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Affinity maturation of the antibody response , a process of antibody affinity increasing over response, is one of the key features of the mammalian immune system. However, the process is incompletely understood in teleost, including channel catfish ( Ictalurus punctaus ). In this study, IgM affinity maturation in channel catfish was investigated by estimating the kinetics of antibody affinity using ELISA and ELISPOT assays . Fish were immunized with a T-cell dependent antigen (TNP-KLH), and individual serum IgM antibody titers and affinities, and IgM + antibody-secreting cells (ASCs) in peripheral blood were analyzed over a period of 14 weeks. A detectable serum anti-TNP response developed by 2-weeks post-immunization, and the maximal antibody production was observed by 6-weeks post-immunization. The average affinity of anti-TNP serum antibody increased consistently and reached the maximum by 10-weeks post-immunization. The increase of antibody affinity beyond the point of optimal antibody titer revealed that the affinity maturation of IgM antibody response occurred in channel catfish. Dissection of dynamics of individual affinity subpopulations indicated that a significant proportion of low affinity subpopulations appeared at early response, and high affinity subpopulations appeared predominantly at later, resulting in a 100-fold increase in affinity over response. Additional, TNP + IgM + ASCs was detected by 2-weeks post-immunization and achieved the maximal number by 6-weeks post-immunization. Using an inhibition ELISPOT assay, the findings of a consistent increase in the average affinity of secreted IgM antibody by peripheral blood ASCs, as the immune response progressed, confirmed the occurrence of the affinity maturation. Taken together, the results of this study indicated that affinity maturation occurred in channel catfish following immunization with a TD antigen TNP-KLH.
Chemokines play crucial roles in the regulation of immune cell migration and development. The CXCL12/CXCR4 axis has been extensively studied in mammals, but its regulatory mechanism in teleost fish remains unclear. In this study, we used Nile tilapia (Oreochromis niloticus) as a teleost model to investigate the mediation of the CXCL12/CXCR4 axis in IgM+ B cells. Our findings demonstrate that the CXCL12/CXCR4 axis exhibits chemotactic activity on IgM+ B cells and promotes the phagocytosis of IgM+ B cells. Blocking CXCR4 severely impairs the chemotaxis and phagocytosis of IgM+ B cells in vitro and reduces the percentages and numbers of IgM+ B cells that migrate to peripheral blood after pathogen infection in vivo. This reduction in migration leads to a decrease in the inflammatory response, an increase in tissue bacterial load, and a decrease in survival rate. We also discovered that the evolutionarily conserved PI3K-AKT signaling pathway and Girdin are involved in the immune response during Streptococcus agalactiae infection. Inhibitors of the PI3K-AKT signaling pathway prevent the chemotaxis and phagocytosis of IgM+ B cells, impair the expression and phosphorylation levels of related proteins in vitro, and prevent IgM+ B cells chemotaxis into the peripheral blood after pathogen infection in vivo. Furthermore, CXCR4 blocking significantly downregulates the expression of AKT and Girdin. Overall, our study reveals the regulatory mechanism of the CXCL12/CXCR4 axis on IgM+ B cells via the PI3K-AKT signaling pathway in tilapia, suggesting that the functions of the CXCL12/CXCR4 axis in B cells may be conserved between mammals and teleost fish.
Activation-induced deaminase (AID) is a key enzyme that catalyzes the deamination of cytosine to uracil in DNA, driving somatic hypermutation (SHM) and class switch recombination (CSR) of antibody genes to promote antibody diversification in the adaptive immunity. The AID gene from Cyprinus carpio (CcAID) has a full-length open reading frame of 633 bp and encodes a protein of 210 amino acids. CcAID shares structural and functional similarities with apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC) family members and contains a conserved catalytic domain characteristic of cytidine deaminases. Notably, the absence of two key phosphorylation sites in CcAID (Ser38 and Tyr184) may be a significant factor contributing to the markedly lower levels of somatic hypermutation (SHM) in teleost fish. A high level of CcAID transcript expression was detected in carp larvae on the sixth day after fertilization. Intraperitoneal injection of T-independent and T-dependent antigens preferentially upregulated CcAID expression in head kidney and splenic lymphocytes. Immunofluorescence analysis confirmed its expression in IgM + spleen lymphocytes. Recombinant CcAID exhibited cytidine deaminase activity. These findings indicate that CcAID is specifically expressed in IgM + spleen lymphocytes, suggesting its potential role in antibody production.
Acute respiratory infections (ARIs) are among the leading causes of morbidity and mortality worldwide, with Mycoplasma pneumoniae (M. pneumoniae) and Chlamydia pneumoniae (C. pneumoniae) being major pathogens responsible for community-acquired respiratory infections. Rapid and accurate diagnosis is critical for improving patient outcomes and effectively controlling disease transmission. Herein, we report a novel dual-channel lateral flow immunoassay (DC-LFIA) platform based on the Co3O4@polydopamine (PDA) nanocomposite with outstanding extinction coefficient, oxidase-like activity, and magnetic properties for the simultaneous detection of specific IgM antibodies (Ab) against these two pathogens in human serum. The magnetic properties of the Co3O4@PDA nanocomposite enable rapid enrichment of trace target Abs from large-volume serum samples, with a high extinction coefficient for direct colorimetric visualization and an excellent oxidase-like catalytic activity for signal enhancement. On the basis of the multifunctional Co3O4@PDA nanocomposite, rapid dual-mode detection of serum Abs was achieved within 12 min. By the direct colorimetric mode, the visual limit of detection (vLOD) for M. pneumoniae Ab and C. pneumoniae Ab reached 2 and 1 ng/mL, respectively, while the oxidase-like catalyzing signal amplification mode further lowered the vLODs to 0.2 and 0.1 ng/mL, accordingly. Additionally, the signal normalization using a 3D-printed device significantly enhanced assay accuracy and reproducibility. When applied to 96 clinical serum samples, compared to commercial kits, this multifunctional Co3O4@PDA-based DC-LFIA demonstrated superior performance, providing a reliable approach for simultaneous antibody detection in respiratory infection diagnosis. Overall, this work contributes a valuable diagnostic tool to the field of rapid detection of ARIs.
B lymphocyte-induced maturation protein 1 (Blimp-1), a transcription factor containing zinc finger , is required and sufficient to trigger terminal differentiation of B cells in mammals. The Blimp-1 ( OnBlimp-1 ) from Nile tilapia ( Oreochromis niloticus ) was identified and characterized its expression pattern during B cell activation and maturation. The cDNA of OnBlimp-1 open reading frame is 2547 bp encoding a protein of 848 amino acids and the predicted molecular weight is 93.36 kDa. OnBlimp-1 contains a SET domain and five ZnF_C2H2 domains, which shares high homology with that of other species. OnBlimp-1 transcription was detected in all examined tissues with high expression in the spleen (SPL). Analysis of sorted lymphocyte populations , including IgM + and IgM − cells from peripheral blood (PBL), SPL and anterior kidney (AK), indicated that the OnBlimp-1 transcription was highly expressed in the IgM + B cells. Upon LPS stimulation, OnBlimp-1 expression was up-regulated in tissues of PBL, SPL and AK significantly. The expression of OnBlimp-1 , as well as the secreted IgM , was significantly up-regulated in the SPL and AK leukocytes stimulated with anti-OnIgM monoclonal antibody and LPS in vitro , respectively. Above results suggest that OnBlimp-1, a cytokine regulating the terminal differentiation of activated B cells to antibody-secreting cells, is likely to play important roles in B cell activation and maturation in Nile tilapia.
Previous researches investigated the adsorption properties and mechanism of clay minerals on antibiotic pharmaceuticals and personal care products (PPCPs) via batch adsorption experiments and X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) characterization, but many PPCPs exhibit different ionization states under different pH conditions; the research on the microscopic mechanism of different ionized PPCPs species adsorbed by clay minerals is very scarce. In this work, as a typical PPCPs contaminant, the common tetracycline (TC) was chosen as the study target; the adsorption micro mechanism of montmorillonite (MMT) on different TC species in aqueous solution was explored in details. Especially outstanding, for the first time, with the help of Multiwfn wavefunction program, quantitative analysis of molecular surface analysis and basin analysis were applied to quantitatively investigate the electrostatic interaction between MMT and different TC species, the possible positions of lone pair electrons. Independent gradient model (IGM) analysis and Hirshfeld surface analysis including local surface analysis were integrated to visualize the binding sites, interaction types, and relative strength between MMT and TC +/− or TC +/2− species, which can insight into the interaction essence from the microscopic perspective at the atomic level.
Most current methods to assess T-cell-dependent antibody responses (TDAR) are semi-quantitative and based on measures of antibody titer generated against a standard antigen like keyhole limpet hemocyanin (KLH). The precision, sensitivity, and convenience of TDAR assays might be improved by applying rapid, sensitive, specific cytometric bead assays (CBA). In the study here, KLH antigen was covalently coupled onto the surface of cytometric beads using immune microsphere technology, and IgM antibody capture spheres were prepared for use in pretreatment processing of samples. The working parameters associated with this novel TDAR-CBA system were optimized in orthogonal experiments. The optimal concentration of the KLH coating solution in this system was 160 μg/ml, that of the anti-KLH IgG capture spheres 6.0 × 105/ml, and the optimal dilution of fluorescein isothiocyanate (FITC)-conjugated Affini-Pure Goat Anti-Mouse IgG (H + L) was 60 μg/ml. Repeated tests indicated that this approach yielded good linearity (r2 = 0.9937) method, with a within-run precision of 3.1–4.9%, and a between-run precision of 4.4–4.9%. This new approach had a limit of detection of 113.43 ng/ml (linear range = 390.63–50 000), and an interference rate of just 0.04–3.51%. Based on these findings, it seems that a new mouse TDAR assay based on CBA can be developed that would appear to be more sensitive, accurate, and precise than the current TDAR assay approaches based on traditional ELISA.
In order to investigate the structural characteristics and immunomodulatory effects of Poria cocos polysaccharides , a water-soluble homogeneous polysaccharide (PCP-2) was isolated by water extraction and alcohol precipitation and further purified by Cellulose DEAE-52 and Sephacryl S-100HR column chromatography . PCP-2 is a heteropolysaccharide composed of glucose, galactose , mannose , and fucose in a molar ratio of 42.0: 35.0: 13.9: 9.1. It exhibits a narrow molecular weight distribution at 2.35 kDa with a branching degree of 37.1 %. The main chain types of PCP-2 include 1,3- β -D-Glc and 1,6- β -D-Glc as the backbone glucans and 1,6- α -D-Gal as the backbone heterogalactan. In vitro experiments demonstrate that PCP-2 directly stimulate RAW264.7 cell proliferation and secretion of inflammatory factors such as NO and TNF- α . In cyclophosphamide (CTX)-induced mice, it promotes the development of thymus and spleen immune organs, elevates the blood levels of IgG, IgA, IgM and CD3 + CD4 + T cells , increases the intestinal villus height/ crypt depth ratio and improves gut barrier dysfunctions. These findings suggest that PCP-2 is a natural fungal polysaccharide with broad spectrum of immunoenhancing effects, which can significantly ameliorate the immunocompromised state.
The Omicron variants of SARS-CoV-2 have been spreading globally and has never disappeared from our sight, indicating that its coexistence with humans has become a fact, and monitoring its evolution and spread remains a current task. Although polymerase chain reaction (PCR) is the most commonly used virus detection method, it requires labor-intensive and time-consuming procedures in a laboratory setting. Herein, a multichannel nanoplasmonic sensing chip based on surface enhanced Raman spectroscopy (SERS) was developed for detecting N and S proteins, as well as IgG and IgM, related to SARS-CoV-2 Omicron variants. Through a self-screening process, specific antibodies for on-site and rapid identification of important variants of concerns (VoCs) were obtained, and their binding was confirmed by protein structure analysis. The use of these S protein specific antibodies can accurately identify Omicron VoCs (BA. 5, BF.7，XBB.1.5) with the detection limit (LoD) at 0.16 pg/mL. Then, the proposed SERS array chip was integrated with a hand-held Raman spectrometer to successfully detect the Omicron subvariants in real saliva samples within only 20 minutes, greatly reducing the detection time of PCR. This sensing technology will provide a powerful and rapid point-of-care testing (POCT) method for virus diagnosis, subtype identification, and post infection antibody level monitoring.
Bacterial disease is one of the important factors leading to economic losses in the turbot ( Scophthalmus maximus ) cultivation industry. T lymphocytes are major components of cellular immunity , whereas B lymphocytes produce immunoglobulins (Ig) that are key elements of humoral immune responses against infection. However, the genomic organization of genes encoding T-cell receptors (TCR) and immunoglobulin heavy chains (IgHs) in turbot remains largely unknown. In this study, abundant full-length transcripts of TCRs and IgHs were sequenced by Isoform-sequencing (Iso-seq), and we investigated and annotated the V, D, J and C gene loci of TCRα, TCRβ, IgT, IgM and IgD in turbot. Furthermore, through single-cell RNA sequencing (scRNA-seq) of blood leukocytes, we confirmed that these identified TCRs and IgHs were highly expressed in T/B cell clusters, respectively. Meanwhile, we also identified the IgM + IgD + B and IgT + B cells with differential gene expression profiles and potential functions. Taken together, our results provide a comprehensive understanding of TCRs and IgHs loci in turbot, which will contribute to evolutionary and functional characterization of T and B lymphocytes in teleost.
Niclosamide (NIC) is a commonly used insecticide and molluscicide in the prevention and treatment of parasitic diseases in fish. The utilization of NIC has the potential to disrupt the microbial community present on the mucosal tissue of fish, leading to localized inflammatory responses. The objective of this study was to evaluate the impact of NIC on the immune system and bacterial populations within the gill and gut of Mylopharyngodon piceus . Fish were subjected to varying concentrations of NIC, including a control group (0 μg/L), a low NIC group (15% 96 h LC50, LNG, 9.8 μg/L), and a high NIC group (80% 96 h LC50, HNG, 52.5 μg/L). Gill and gut samples were collected 28 days post-exposure for analysis. The findings revealed that the 96-h LC 50 for NIC was determined to be 65.7 μg/L, and histopathological examination demonstrated that exposure to NIC resulted in gill filament subepithelial edema, exfoliation, degeneration, and a decrease in gill filament length. Furthermore, the gut exhibited apical enterocyte degeneration and leucocyte infiltration following NIC exposure. Additionally, NIC exposure led to a significant elevation in the levels of immunoglobulin M (IgM), complement component 3 (C3), and complement component 4 (C4) in both gill and gut tissues. Moreover, the activity of lysozyme (LYZ) was enhanced in the gill, while the activities of peroxidase (POD) and immunoglobulin T (IgT) were increased in gut tissue. The exposure to NIC resulted in enhanced mRNA expression of c3 , c9 , tnfα , il6 , il8 , and il11 in the gill tissue, while decreasing c3 and il8 expression in the gut tissue. Furthermore, the natural resistance-associated macrophage protein ( nramp ) mRNA increased, and liver-expressed antimicrobial peptide 2 ( leap2 ) mRNA decreased in gill and gut tissues. And hepcidin ( hepc ) mRNA levels rose in gill but fell in gut tissue. NIC exposure also led to a decrease in gill bacterial richness and diversity, which significantly differed from the control group, although this separation was not significant in the gut tissue. In conclusion, the administration of NIC resulted in alterations in both the immune response and mucosal microbiota of fish. Furthermore, it was noted that gills displayed a heightened vulnerability to sublethal effects of NIC in comparison to gut tissues.
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