Search Results (468)

Sepsis is a life-threatening syndrome characterized by dysregulated host responses to infection. In addition to early hyperinflammation, many patients develop profound immune suppression, and multiple targeted immunotherapies have failed to improve outcomes, highlighting the need for actionable biomarkers and new therapeutic strategies. Here, we integrated metabolomic and transcriptomic profiling of peripheral blood mononuclear cells (PBMCs) and splenocytes in rat models of polymicrobial sepsis to identify metabolites associated with immune dysfunction. Candidate findings were validated using in vivo supplementation studies and in vitro functional assays, and clinical relevance was assessed in PBMCs from patients with sepsis and healthy volunteers. Across omics datasets, intracellular aspartate (ASP) was consistently reduced in immune cells during sepsis and was associated with features of immune paralysis. Supplementation with L-ornithine L-aspartate (LOLA), an ASP source, improved survival in septic rats, enhanced bacterial clearance, and mitigated acute kidney injury. In vitro, pharmacologic or genetic disruption of ASP production impaired phagocytosis and cytokine responses, which were partially rescued by ASP supplementation. Consistently, patients with sepsis exhibited lower intracellular ASP levels in PBMCs than healthy volunteers. Together, these results support a critical role for ASP in maintaining immune competence during sepsis and suggest that intracellular ASP may serve as a biomarker of immune suppression and a potential therapeutic target. Full article

Fermented soybean-based foods contain diverse bioactive compounds with recognized health benefits. Among them, doenjang is widely consumed in East Asia and has been associated with protective effects against several disorders, including immunosuppression. This study evaluated the immunoenhancing effects of doenjang sourced from four regions of Korea in cyclophosphamide (CP)-induced immunosuppressed rats. Four-week doenjang administration restored spleen weight and improved hematological parameters, including white blood cell, lymphocyte, neutrophil, and monocyte counts. Additionally, doenjang intake enhanced immune function, as evidenced by increased splenic natural killer cell activity, increased splenocyte proliferation under lipopolysaccharide- and concanavalin A-stimulated conditions, and higher levels of interleukin (IL)-2, IL-12, interferon-γ, and immunoglobulin G. Furthermore, the suppressed phosphorylation of mitogen-activated protein kinases/nuclear factor kappa B signaling was recovered, accompanied by improved splenic structure. Collectively, our findings demonstrate that the regional varieties of doenjang effectively mitigate CP-induced immune dysfunction, indicating their potential as functional dietary interventions. Full article

Background: Schistosomiasis affects more than 250 million people, and praziquantel remains the only drug available for treatment; however, its activity is restricted to adult worms. Previously, our group evaluated six thiazole derivatives (PBT1–PBT6) in vitro against adult Schistosoma mansoni, identifying PBT2, PBT5, and PBT6 as the most active compounds. The present study aimed to evaluate the in vitro activity of PBT2, PBT5, and PBT6 against schistosomula and juvenile worms, as well as their in vivo efficacy against adult S. mansoni. Methods: Mechanically transformed schistosomula and juvenile worms recovered from mice (21 days post-infection) were incubated with the compounds (12.5–200 μM). Cytotoxicity was assessed using murine splenocytes and peritoneal macrophages exposed to the same concentration range. For in vivo evaluation, infected mice were orally treated with compounds (50, 100, or 200 mg/kg) for five consecutive days. Results: All compounds induced 100% mortality in schistosomula and juvenile worms within 3 h of exposure at 100 and 200 μM. Parasite cell viability was markedly reduced (>90%) at concentrations between 50 and 200 μM. The LC₅₀ values ranged from 15.3 to 30.9 μM for schistosomula and from 27.8 to 34.9 μM for juvenile worms, with low cytotoxicity observed in mammalian cells (CC₅₀ ≥ 193.9 μM). In vivo treatment resulted in significant reductions in fecal egg counts (~80% at 200 mg/kg), total worm burden (~60%), and egg loads in liver and intestinal tissues, in addition to an increased proportion of dead eggs in the intestine. Conclusions: The evaluated thiazole derivatives demonstrated potent in vitro activity against immature stages of S. mansoni and significant in vivo efficacy against adult parasites, accompanied by favorable changes in key parasitological parameters. These findings reinforce the potential of thiazole-based compounds as promising multistage schistosomicidal candidates. Full article

Porcine epidemic diarrhea (PED), caused by the porcine epidemic diarrhea virus (PEDV), is an acute and highly contagious intestinal disease that inflicts substantial economic losses on the global swine industry. The nucleocapsid (N) protein of PEDV plays a critical role during viral infection and replication. In this study, the full-length N gene was cloned and expressed using the prokaryotic expression vector pET-32a (+). The purified recombinant N protein was used to immunize BALB/c mice. Subsequently, splenocytes from the immunized mice were fused with SP2/0 cells, and hybridoma cell lines secreting monoclonal antibodies (mAbs) against N protein were screened via indirect ELISA. The linear B-cell epitopes recognized by the mAbs were mapped using truncated N protein fragments. Results showed that three stable hybridoma cell lines (1A3, 1G1 and 1A10) secreting N protein-specific mAbs were obtained. Epitope mapping revealed that mAbs 1A3 and 1G1 recognized the epitope ⁷¹SNWHF⁷⁵, whereas mAb 1A10 recognized ⁶⁶RIEQP⁷⁰. Bioinformatics analysis indicated that these epitopes are highly conserved among the analyzed PEDV strains and show no cross-reactivity with the N proteins of other coronaviruses. These findings could provide valuable experimental materials for further investigation of the N protein’s structure and function and support the development of diagnostic assays and subunit antigen vaccine for PEDV. Full article

Systemic lupus erythematosus (SLE) is a severe autoimmune disease that is challenging to treat due to poor understanding of its pathogenesis and etiology. Clearly understanding and dissecting the therapeutic effects of potential treatment in animal models are important. It has been shown that human alpha-1 antitrypsin (hAAT) holds therapeutic potential for the treatment of autoimmune diseases including lupus. However, the mechanism underlying its protective effect requires further investigation. In the present study, we used a chronic graft-versus-host disease-induced lupus mouse model to test the effect of hAAT on lupus development. We performed adoptive transfer of MHC I-aβ mismatched bm12 splenocytes into hAAT transgenic mice and showed that hAAT significantly blocked the production of anti-dsDNA IgG autoantibodies. Mechanistically, hAAT inhibited T cell activation and proliferation, including that of effector memory T (Tem) and T follicular helper (Tfh) cells. In addition, hAAT suppressed germinal center formation and functions. These results advanced the current understanding of hAAT functions and provide a new insight for the treatment of SLE. Full article

Background/Objectives: Schlafen (SLFN) 8 and SLFN9 are mouse members of the Schlafen protein family, believed to have arisen through a gene duplication event. The physiological roles of these proteins remain poorly defined, in part due to the absence of reliable, commercially available antibodies for their detection. Methods: To develop specific antibodies, we performed an amino acid sequence alignment of these proteins and identified a thirteen amino acids long peptide predicted by AlphaFold modeling and hydropathicity analysis to be surface-exposed in both SLFN proteins. The SLFN8 peptide was conjugated to KLH and used to immunize mice, employing Poly(I:C) as an adjuvant. Results: We verified the anti-SLFN8 antibody specificity in mouse tissues, engineered human cells, and recombinant proteins by different immunodetection techniques, including Western blotting, immunoprecipitation, immunohistochemistry, and ELISA. Furthermore, splenocytes from immunized mice were used to generate hybridomas that secreted IgG antibodies with SLFN8-peptide specificity, as assumed by ELISA. Conclusions: Our results demonstrate that the identified peptide is highly immunogenic and capable of eliciting antibodies that distinguish between these two exceedingly similar proteins in a broad group of immunodetection techniques. Full article

Nutritional strategies based on sourdough fermented breads with wholemeal ancient grains and legumes are emerging as promising modulators of (neuro)immune processes. This study investigated whether prolonged consumption of a sourdough bread enriched with a mixture of ancient cereals and legumes, commercially available in Italy (Primus^® bread, P^®B), modulates neuroimmune systemic responses to repeated lipopolysaccharide (LPS) challenge in mice. For this study, male C57BL/6J mice were fed for 14 days with either a standard diet (SD) or P^®B. Animals then received intraperitoneal LPS (3 mg/kg/day for 3 days) or vehicle. Body weight and food intake were monitored throughout. Pain-like behaviours were assessed by von Frey, plantar and tail flick tests, and plasma cytokine (32-plex panel), splenocyte and peritoneal macrophage cytokine expression, and expression of pro-inflammatory cytokines in sciatic nerves, dorsal root ganglia (DRG) and the spinal cord were analyzed by Reverse Transcription–quantitative Polymerase Chain Reaction (RT-qPCR). P^®B prevented LPS-induced body weight loss and reduced splenomegaly. Unlike SD mice, which exhibited widespread plasmatic cytokine upregulation, P^®B-fed mice displayed only limited increases Interleukin (IL)-1β, IL-12p40 and Tumor Necrosis Factor (TNF)α. Ex vivo cultures of splenocytes and macrophages confirmed attenuated cytokine overexpression. LPS-induced hypersensitivity to mechanical, thermal and nociceptive stimuli was significantly reduced in P^®B mice. Molecular analyses revealed that the P^®B diet blunted the pro-inflammatory cytokine expression present after LPS challenge in the sciatic nerves and DRG, with partial attenuation in the spinal cord. Our findings highlight the great potential of functional foods as affordable dietary strategies to mitigate systemic immune and neuroimmune dysregulation. Full article

Background: Experimental autoimmune thyroiditis is an important animal model for studying Hashimoto’s thyroiditis. Our aim was to develop the model using CBA/J-DR3 mice expressing human HLA-DR3, which is associated with autoimmune thyroiditis in humans, to better simulate human autoimmune thyroiditis. Such a humanized model can be used to test specific antigen therapies for autoimmune thyroiditis. Methods: CBA/J-DR3 mice were produced by back-crossing B6-DR3 mice to the CBA/J background. Female CBA/J-DR3 mice were immunized with human thyroglobulin (Tg) in complete Freund’s adjuvant on days 0 and 7. On day 21, mice were sacrificed, blood collected, spleen and thyroid harvested for analysis. Splenocytes were analyzed for T cell responses to Tg and its major T-cell epitope in human autoimmune thyroiditis, Tg.2098. Serum anti-thyroglobulin antibodies were measured by ELISA, and thyroid-stimulating hormone was measured using the Luminex assay. Thyroid histology and immunohistochemistry were examined. Results: Immunized CBA/J-DR3 mice showed significant T cell proliferation in response to Tg (stimulation index 3.4 ± 4.5) and Tg.2098 (1.5 ± 0.7). Anti-thyroglobulin antibody levels were elevated in immunized mice when compared to control mice (2.05 ± 0.75 vs. 0.15 ± 0.06, p < 0.0001). T cells demonstrated higher reactivity to thyroid antigens by enhanced production of pro-inflammatory cytokines. Thyroid immunohistochemistry revealed mild CD3-positive T-cell infiltration. Conclusions: This novel humanized CBA/J-DR3 mouse model of Hashimoto’s thyroiditis demonstrates key features of human autoimmune thyroiditis. The HLA-DR3 background and the immune response to Tg and Tg.2098 enhance translational relevance, making this a valuable model for studying thyroid disease pathogenesis and testing targeted immune-modifying therapies. Full article

Heat-killed Enterococcus faecalis EF-2001 (EF-2001) is a postbiotic preparation reported to modulate host immunity. However, its specific impact on host immune responses and virological outcomes during the early phase of influenza infection remains insufficiently characterized. Female BALB/c mice received oral EF-2001 (16 mg/kg/day) for either 4 days or 14 days prior to intranasal inoculation with influenza A/H3N2 (A/Aichi/2/68). On day 2 post-infection, splenic T-cell subsets (CD3⁺, CD4⁺, CD8⁺) were quantified by flow cytometry. Cytokines released from PMA/ionomycin-stimulated splenocytes were measured using a cytometric bead array assay to assess functional polarization. Lung viral titers (TCID₅₀) and interferon-α (IFN-α) concentrations were assessed to evaluate local antiviral efficacy. EF-2001 administration significantly increased the proportions of splenic CD3⁺ T cells, including both CD4⁺ and CD8⁺ subsets, compared to controls. The 14-day pretreatment regimen significantly enhanced IFN-γ production while reducing IL-10, IL-4, and IL-2 secretion, consistent with a distinct systemic Th1-skewed immune activation. In contrast to these systemic effects, EF-2001 did not significantly reduce lung viral titers (difference < 0.2 log₁₀ TCID₅₀) and did not increase lung IFN-α concentrations at day 2 post-infection. Oral EF-2001 pretreatment promoted systemic immune activation characterized by T-cell expansion and a Th1-biased cytokine profile. However, this systemic priming showed no detectable antiviral effect on lung viral burden at the early evaluation time point. EF-2001 may be better positioned as an adjunctive immunomodulatory approach rather than a direct antiviral agent, warranting further studies that include clinical outcomes and multi-time-point antiviral and mucosal immune assessments. Full article

Coxsackieviruses B are single-stranded RNA viruses belonging to the Enterovirus genus and are associated with various clinical outcomes, ranging from acute infections to chronic diseases, such as type 1 diabetes (T1D). It was previously shown that inoculation of Swiss albino mice with CVB4 by the intraperitoneal route induced both anti-CVB4 neutralizing and enhancing activities of serum. This study aimed to investigate the humoral immune response of mice inoculated with CVB4 by the mucosal route. Mice were inoculated orally or intranasally with CVB4, and the anti-CVB4 neutralizing activity of serum and saliva was assessed by a cell culture neutralization assay. Anti-enterovirus (EV) IgG and IgA antibodies were detected in serum and saliva, respectively, by ELISA. The serum-dependent enhancement of CVB4 infection in cultures of murine splenocytes was evaluated by detecting intracellular viral RNA using RT-qPCR. At day 45 post-inoculation, an anti-CVB4 neutralizing activity, the extent of which depends on the amount of inoculated infectious particles, was detected in the serum of mice exposed orally or intranasally. An increase in anti-CVB4 neutralizing activity was observed in the saliva of mice inoculated orally or intranasally during the follow-up. Oral or intranasal inoculation of CVB4 induced a systemic IgG and mucosal IgA response. In addition, serum from these mice harbored an anti-CVB4 enhancing activity in vitro. These data indicate that Swiss albino mice exposed to CVB4 via the mucosal route constitute a potentially useful model for testing strategies to promote the production of protective mucosal and systemic anti-CVB4 antibodies and for verifying whether or not enhanced antibodies are produced. Full article

Background: Dengue virus (DENV) is becoming a global public health problem, but the immunogenicity of DENV structural proteins is not fully understood. Methods: We predicted the epitope-based immunogenicity of DENV proteins from four serotypes in silico and evaluated their efficacy in vitro (T-cell proliferation assays) and in vivo (ELISpot, qRT-PCR, and plaque reduction neutralization tests using murine splenocytes). We focused on the envelope protein, which contains envelope domain III. Immunogenic B-cell epitopes were predicted using BepiPred-2.0, and regions that induce T cell-mediated immune responses were analyzed using the immune epitope database (IEDB), which validates peptides presented on HLA class I. Results: Nine-amino-acid peptide candidates were selected based on a score of >0.1. The best peptide candidates were tested in T-cell proliferation assays to confirm the in silico data. Subsequently, BALB/c mice were vaccinated with candidate peptides showing immunity in the proliferation assay, and their splenocytes were analyzed. ELISpot and qRT-PCR data showed that some candidate peptides highly regulated cytokines, including interferon-γ, tumor necrosis factor-α, and interleukin-4. Murine sera were collected after peptide boosting 2 weeks apart. Stimulation of cellular immunity was confirmed for some candidates in plaque reduction neutralization tests. Full article

Background: The apicomplexan parasite Toxoplasma gondii causes serious diseases in animals and humans. The in vitro efficacy of the antimicrobial peptide mixture tyrothricin, composed of tyrocidines and gramicidins, against T. gondii tachyzoites was investigated. Methods: Effects against T. gondii were determined by monitoring inhibition of tachyzoite proliferation and electron microscopy, host cell and splenocyte toxicity was measured by Alamar blue assay, and early embryo toxicity was assessed using zebrafish embryos. Differential affinity chromatography coupled to mass spectrometry and proteomics (DAC-MS-proteomics) was employed to identify potential molecular targets in T. gondii cell-free extracts. Results: Tyrothricin inhibited T. gondii proliferation at IC₅₀s < 100 nM, with tyrocidine A being the active and gramicidin A the inactive component. Tyrothricin also impaired fibroblast, T cell and zebrafish embryo viability at 1 µM. Electron microscopy carried out after 6 h of treatment revealed cytoplasmic vacuolization and structural alterations in the parasite mitochondrion, but these changes appeared only transiently, and tachyzoites recovered after 96 h. Tyrothricin also induced a reduction in the mitochondrial membrane potential. DAC-MS-proteomics identified 521 proteins binding only to tyrocidine A. No specific binding to gramicidin A was noted, and four proteins were common to both peptides. Among the proteins binding specifically to tyrocidine A were several SRS surface antigens and secretory proteins, mitochondrial inner and outer membrane proteins associated with the electron transfer chain and porin, and several calcium-binding proteins putatively involved in signaling. Discussion: These results suggest that tyrocidine A potentially affected multiple pathways important for parasite survival and development. Full article

Signal transducer and activator of transcription 2 (STAT2) is a key component of the type I interferon (IFN-I/III) signaling pathway, which is pivotal in host defense against cancer and viral infections and in shaping immune responses. Building on our previously reported conditional Stat2 knockout (KO) mouse, we expand its utility by validating additional tissue-specific models and exploring novel functional contexts. Mice carrying loxP-flanked Stat2 alleles were crossed with CMV-Cre, Cdx2-Cre or CD11c-Cre mice. Deletion of STAT2 was validated by PCR genotyping and western blotting in the relevant tissues. To confirm defective IFN-I signaling with STAT2 deletion, IFN-β stimulation of splenocytes from CMV-Cre Stat2 KO mice showed a lack of induction of canonical IFN-I target genes, confirming functional disruption of the pathway. In vivo, global Stat2 deletion significantly impaired the antitumor efficacy of IFN-β treatment. Similarly, lung fibroblasts isolated from globally deleted Stat2 KO mice showed defective antiviral responses to IFN-β. Tissue-specific Cre models demonstrated selective ablation of STAT2 in target compartments without affecting its expression in non-target tissues. Together, these studies expand our published conditional Stat2 KO findings and highlight the value of this model as a versatile platform for dissecting STAT2-dependent signaling pathways in a tissue- and disease-specific manner. Full article

Background: Developing an effective vaccine is crucial for the prevention and control of AIDS. Viral vector-based vaccines, particularly those utilizing homologous or heterologous prime-boost strategies, represent an important direction in current HIV vaccine research. Methods: In this study, replication-defective chimeric adenovirus Ad5F35 and modified vaccinia virus Ankara (rMVA) vector vaccines expressing the HIV-1 AEgp145 were successfully constructed, designated as Ad5F35-AEgp145 and rMVA-AEgp145, respectively. Sixty BALB/c mice were randomly divided into three groups: Ad5F35 alone, rMVA prime/Ad5F35 boost, and PBS control. The mice were immunized intramuscularly at weeks 0 and 3, and humoral and cellular immune responses were assessed at 4, 8, 12, and 16 weeks after the initial immunization. Results: The homologous Ad5F35 and heterologous rMVA/Ad5F35 vaccination regimens elicited comparable levels of HIV Env-specific cellular immune responses, peaking at 2100 ± 222 SFCs/million splenocytes and 2200 ± 619 SFCs/million splenocytes, respectively (p > 0.05). Compared to the heterologous regimen, the homologous Ad5F35 regimen induced significantly higher levels of gp120-binding antibodies at weeks 4 and 8 post-initial immunization, with geometric mean titers of 1:25,600 ± 7011 versus 1:1280 ± 150.7 and 1:10,240 ± 4048 versus 1:2560 ± 391.9, respectively. Furthermore, neutralizing activity at week 8 was significantly higher in the homologous group, with a 50% neutralization titers of 1:45 compared to 1:12 in the heterologous group (p < 0.01). Conclusion: This study demonstrates that the Ad5F35-AEgp145 vaccine, whether administered alone or in combination with rMVA-AEgp145, effectively induces strong and comparable cellular immune responses targeting HIV-1 Env in mice. While both regimens are effective, homologous immunization elicits moderately higher levels of antibody responses. These findings provide an important foundation for the further investigation of vector-based HIV vaccine formulations. Full article

Background: A quadrivalent HPV vaccine (BPV) has been developed to prevent diseases caused by HPV types 6, 11, 16, and 18 for the first time in Iran. The BPV is composed of the papillomavirus major capsid protein L1, which serves as the primary target in the design of the prophylactic HPV vaccines. To enhance immunogenicity, BPV was formulated with an amorphous aluminum hydroxy phosphate sulfate adjuvant. Methods: The immunogenicity and safety of BPV were assessed through analyses of both humoral and cell-mediated immunity, single and repeated doses, and reproductive effects using animal models. Results: Acute toxicity assessments showed no abnormalities in ophthalmic examinations, biochemical profiles, hematological parameters, and gross pathology findings. Additionally, no mortality or abnormal clinical signs were observed during a 90-day repeated-dose toxicity study. While some inflammatory reactions were noted at the injection sites and in the liver tissues of BPV-treated groups, these reactions were resolved by day 90 after the initial BPV administration. Furthermore, no signs of toxicity were detected in F1 offspring, and no adverse effects were identified in maternal reproductive performance, fertility, or hematological or biochemical parameters throughout the study duration. The BPV candidate successfully induced T-cell proliferation and increased the proportions of CD₃⁺ CD₄⁺ and CD₃⁺ CD₈⁺ T cells. It also stimulated the secretion of both interferon gamma (IFN-γ) and interleukin-4 (IL-4) cytokines in splenocytes isolated from animal models after the third dose. Moreover, anti-HPV L1 IgG antibody production was confirmed on day 14 after administration of each of the three BPV vaccine doses. Conclusions: The findings suggest that BPV is a vaccine candidate that stimulates both cellular and humoral immunity and demonstrate its safety profile in animal models. Full article