BACKGROUND: The Atlantic cod is a prolific species in the Atlantic, despite its inconsistent specific antibody response. It presents a peculiar case within vertebrate immunology due to its distinct immune system, characterized by the absence of MHCII antigen presentation pathway, required for T cell-dependent antibody responses. Thorough characterisation of immunoglobulin loci and analysis of the antibody repertoire is necessary to further our understanding of the Atlantic cod\'s immune response on a molecular level.
RESULTS: A comprehensive search of the cod genome (gadmor3.0) identified the complete set of IgH genes organized into three sequential translocons on chromosome 2, while IgL genes were located on chromosomes 2 and 5. The Atlantic cod displayed a moderate germline V gene diversity, comprising four V gene families for both IgH and IgL, each with distinct chromosomal locations and organizational structures. 5\'RACE sequencing revealed a diverse range of heavy chain CDR3 sequences and relatively limited CDR3 diversity in light chains. The analysis highlighted a differential impact of V-gene germline CDR3 length on receptor CDR3 length between heavy and light chains, underlining different recombination processes.
CONCLUSIONS: This study reveals that the Atlantic cod, despite its inconsistent antibody response, maintains a level of immunoglobulin diversity comparable to other fish species. The findings suggest that the extensive recent duplications of kappa light chain genes do not result in increased repertoire diversity. This research provides a comprehensive view of the Atlantic cod\'s immunoglobulin gene organization and repertoire, necessary for future studies of antibody responses at the molecular level.

Leucocytes, especially neutrophils featuring pro- and anti-cancerous characteristics, are involved in nearly every stage of tumorigenesis. Phenotypic and functional differences among mature and immature neutrophil fractions are well reported, and their correlation with tumor progression and therapy has emerging implications in modern oncology practices. Technological advancements enabled modern hematology analyzers to generate extended information (research parameters) during complete blood cell count (CBC) analysis. We hypothesized that neutrophil and lymphocyte fractions-related extended differential leucocytes count (DLC) parameters hold superior diagnostic utility over routine modalities. The present study was carried out over a four-and-a-half-year period wherein extended neutrophil (immature granulocyte [IG] and mature neutrophil [NEUT#&]), and lymphocyte (activated/high fluorescence lymphocyte count [HFLC] and resting lymphocyte [LYMP#&]) parameters were challenged over routine neutrophil [NEUT#] and lymphocyte [LYMP#] items in a study population of 1067 hematological neoplasm patients. Extending the classical statistical approaches, machine-learning-backed data visualization was used to explore trends in the study parameters. As a whole, extended neutrophil and lymphocyte count outperformed and was diagnostically more relevant than routine neutrophil and lymphocyte parameters by showing the least difference from their respective (gold-standard) manual DLC counts. The mature neutrophil count was compared to IG, and resting lymphocyte count was compared to HFLC by calling the function \'correlation\' as a \'clustering function\' for heatmap based visualization. The aforementioned study parameters displayed close clustering (rearrangement) for their respective study items by presenting distinct trends of equally valuable weights (deviated values), advocating fractions-based extended DLC reporting. Importantly, using a Bland and Altman analysis analogously to a manual neutrophil count, the mature neutrophil count [NEUT#&] remained unbiased since a routine neutrophil count [NEUT#] was found to be a negatively biased. The extended DLC-parameter-driven fractions-based reporting has superior diagnostic utility over classical routine approaches; this finding can largely minimize labor-intensive manual DLC practices, especially in hematology-oncology departments.

Use of adaptive immune receptor repertoire sequencing (AIRR-seq) has become widespread, providing new insights into the immune system with potential broad clinical and diagnostic applications. However, like many high-throughput technologies, it comes with several problems, and the AIRR Community was established to understand and help solve them. We, the AIRR Community\'s Biological Resources Working Group, have surveyed scientists about the need for standards and controls in generating and annotating AIRR-seq data. Here, we review the current status of AIRR-seq, provide the results of our survey, and based on them, offer recommendations for developing AIRR-seq standards and controls, including future work.

Ig is a Y-shaped protein produced by plasma cells and exerts multiple functions in humoral immunity. There are five groups of Igs including IgA, IgD, IgE, IgG, and IgM, which differ in their heavy chain class. The primary function of Igs includes the neutralization of extrinsic pathogens, agglutination of foreign cells for phagocytosis, precipitation of soluble antigens in serum, and complement fixation. The B cells activated by antigen(s) can differentiate into antibody-producing cells that are called plasma cells and usually matured in the germinal center (GC). Follicular T helper (Tfh) cells crosstalk with antigen-presenting cells and play a crucial role in the development of the GC. Moreover, Tfh cells regulate trafficking through the GC to allow formative interaction with GC B cells that ultimately results in affinity maturation, B-cell memory, and Ig class switching. The B7 family is a series of number of structurally related membrane proteins that bind with a specific receptor to deliver costimulatory or co-inhibitory signals that regulate the activation of T cells in GC. Here, we review and summarize the recent advance of the effects of B7 family members on Ig production and relative diseases.

BACKGROUND: COVID-19 is an infectious disease caused by a novel coronavirus (SARS-CoV-2). The immunopathogenesis of the infection is currently unknown. Healthcare workers (HCWs) are at highest risk of infection and disease. Aim of the study was to assess the sero-prevalence of SARS-CoV-2 in an Italian cohort of HCWs exposed to COVID-19 patients.
METHODS: A point-of-care lateral flow immunoassay (BioMedomics IgM-IgG Combined Antibody Rapid Test) was adopted to assess the prevalence of IgG and IgM against SARS-CoV-2. It was ethically approved (\"Milano Area 1\" Ethical Committee prot. n. 2020/ST/057).
RESULTS: A total of 202 individuals (median age 45 years; 34.7% males) were retrospectively recruited in an Italian hospital (Milan, Italy). The percentage (95% CI) of recruited individuals with IgM and IgG were 14.4% (9.6-19.2%) and 7.4% (3.8-11.0%), respectively. IgM were more frequently found in males (24.3%), and in individuals aged 20-29 (25.9%) and 60-69 (30.4%) years. No relationship was found between exposure to COVID-19 patients and IgM and IgG positivity.
CONCLUSIONS: The present study did show a low prevalence of SARS-CoV-2 IgM in Italian HCWs. New studies are needed to assess the prevalence of SARS-CoV-2 antibodies in HCWs exposed to COVID-19 patients, as well the role of neutralizing antibodies.

The present study investigated the genetic etiology and possible immunological pathogenesis of recurrent spontaneous abortion by analyzing chromosome abnormalities, and the balance between T helper 17 (Th17) and regulatory T (Treg) cells. A total of 54 patients with recurrent spontaneous abortion were selected. The villus and decidual tissues, and peripheral venous blood were collected from each patient. Villus chromosome analysis was performed by high-throughput gene sequencing. Flow cytometry was used to detect Th17 and Treg cells in patients without chromosome abnormalities (n=30) and the control group (normal pregnancy; n=32). Immunoglobulin (IG) combined with human chorionic gonadotropin hormone (HCG) treatment was given to patients without chromosome abnormalities (n=30). Changes in the expression levels of Th17 and Treg cells before and after treatment were compared with patients with successful pregnancy (n=18). Before treatment, compared with the control group, the proportion of Th17 cells in peripheral blood and decidual tissue was increased and the proportion of Treg cells decreased. After treatment, compared with patients before treatment, the proportion of Th17 cells decreased and Treg cells increased, and the Th17 and Treg cells balance was reversed with a biased towards Treg cells. The present results suggested that the Th17 and Treg cell immune imbalance may be an important immune factor in recurrent spontaneous abortion. IG combined with HCG therapy may improve pregnancy outcomes by reversing the imbalance between Th17 and Treg cells.

Immunoglobulin (Ig) diversification occurs via somatic hypermutation (SHM) and class switch recombination (CSR), and is initiated by activation-induced deaminase (AID), which converts cytosine to uracil. Variable (V) region genes undergo SHM to create amino acid substitutions that produce antibodies with higher affinity for antigen. The conversion of cytosine to uracil in DNA promotes mutagenesis. Two distinct DNA repair mechanisms regulate uracil processing in Ig genes. The first involves base removal by the uracil DNA glycosylase (UNG), and the second detects uracil via the mismatch repair (MMR) complex. Methyl binding domain protein 4 (MBD4) is a uracil glycosylase and an intriguing candidate for involvement in somatic hypermutation because of its interaction with the MMR MutL homolog 1 (MLH1). We found that the DNA uracil glycosylase domain of MBD4 is highly conserved among mammals, birds, shark, and insects. Conservation of the human and chicken MBD4 uracil glycosylase domain structure is striking. Here we examined the function of MBD4 in chicken DT40 B cells which undergo constitutive SHM. We constructed structural variants of MBD4 DT40 cells using CRISPR/Cas9 genome editing. Disruption of the MBD4 uracil glycosylase catalytic region increased SHM frequency in IgM loss assays. We propose that MBD4 plays a role in SHM.

Endogenous rhythmic behaviors are evolutionarily conserved and essential for life. In mammalian and invertebrate models, well-characterized neuronal circuits and evolutionarily conserved mechanisms regulate circadian behavior and sleep [1-4]. In Drosophila, neuronal populations located in multiple brain regions mediate arousal, sleep drive, and homeostasis (reviewed in [3, 5-7]). Similar to mammals [8], there is also evidence that fly glial cells modulate the neuronal circuits controlling rhythmic behaviors, including sleep [1]. Here, we describe a novel gene (CG14141; aka Nkt) that is required for normal sleep. NKT is a 162-amino-acid protein with a single IgC2 immunoglobulin (Ig) domain and a high-quality signal peptide [9], and we show evidence that it is secreted, similar to its C. elegans ortholog (OIG-4) [10]. We demonstrate that Nkt-null flies or those with selective knockdown in either neurons or glia have decreased and fragmented night sleep, indicative of a non-redundant requirement in both cell types. We show that Nkt is required in fly astrocytes and in a specific set of wake-promoting neurons-the mushroom body (MB) α\'β\' cells that link sleep to memory consolidation [11]. Importantly, Nkt gene expression is required in the adult nervous system for normal sleep, consistent with a physiological rather than developmental function for the Ig-domain protein.

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IMGT®, the international ImMunoGeneTics information system® ( http://www.imgt.org ), was created in 1989 in Montpellier, France (CNRS and Montpellier University) to manage the huge and complex diversity of the antigen receptors, and is at the origin of immunoinformatics, a science at the interface between immunogenetics and bioinformatics. Immunoglobulins (IG) or antibodies and T cell receptors (TR) are managed and described in the IMGT® databases and tools at the level of receptor, chain and domain. The analysis of the IG and TR variable (V) domain rearranged nucleotide sequences is performed by IMGT/V-QUEST (online since 1997, 50 sequences per batch) and, for next generation sequencing (NGS), by IMGT/HighV-QUEST, the high throughput version of IMGT/V-QUEST (portal begun in 2010, 500,000 sequences per batch). In vitro combinatorial libraries of engineered antibody single chain Fragment variable (scFv) which mimic the in vivo natural diversity of the immune adaptive responses are extensively screened for the discovery of novel antigen binding specificities. However the analysis of NGS full length scFv (~850 bp) represents a challenge as they contain two V domains connected by a linker and there is no tool for the analysis of two V domains in a single chain.
The functionality \"Analyis of single chain Fragment variable (scFv)\" has been implemented in IMGT/V-QUEST and, for NGS, in IMGT/HighV-QUEST for the analysis of the two V domains of IG and TR scFv. It proceeds in five steps: search for a first closest V-REGION, full characterization of the first V-(D)-J-REGION, then search for a second V-REGION and full characterization of the second V-(D)-J-REGION, and finally linker delimitation.
For each sequence or NGS read, positions of the 5\'V-DOMAIN, linker and 3\'V-DOMAIN in the scFv are provided in the \'V-orientated\' sense. Each V-DOMAIN is fully characterized (gene identification, sequence description, junction analysis, characterization of mutations and amino changes). The functionality is generic and can analyse any IG or TR single chain nucleotide sequence containing two V domains, provided that the corresponding species IMGT reference directory is available.
The \"Analysis of single chain Fragment variable (scFv)\" implemented in IMGT/V-QUEST and, for NGS, in IMGT/HighV-QUEST provides the identification and full characterization of the two V domains of full-length scFv (~850 bp) nucleotide sequences from combinatorial libraries. The analysis can also be performed on concatenated paired chains of expressed antigen receptor IG or TR repertoires.