Search Results (1,121)

Inhibin, a water-soluble protein emitted by the gonads, plays a pivotal role in regulating the release of follicle-stimulating hormone (FSH) from the pituitary gland, which, in turn, influences follicular growth, gamete production, and the secretion of associated hormones. We performed high-throughput sequencing of the nanobody gene in the lymphocytes of Bactrian camels before and after inhibin α protein immunization followed by mass spectrometry analysis of specific antibodies to this protein in the serum following immunization to screen for inhibin α subunit-specific nanobodies. Seven inhibin α-specific nanobodies, namely Nb-1712, Nb-1971, Nb-2000, Nb-799, Nb-2004, Nb-1737, and Nb-338, were identified through high-throughput sequencing and mass spectrometry. Following the construction and expression of a prokaryotic expression vector, five of these nanobody proteins were successfully produced. These proteins demonstrated high affinity for inhibin α in the indirect enzyme-linked immunosorbent assay. Notably, nanobodies Nb-1737, Nb-1971, and Nb-2004 significantly downregulated Inha and upregulated Fshb gene expression, enhancing follicle-stimulating hormone secretion. In female mice, these three nanobodies promoted follicular development and led to a numerical increase in litter size (average ~10%, with Nb-2004 showing a 14.93% increase), although the differences were not statistically significant. These findings demonstrate their potential to regulate reproductive function. We identified 7 inhibin α subunit-specific nanobody genes from a Xinjiang Bactrian camel’s lymphocyte genome through high-throughput sequencing and mass spectrometry. We also compared their relative binding affinities and characterized their biological functions, thereby providing key theoretical guidance and technical support for increasing FSH levels. Full article

Mucosal immunity in the respiratory tract provides the first line of defense against airborne pathogens, yet most current vaccines fail to induce strong and durable immune responses at these sites. Respiratory viruses, including respiratory syncytial virus (RSV), influenza viruses, and coronaviruses, remain major global health threats, in part due to their ability to evade long-term mucosal protection. Although systemic vaccination generates robust circulating immunity, it induces limited local responses, particularly secretory immunoglobulin A (IgA), which is critical for preventing viral entry and transmission at the airway surface. The mechanisms regulating B cell responses within the airway mucosa are not fully understood. B cell–activating factor (BAFF), a member of the tumor necrosis factor (TNF) superfamily, has emerged as an important context-dependent regulator of mucosal B cell immunity. BAFF is produced by airway epithelial cells and multiple myeloid populations, including dendritic cells and neutrophils, and is rapidly induced during respiratory viral infection through type I interferon–dependent pathways. Functionally, BAFF supports B cell survival, differentiation, and class-switch recombination, promoting the generation of antibody-secreting plasma cells and enhancing IgA production. In the lung, these effects align with early, intermediate, and late stages of the response, supporting initial local antibody production, the formation of inducible bronchus-associated lymphoid tissue (iBALT), and the development of tissue-resident memory B cells that sustain long-term immunity. Although BAFF plays an essential role in mucosal immunity, its activity requires tight regulation to maintain immune balance. Current evidence supports BAFF as a promising immunomodulatory component and highlights its potential as an adjuvant platform for enhancing mucosal vaccine efficacy, warranting further investigation as a potential adjuvant in this context. Full article

Immunoglobulins exhibit important biological functions, including the neutralization of cytotoxins, enhancement of phagocytic activity, and activation of the complement system, which have driven their widespread application in both the food and pharmaceutical industries. Due to their low cost and short production cycles, microbial expression systems such as bacteria and yeast have been increasingly developed in recent years for immunoglobulin production. However, microbial systems face considerable challenges in ensuring proper protein folding, accurate chain assembly, and the soluble expression of full-length immunoglobulins. Recent optimization strategies have focused on host engineering (e.g., modulating secretion pathways and chaperone proteins), the coordinated regulation of expression elements (e.g., optimizing the light-to-heavy chain ratio), and regulation of fermentation processes. In addition to summarizing the above strategies, this review discusses the progress made in expressing both full-length immunoglobulins and antibody fragments across different microbial hosts, analyzes the advantages and limitations of each system, and explores potential future directions, aiming to provide a reference for the efficient heterologous expression of immunoglobulins. Full article

This study aimed to determine whether intranasal hinokitiol modulates short-term salivary secretory IgA (SIgA) secretion dynamics and IgA antibody-forming cell (AFC) activity in the submandibular glands of aged mice, a model of age-associated mucosal immune decline. Aged BALB/c mice received intranasal hinokitiol (50 μg) once weekly for 4 weeks. Saliva was collected on days 0, 7, 14, and 21 at baseline, 0.5 h, 1.5 h, 3 h, and 6 h after each administration. SIgA levels were measured using an enzyme-linked immunosorbent assay. On day 21, IgA AFCs were enumerated using an enzyme-linked immunosorbent spot assay, and their viability and proliferative activity were assessed using the MTT assay. Salivary SIgA rose transiently after each dose, peaking at 1.5 h and returning to baseline by 6 h. By day 21, baseline SIgA secretion was significantly higher than at day 0, indicating a cumulative effect. IgA AFCs were unchanged in number, but viability and proliferation increased at 0.5 and 1.5 h, coinciding with SIgA peaks. Flow cytometry revealed significant expansion of B220⁺CD38⁺ memory B-cells; B220⁺CD138⁺ plasma cells were unaffected. Intranasal hinokitiol transiently enhances salivary SIgA secretion in aged mice, likely through short-term modulation of salivary gland immune activity. This non-invasive approach may aid mucosal defense in aging populations. These findings suggest that intranasal HNK may represent a novel non-invasive approach for enhancing mucosal immune function during aging and may provide a basis for future preventive strategies against oral and respiratory infections. Full article

Acute kidney injury (AKI) caused by ischemia–reperfusion injury (IRI) involves rapid activation of innate immune responses, in which myeloid-derived immune cells critically shape injury severity. Y-box binding protein 1 (YB-1) regulates pro-inflammatory gene expression intracellularly and can be secreted to function extracellularly, yet how these two compartments jointly influence early IRI pathology remains poorly understood. To dissect the roles of intracellular myeloid versus extracellular YB-1, we subjected myeloid-specific Ybx1 knockout, Ybx1^fl/fl × LysM^cre, mice and wild-type (WT) littermates to unilateral renal IRI following administration of either a neutralizing anti-YB-1 antibody or control IgG. Kidney injury, inflammation, immune cell recruitment, neutrophil extracellular trap (NET) formation, antibody localization, and Fcγ receptor expression were assessed by qRT-PCR, histology, immunostaining, Western blotting, and flow cytometry. Myeloid-specific knockout of Ybx1 markedly reduced renal inflammation, neutrophil infiltration, NET formation, and tubular injury. This protective phenotype was lost when extracellular YB-1 was simultaneously reduced: anti-YB-1 treatment in knockout mice restored pro-inflammatory cytokine expression, increased tubular damage markers such as NGAL and KIM-1, exacerbated neutrophil recruitment and NET formation, and led to luminar accumulation of YB-1/anti-YB-1 immune complexes in tubular cells. Mechanistically, Ybx1-deficient myeloid cells exhibited significantly reduced CD16 expression, pointing to impaired Fcγ receptor-mediated phagocytosis as the cause of defective immune complex clearance. In contrast, wild-type mice efficiently cleared extracellular YB-1 complexes and showed no injury aggravation upon antibody treatment. Our findings identify myeloid YB-1 as a central regulator of early inflammatory injury in renal IRI and reveal that its protective depletion becomes pathogenic when extracellular YB-1 is simultaneously neutralized, likely due to unmasked defects in immune complex clearance. Full article

Concurrent multiple myeloma (MM) and AL amyloidosis is associated with the poorest outcomes among plasma cell dyscrasias and has dramatically reduced median overall survival. Despite their clinical significance, comprehensive systematic histopathological studies, characterizing multiorgan involvement and lesion severity are remarkably scarce. This study includes 24 autopsies (of 18 women and six men; median age—68 years) with MM-AL. Immunohistochemical (IHC) typing was performed with an expanded antibody panel targeting the amyloid precursor protein; anti-human CD138 antibody was used to identify plasma cells in bone marrow sections. Clinical diagnosis of MM with monoclonal G-lambda secretion, Durie–Salmon Stages II–III, was established in 17 (71%) patients; MM with monoclonal G-kappa secretion, Stage III, in five (21%); and non-secreting MM in two (8%). Systemic amyloidosis was revealed during life in only 15 (62.5%) patients. In all cases, extensive amyloid deposits were observed in the myocardium, lungs and kidneys, establishing the morphological basis for multiorgan failure. IHC typing of amyloids confirmed 18 (75%) cases of AL-lambda amyloidosis and six (25%) of AL-kappa amyloidosis. Our results clarify MM-AL morphogenesis and underscore that AL is frequently underdiagnosed in MM patients. Comprehensive histopathological studies with IHC typing are necessary to confirm the diagnosis, refine the prognosis, and optimize the therapeutic strategies. Full article

Annexin A6 (AnxA6) is a predominantly intracellular calcium-dependent membrane-binding multifunctional protein that is also detected extracellularly and in small extracellular vesicles (exosomes). We previously demonstrated that lapatinib resistance in triple-negative breast cancer (TNBC) cells is associated with AnxA6 upregulation and accumulation of cholesterol in late endosomes. Here, we investigated the fate of AnxA6 and cholesterol in lapatinib-resistant (LAP-R) cells and whether extracellular AnxA6 influences TNBC cell survival. We demonstrate that reduced expression of AnxA6 in LAP-R cells decreased the secretion of MCP-1/CCL2, CCL8/IL-8, DKK1, TSP-1, and OPN by antibody arrays. The secretion of exosomes was also markedly reduced in AnxA6-depleted LAP-R cells, while AnxA6 upregulation stimulated the release of MCP-1 and exosomes. Compared to the respective controls, exosome-associated AnxA6, Rab7, and cholesterol levels were increased in exosomes isolated from AnxA6-expressing LAP-R cells. Mechanistically, we demonstrated by co-immunoprecipitation, GST pulldown, and proximity ligation assays that AnxA6 interacts with SNAP23, a component of the membrane fusion machinery. Finally, blocking extracellular AnxA6 with neutralizing antibodies reduced the viability of AnxA6-low TNBC cells but had little effect on AnxA6-high cells. These findings suggest that extracellular AnxA6 is critical for the survival of highly proliferative AnxA6-low basal-like breast cancer cells and that AnxA6 influences TNBC progression by facilitating the secretion of pro-inflammatory cytokines and cholesterol-enriched exosomes. Full article

Background/Objectives: The induction of anti-SARS-CoV-2 antibodies by COVID-19 vaccination reduces morbidity and mortality, but immune responses may be compromised in people living with HIV (PLWH). The aims of the current study were to determine whether viral suppression (VS) or immune reconstitution (IR) in PLWH directly affected their ability to produce effective levels of anti-SARS-CoV-2 antibodies in mucosal secretions or blood induced by vaccination. Methods: Anti-SARS-CoV-2 spike IgG, IgA and secretory IgA (SIgA) antibodies and their avidities were measured by ELISA in HIV-negative healthy controls (HC; n = 49) and PLWH (n = 94) using stimulated oral fluid (SOF) and serum. Frequencies of CD4/CD8 T cells and their expression of exhaustion/senescence were determined by flow cytometry. Cytokine levels were measured by cytokine bead arrays. Results: We showed that higher HIV burden negatively impacted the levels of systemic and mucosal anti-SARS-CoV-2 spike IgG antibodies produced. This differential IgG antibody production was unaffected by IR status, antiretroviral therapy duration or T cell exhaustion/senescence. PLWH elicited higher anti-SARS-CoV-2 spike IgA antibodies both in peripheral blood and oral mucosa and highr secretory IgA (SIgA) antibodies in the oral mucosa. PLWH with higher HIV RNA copies elicited lower IgG avidity but the IgA avidity indices remained unaffected. PLWH expressed higher levels of innate immunity cytokines in the oral mucosa, irrespective of the HIV RNA copies. Conclusions: Significantly fewer breakthrough infections in PLWH compared with HC, along with high IgA/SIgA antibodies and increased innate immunity cytokines in the SOF, suggest a potential role for mucosal immunity in the immunopathogenesis of COVID-19. Full article

Interleukin-6 (IL-6) is a cytokine with multiple biological effects. It plays a complex and seemingly paradoxical central role in both the physiological homeostasis and pathological processes of skeletal muscle. Under physiological conditions, particularly during acute exercise, IL-6 produced and secreted by the contracting skeletal muscle itself acts as an important “myokine.” It operates in an autocrine, paracrine, or endocrine manner to regulate systemic energy metabolism, insulin sensitivity, muscle regeneration, and adaptive hypertrophy. This function is crucial for the health benefits conferred by exercise. However, under various pathological conditions—such as cancer cachexia, sepsis, muscular dystrophy, denervation, disuse atrophy, and chronic inflammatory diseases—persistently elevated systemic or local IL-6 levels become a key mediator driving skeletal muscle atrophy, metabolic disorders, and functional decline. This review systematically elaborates on the dual role of IL-6 in skeletal muscle. It provides an in-depth analysis of its downstream signaling pathways (e.g., JAK/STAT, gp130, MAPK, PI3K-Akt) and upstream regulatory mechanisms (e.g., the Piezo1/KLF15 axis, calcium signaling, mitochondrial function, oxidative stress). A particular focus is placed on discussing the distinct biological effects of classical IL-6 signaling versus trans-signaling. Furthermore, we address current challenges in research and practice, including the cell specificity of IL-6 signaling, the complexity of its temporal regulation, the definition of physiological versus pathological concentrations, discrepancies between animal models and human diseases, and the plasticity of its function across different pathological contexts. Finally, this review explores the potential of targeting the IL-6 signaling pathway as a therapeutic strategy for skeletal muscle atrophy and related metabolic diseases. Potential interventions include IL-6/IL-6R monoclonal antibodies, JAK/STAT inhibitors, gp130 modulators, exercise interventions, and nutritional strategies. This aims to provide a theoretical foundation and novel perspectives for future translational research and clinical interventions. Full article

Monoclonal antibody (mAb) discovery has been transformed by advances in single-cell technologies, microfluidics, high-throughput sequencing, and computational design. Modern platforms enable the interrogation of large numbers of individual B cells, directly linking antibody sequence with antigen specificity and functional activity. Microfluidic and optofluidic systems now support high-throughput compartmentalisation and functional screening of antibody-secreting cells, while sequencing-based approaches allow parallel recovery of paired heavy- and light-chain sequences. These developments have shifted antibody discovery from binding-based selection toward function-first paradigms, enabling the rapid identification of diagnostic and therapeutically relevant antibodies. Integration with computational tools, including machine learning and structure-based modelling, has further enabled the emergence of closed-loop discovery pipelines, in which experimental and in silico methods iteratively refine candidates. This review summarises key advances in single-cell microtools over the last decade and highlights how the convergence of experimental and computational technologies is reshaping antibody discovery toward scalable, data-driven, and increasingly automated platforms. Full article

The alkaline pectate lyase A from Paenibacillus barcinonensis, encoded by pelA (GenBank accession no. CAB40884), is an enzyme with high activity on pectin and potential application in sustainable industrial biotechnology. In this study, pelA was expressed in Saccharomyces cerevisiae by using different domains of the cell wall protein Pir4 as translational fusion partners. Given the presence of five potential N-glycosylation sites in the amino acid sequence coded by pelA, and two of them in conserved regions of class III pectate lyases, the effect of glycosylation on the enzymatic activity of the recombinant enzyme was investigated by expressing the recombinant fusion proteins in both standard and glycosylation deficient strains of S. cerevisiae. The correct targeting of the recombinant fusion proteins was confirmed by Western blot analysis using Pir-specific antibodies, whilst enzymatic activity on polygalacturonic acid was demonstrated on both plate assays and colorimetric assays. Maximum activities were over two and a half times higher when the enzyme was expressed in the glycosylation deficient strain, suggesting a better adaptation of this strain to the secretion of the functional enzyme. Full article

Background: Glioblastoma (GBM) is characterized by pronounced transcriptional plasticity and a highly structured immune microenvironment, yet the molecular features associated with tumor-state transitions and immune remodeling remain incompletely understood. Methods: We used an integrative multi-omics framework to examine how secreted phosphoprotein 1 (SPP1) relates to tumor microenvironment organization in human gliomas. Results: Single-cell analyses associated SPP1 with myeloid populations, mesenchymal-like (MES-like) malignant states, inflammatory regulatory programs, and inferred ligand–receptor co-expression patterns involving SPP1–CD44 and SPP1–integrin pairs. Spatial transcriptomic analyses showed that SPP1-high regions were enriched for estimated myeloid abundance, MES-like tumor signal, and ECM/angiogenic programs, supporting an SPP1-associated spatial mesenchymal–myeloid program in GBM. Computational perturbation analyses provided network-level support for SPP1–CD44-associated stress-responsive programs. HPA immunohistochemistry provided tissue-level protein context for SPP1 and related mesenchymal/receptor-associated components. Ivy GAP analysis showed enrichment of SPP1-associated features in core-like anatomic compartments, and CODEX spatial protein imaging provided antibody-panel-based contextual support for mesenchymal–myeloid-associated features. In the TCGA-GBM cohort, elevated SPP1 expression and an SPP1-associated mesenchymal signature were associated with poorer overall survival. Conclusions: These findings support an inferential model in which SPP1 is associated with spatial mesenchymal–myeloid organization in GBM and nominate SPP1-associated programs as candidate readouts of tumor plasticity, inflammatory myeloid remodeling, and spatial tumor microenvironment organization. Full article

Background/Objectives: Lactobacillus rhamnosus GG (LGG) is one of the most widely utilized probiotic strains with a variety of biological functions including prevention and treatment of gastro-intestinal infections and regulation of immune responses. Methods: Here, we explored the role of LGG in regulating the differentiation of naïve CD4⁺ T cells and its effect on alleviating the dextran sulfate sodium (DSS)-induced colitis in mice. Results: In vitro, we showed that LGG-derived metabolites not only promoted the differentiation of naive CD4⁺ T cells into T-helper 17 cells (Th17 cells), but also selectively upregulated the expression of lactate-specific transporter solute carrier family 5 member 12 (SLC5A12). Interestingly, we manipulated a CD4⁺ T cell-monocytes co-culture and found that heated LGG-loaded monocytes modulate naive CD4⁺ T cells to differentiate preferentially into Treg cells rather than Th17 cells. To explain the above-mentioned contradiction, we used an experimental colitis model and found that LGG administration alleviated the DSS-induced colitis in mice, as indicated by decreases in weight loss and disease activity index. Moreover, SLC5A12 blockade (using a specific antibody) further reduced the colonic histological inflammatory score and decreased secretion of proinflammatory cytokines such as IFN-γ, IL-6, IL-17F, and IL-21. Notably, SLC5A12 blockade abolished the LGG-induced differentiation of the IL-17⁺CD4⁺ T (Th17) cells but significantly increased the frequency of Foxp3⁺CD4⁺ T (Treg) cells in the colonic lamina propria. Furthermore, a higher intracellular lactate concentration was observed in the colonic CD4⁺ T cells isolated from the LGG-treated colitic mice compared with other groups. Additionally, we also found elevated levels of oxidative stress indicators such as MDA and H₂O₂, as well as excessive reactive oxygen species (ROS) in colonic tissue of DSS-treated only mice, while LGG can scavenge ROS by inducing nuclear factor-erythroid 2-related factor 2 (Nrf2) expression in enterocytes. Conclusions: Altogether, these results indicate that LGG might alleviate preclinical colitis by modulating the Th17/Treg balance, and SLC5A12 blockade appears to enhance the anti-inflammatory properties of LGG. Full article

Differentiation therapy holds significant potential for the treatment of multiple myeloma (MM). We previously identified that the aminopeptidase N (APN) inhibitor Bestatin promotes MM cell differentiation. Herein, we elucidate the underlying molecular mechanisms of this process. Utilizing MM1.S, U266, and RPMI-8226 cell lines, a combination of CCK-8 assays, flow cytometry, Wright–Giemsa staining, Western blotting, qRT-PCR, ELISA, APN enzymatic activity analysis, SA-β-gal staining, and bioinformatic analyses revealed elevated APN expression across all cell types. Bestatin treatment induced MM cell differentiation in a concentration-dependent manner, which was accompanied by the upregulation of the differentiation marker CD49e, increased immunoglobulin light chain secretion, elevated cellular senescence, and a concomitant suppression of cell proliferation and APN enzymatic activity. Mechanistically, Bestatin exerts its effects by downregulating the CD79B/BTK signaling pathway, thereby activating the downstream transcription factor STAT3. Consistent with this axis, direct inhibition of CD79B/BTK alone was sufficient to induce differentiation, while blockade of STAT3 completely abrogated the differentiation-promoting effect of Bestatin. The APN-neutralizing antibody (WM15) yielded consistent results with Bestatin, further validating this regulatory axis. Furthermore, both the CD79B/BTK inhibitor Ibrutinib and the STAT3 agonist GCDA potentiated the cytotoxicity of the clinical MM drug Ixazomib. Bestatin itself synergized with Ixazomib and enhanced the anti-proliferative effect of IL-6. In summary, our findings establish that the APN inhibitor Bestatin induces MM cell differentiation via the CD79B/BTK-STAT3 signaling axis. Targeting this pathway represents a promising strategy to enhance the efficacy of Ixazomib, providing a compelling rationale for novel combination therapies in MM. Full article

Characterizing the specific interactions of Leishmania species with different host systems is essential for the development and validation of experimental infection models and for identifying potential therapeutic targets. Leishmania parasites elicit diverse host immune responses that result in different levels of disease severity. Here, we developed a murine model of L. panamensis infection and compared the responses of BALB/c and C57BL/6 mice following intradermal ear inoculation. BALB/c mice developed progressive ulcerative lesions associated with high parasite burden, whereas C57BL/6 mice exhibited a transient edema and maintained low parasite levels detected only at early stages of infection. C57BL/6 mice displayed early production of IL-13, IL-4, and IL-10, followed by delayed IFN-γ secretion. In contrast, BALB/c mice showed a mixed Th1/Th2 response at later stages of infection. Humoral responses also differed between strains, with BALB/c mice developing an early and sustained IgG1-dominated response, while C57BL/6 mice exhibited weak and delayed antibody production. These findings suggest that resistance to L. panamensis infection in C57BL/6 mice is associated with an early and transient Th2/regulatory response accompanied by a weak and delayed antibody production. Full article