MCL, can be used in combination with a reverse primer placed in the IGH joining region of chromosome 14. PCR-based methods using allele-specific primers can reach a high sensitivity of up to 10-5. This chapter provides all relevant background information and technical aspects for the complete laboratory process from detection of the clonal IG gene rearrangements and the chromosomal translocations at diagnosis to the actual MRD measurements in clinical follow-up samples of B-NHL. However, it should be noted that MRD diagnostics for clinical treatment protocols has to be accompanied by regular international quality control rounds to ensure the reproducibility and reliability of the MRD results. This is available by the EuroMRD network ( https://euromrd.org ), a subgroup of ESHLO ( https://eslho.org ).

On the humanization of VHHs: Prospective case studies, experimental and computational characterization of structural determinants for functionality.

The humanization of camelid-derived variable domain heavy chain antibodies (VHHs) poses challenges including immunogenicity, stability, and potential reduction of affinity. Critical to this process are complementarity-determining regions (CDRs), Vernier and Hallmark residues, shaping the three-dimensional fold and influencing VHH structure and function. Additionally, the presence of non-canonical disulfide bonds further contributes to conformational stability and antigen binding. In this study, we systematically humanized two camelid-derived VHHs targeting the natural cytotoxicity receptor NKp30. Key structural positions in Vernier and Hallmark regions were exchanged with residues from the most similar human germline sequences. The resulting variants were characterized for binding affinities, yield, and purity. Structural binding modes were elucidated through crystal structure determination and AlphaFold2 predictions, providing insights into differences in binding affinity. Comparative structural and molecular dynamics characterizations of selected variants were performed to rationalize their functional properties and elucidate the role of specific sequence motifs in antigen binding. Furthermore, systematic analyses of next-generation sequencing (NGS) and Protein Data Bank (PDB) data was conducted, shedding light on the functional significance of Hallmark motifs and non-canonical disulfide bonds in VHHs in general. Overall, this study provides valuable insights into the structural determinants governing the functional properties of VHHs, offering a roadmap for their rational design, humanization, and optimization for therapeutic applications.

Core enhancers of the 3'RR optimize IgH nuclear position and loop conformation for successful oriented class switch recombination.

In B lymphocytes, class switch recombination (CSR) is an essential process that adapts immunoglobulin (Ig) subtypes to antigenresponse. Taking place within the Ig heavy chain (IgH) locus, CSR needs controlled transcription of targeted regions governed by the IgH 3'regulatory region (3'RR). This super-enhancer is composed of four core enhancers surrounded by inverted repeated sequences, forming a quasi-palindrome. In addition to transcription, nuclear organization appears to be an important level in CSR regulation. While it is now established that chromatin loop extrusion takes place within IgH locus to facilitate CSR by bringing the donor and acceptor switch regions closer together, the underlying mechanism that triggers CSR loop formation remains partially understood. Here, by combining DNA 3Dfluorescence in situhybridizationwith various high-throughput approaches, we deciphered critical functions for the 3'RR core enhancer element in nuclear addressing, accessibility and chromatin looping of the IgH locus. We conclude thatthe 3'RR core enhancers are necessary and sufficient to pre-organize the position and conformation of IgH loci in resting B-cell nuclei to enable the deletional recombination events required for productive successful CSR in activated B-cell nuclei.

Genetic Profiling in Chronic Lymphocytic Leukemia: The Role of TP53 Mutations and IGHV Status in Treatment Decision-Making – A Case Report

Introduction. The TP53 gene encodes the p53 protein, crucial for DNA damage response, apoptosis, and cell cycle regulation. In chronic lymphocytic leukemia (CLL), TP53 loss due to 17p deletion or mutation leads to poor chemoimmunotherapy response and shorter survival. Patients with unmutated immunoglobulin heavy chain variable (IGHV) status exhibit a more aggressive disease course, poorer outcomes, and reduced response to standard chemoimmunotherapy. These genetic variations significantly affect disease progression, treatment choice, and response to therapy. Case Report. In November 2023, a 61-year-old man presented to our clinic with complaints of generalized weakness and rapid fatigue since October 2023. CLL was diagnosed in 2018 through pathohistological and immunohistochemical analysis of the bone marrow. The patient received no therapy until 2023 due to the absence of active disease signs. From March 28, 2023, to July 25, 2023, the patient received chemoimmunotherapy with the bendamustine and rituximab (BR) regimen. Five courses of therapy were administered; however, further treatment was discontinued due to side effects that occurred during the fifth course of bendamustine administration. A subsequent diagnostic evaluation aimed at assessing treatment efficacy, prognosis, and genetic profiling included a cytomorphological analysis, which revealed that lymphocytes comprised 80% of the cellular composition. Immunocytological analysis confirmed the presence of monoclonal atypical lymphocytes, accounting for 70% of the cell population. Next-generation sequencing identified a pathogenic TP53 mutation, and fragment analysis confirmed an unmutated IGHV status. The presence of the TP53 mutation and unmutated IGHV status categorizes the patient as being in the very high-risk group, for which the use of Bruton’s tyrosine kinase inhibitors (BTKIs) and/or B-cell lymphoma-2 (BCL-2) inhibitors is recommended. Conclusions. This case highlights the significance of comprehensive genetic profiling in CLL patients using techniques such as fluorescence in situ hybridization, polymerase chain reaction, and next-generation sequencing. Such profiling detects molecular genetic alterations, facilitating personalized and effective treatment.

An anti-Shiga toxin VHH nanobody multimer protects mice against fatal toxicosis when administered intramuscularly as repRNA.

Hemolytic uremic syndrome (HUS) is a systemic sequelae from gastrointestinal infection with Shiga toxin (Stx) producing Escherichia coli (STEC) that can result in acute kidney injury, lasting renal disease, and death. Despite a window for intervention between hemorrhagic diarrhea and onset of HUS, no specific therapies exist to prevent or treat HUS following STEC infection. Furthermore, there is no way to predict which patients with STEC will develop HUS or any rapid way to determine which Stx variant is present. To address this, we have broadened the therpay to neutralize additional toxin variants. It contains a multimer of nanobodies derived from camelid heavy chain antibody fragments (VHHs). An improved VHH-based neutralizing agent (VNA2) is delivered intramuscularly as RNA combined with LION nanoparticles rather than mRNA, that replicates on administration (repRNA), resulting in a rapidly circulating VNA that can bind systemic toxin. The RNA/VNA2-Stx administered intramuscularly prevents toxicity and death in a mouse model of acute Stx toxicity.

B cell immune repertoire sequencing in tobacco cigarette smoking, vaping, and chronic obstructive pulmonary disease in the COPDGene cohort.

Cigarette smoking (CS) impairs B cell function and antibody production, increasing infection risk. The impact of e-cigarette use ('vaping') and combined CS and vaping ('dual-use') on B cell activity is unclear. To examine B cell receptor sequencing (BCR-seq) profiles associated with CS, vaping, dual-use, COPD-related outcomes, and demographic factors. BCR-seq was performed on blood RNA samples from 234 participants in the COPDGene study. We assessed multivariable associations of B cell function measures (immunoglobulin heavy chain (IGH) subclass expression and usage, class-switching, V-segment usage, and clonal expansion) with CS, vaping, dual-use, COPD severity, age, sex, and race. We adjusted for multiple comparisons using the Benjamini-Hochberg method, identifying significant associations at 5% FDR and suggestive associations at 10% FDR. Among 234 non-Hispanic white (NHW) and African American (AA) participants, CS and dual-use were significantly positively associated with increased secretory IgA production, with dual-use showing the strongest associations. Dual-use was positively associated with class switching and B cell clonal expansion, indicating increased B cell activation, with similar trends in those only smoking or only vaping. We observed significant associations between race and IgG antibody usage. AA participants had higher IgG subclass proportions and lower IgM usage compared to NHW participants. CS and vaping additively enhance B cell activation, most notably in dual-users. Self-reported race was strongly associated with IgG isotype usage. These findings highlight associations between B cell activation and antibody transcription, suggesting potential differences in immune and vaccine responses linked to CS, vaping, and race.

Diversification of Antibodies: From V(D)J Recombination to Somatic Exon Shuffling.

Antibodies that gain specificity by a large insert encoding for an extra domain were described for the first time in 2016. In malaria-exposed individuals, an exon deriving from the leukocyte-associated immunoglobulin-like 1 (LAIR1) gene integrated via a copy-and-paste insertion into the immunoglobulin heavy chain encoding region. A few years later, a second example was identified, namely a dual exon integration from the leukocyte immunoglobulin-like receptor B1 (LILRB1) gene that is located in close proximity to LAIR1. A dedicated high-throughput characterization of chimeric immunoglobulin heavy chain transcripts unraveled, that insertions from distant genomic regions (including mitochondrial DNA) can contribute to human antibody diversity. This review describes the modalities of insert-containing antibodies. The role of known DNA mobility aspects, such as genomic translocation, gene conversion, and DNA fragility, is discussed in the context of insert-antibody generation. Finally, the review covers why insert antibodies were omitted from the past repertoire analyses and how insert antibodies can contribute to protective immunity or an autoreactive response.

B-227 Customized molecular tests for minimal residual disease: a tool for therapeutic decisions in follicular lymphoma patients

Abstract Background Monitoring of minimal residual disease (MRD) is based on accurate laboratory tests which can recognize a small fraction of malignant cells that survived the course of treatment. Several studies of the B-non-Hodgkin lymphoma group have been presented that MRD positivity at the end of treatment is a negative prognostic index. Follicular lymphoma (FL) is a chronic indolent malignancy, which response highly to immuno-chemotherapy followed by prolonged remissions but with inevitable relapse in most patients. The GALLIUM study showed that immuno-chemotherapy combinations, 6 cycles of bendamustine-obinutuzumab (BO), is the most efficacious treatment, but with severe side effects .In addition, 90% of patients achieved negative MRD from peripheral blood (PB) or bone marrow (BM) after only 3 BO cycles. These findings raise the possibility for MRD based treatment approach, where the duration of chemotherapy could be decided according to MRD status at mid-induction (MI). Identification of a marker in the PB and/or BM of each FL patient which will be use to the design of a sensitive RQ-PCR test for MRD detection for follow-up. Methods Qualitative PCR for translocation 14:18 and for the VDJ rearrangement sequence of the immunoglobulin heavy chain was used for marker identification. MRD monitoring was done by RQ-PCR. MRD monitoring for VDJ was designed specifically to each patients based on his VDJ sequence. MRD was assessed on PB and BM at MI. Results Marker for monitoring was identified at 11/21 patients in this study. After 3 combined therapy cycles, 7 patients were found to have MRD negativity by RQ-PCR therefore continue with immuno-therapy only. MRD positivity was assessed in BM of 3 patients (0.11%, 0.022%, and 0.133%). These 3 patients continued with the combined therapy. RQ-PCR sensitivity was 10-4-10-5. Conclusions This study will reveal the ability to use sensitive and specific MRD that has to be designed for each patient according to his marker and enable the establishment of a treatment with a high safety profile for FL patients. Correlation between MRD status and clinical and safety parameters will be assessed at the end of the study, 30 months after the first treatment, taking into account MRD status from different time point of the follow-up period.

Functional characterization of a nanobody-based glycoprotein VI-specific platelet agonist

BackgroundGlycoprotein (GP)VI is a platelet-specific collagen receptor required for platelet activation during hemostasis. Platelet reactivity toward collagen is routinely assessed during diagnostic workup of platelet disorders. GPVI can be activated by inducing receptor clustering with suspensions of fibrillar collagen or synthetic cross-linked collagen-related peptide (CRP-XL). However, these suspensions are poorly standardized or difficult to produce. Nanobodies are small recombinant camelid-derived heavy-chain antibody variable regions. They are highly stable, specific, and ideal candidates for developing a stable GPVI agonist for diagnostic assays. ObjectivesDevelop a stable nanobody-based GPVI agonist. MethodsNanobody D2 (NbD2) was produced as dimers and purified. Tetramers were generated via C-terminal fusion of dimers with click chemistry. Nanobody constructs were functionally characterized with light transmission aggregometry (LTA) in platelet-rich plasma and whole blood flow cytometry. Diagnostic performance was assessed in patients with inherited platelet function disorders with LTA and flow cytometry. ResultsNbD2 was specific for human platelet GPVI. Dimers did not result in platelet activation in LTA or flow cytometry settings and fully inhibited CRP-XL-induced P-selectin expression and fibrinogen binding in whole blood and attenuated collagen-induced platelet aggregation in platelet-rich plasma. However, NbD2 tetramers caused full platelet aggregation, as well as P-selectin expression and fibrinogen binding. NbD2 tetramers were able to discriminate between inherited platelet function disorder patients and healthy controls based on fibrinogen binding, similar to CRP-XL. ConclusionNanobody tetramers to GPVI induce platelet activation and can be used to assess the GPVI pathway in diagnostic assays.

Overlapping Gene Expression and Molecular Features in High-Grade B-Cell Lymphoma

Aggressive B-cell lymphoma encompasses Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), and, as per the 2016 WHO classification, high-grade B-cell lymphoma (HGBL) not otherwise specified (NOS) and HGBL double/triple hit (DH/TH). However, the diagnostic distinction of HGBL from BL and DLBCL is difficult by means of histology/immunostaining in a substantial number of patients. This study aimed to improve subtyping by the identification of molecular features of aggressive B-cell lymphomas, with a specific focus on HGBL. To this end, we performed a comprehensive gene expression and mutational pattern analysis as well as the detection of B-cell clonality of 34 cases diagnosed with BL (n = 4), DLBCL (n = 16), HGBL DH (n = 8), and HGBL NOS (n = 6). Three distinct molecular subgroups were identified based on gene expression, primarily influenced by MYC expression/translocation and cell proliferation. In HGBL, compared to BL, there was an upregulation of PRKAR2B and TERT. HGBL DH exhibited elevated expression of GAMT and SMIM14, while HGBL NOS showed increased expression of MIR155HG and LZTS1. Our gene mutation analysis revealed MYC, ARID1A, BCL2, KMT2D, and PIM1 as the most affected genes in B-cell lymphoma, with BCL2 and CREBBP predominant in HGBL DH, and MYC and PIM1 in HGBL NOS. Clonality analysis of immunoglobulin heavy and light chain rearrangements did not show distinguishable V- or J-usage between the diagnostic subgroups.

Effect of Inhibition against Bovine Rotavirus Infection in Calves by Oral Administration of Bovine Rotavirus-Specific Single Variable Domain of Heavy-chain Antibodies

Effect of Inhibition against Bovine Rotavirus Infection in Calves by Oral Administration of Bovine Rotavirus-Specific Single Variable Domain of Heavy-chain Antibodies

Mitochondrial genome-derived circRNAs: Orphan epigenetic regulators in molecular biology

Mitochondria are vital for cellular activities, influencing ATP production, Ca2+ signaling, and reactive oxygen species generation. It has been proposed that nuclear genome-derived circular RNAs (circRNAs) play a role in biological processes. For the first time, this study aims to comprehensively explore experimentally confirmed human mitochondrial genome-derived circRNAs (mt-circRNAs) via in-silico analysis. We utilized wide-ranging bioinformatics tools to anticipate their roles in molecular biology, involving miRNA sponging, protein antagonism, and peptide translation. Among five well-characterized mt-circRNAs, SCAR/mc-COX2 stands out as particularly significant with the potential to sponge around 41 different miRNAs, which target several genes mostly involved in endocytosis, MAP kinase, and PI3K-Akt pathways. Interestingly, circMNTND5 and mecciND1 specifically interact with miRNAs through their unique back-splice junction sequence. These exclusively targeted miRNAs (has-miR-5186, 6888-5p, 8081, 924, 672-5p) are predominantly associated with insulin secretion, proteoglycans in cancer, and MAPK signaling pathways. Moreover, all mt-circRNAs intricately affect the P53 pathway through miRNA sequestration. Remarkably, mc-COX2 and circMNTND5 appear to be involved in the RNA’s biogenesis by antagonizing AGO1/2, EIF4A3, and DGCR8. All mt-circRNAs engaged with IGF2BP proteins crucial in redox signaling, and except mecciND1, they all potentially generate at least one protein resembling the immunoglobulin heavy chain protein. Given P53′s function as a redox-sensitive transcription factor, and insulin’s role as a crucial regulator of energy metabolism, their indirect interplay with mt-circRNAs could influence cellular outcomes. However, due to limited attention and infrequent data availability, it is advisable to conduct more thorough investigations to gain a deeper understanding of the functions of mt-circRNA.

Neutralizing chimeric heavy-chain antibody targeting the L-HN domain of Clostridium botulinum neurotoxin type F.

Botulinum toxin (BoNT) from Clostridium botulinum is the most toxic biotoxin known and is also an important bioterrorism agent. After poisoning, the only effective treatment is injection of antitoxin. However, neutralizing nanoantibodies are safer and more effective, representing a promising therapeutic approach. Therefore, it is important to obtain effective neutralizing nanoantibodies. Hence, the present study aimed to construct a phage antibody library by immunizing a camel and screening specific clones that bind to the L-HN domain of BoNT/F and constructing chimeric heavy-chain antibodies by fusing them with a human Fc fragment. The antibodies' affinity and in vivo neutralizing activities were evaluated. The results showed that 2µg of F20 antibody could completely neutralize 20 × the median lethal dose (LD50) of BoNT/F in vitro. Injection of 5mg/kg F20 at 1h, 2h, 3h, and 4h into mice after BoNT/F challenge resulted in complete survival in vivo. Overall, the antibody might be a candidate for the development of new drugs to treat botulism.