Background/Objectives: Freund’s adjuvants induce different immunomodulatory effects, but their underlying molecular mechanisms are unclear. In this study, we investigated whether the immune-stimulating effects of the complete Freund’s adjuvant (CFA) involve the mechanisms of trained immunity (TI). Methods: We examined bone marrow cells (BMCs) isolated from CFA-immunized A/J mice to address this question. Incomplete Freund’s adjuvant (IFA) and Mycobacterium tuberculosis var. bovis Bacillus Calmette-Guérin (BCG) served as negative and positive controls, respectively. We evaluated cytokine profiles, metabolic, and epigenetic changes. Results: First, BMCs from all groups except saline showed varied levels of IL-1β, IL-6, and TNF-α. But expression of CCL5 and CXCL10 was significantly elevated only in the CFA and BCG groups. Transcriptionally, significant elevations were noted for TNF-α and IL-1β in the CFA and BCG groups, whereas CXCL10, IL-6, and IL-10 were upregulated in the CFA and BCG groups, respectively. Second, while BMCs from the BCG group expressed the markers of both the M1 and M2 macrophages, no clear trends were noted in the CFA and IFA groups. Third, cell lysates from the CFA group revealed metabolic reprogramming in the BMCs. Specifically, we observed an increased level of lactate, indicative of aerobic glycolysis, which is implicated in TI, and this was also detected in the IFA group. Fourth, epigenetic analysis revealed histone enrichment in the promoter region of TNF-α, in the CFA group, but to a lesser degree than the BCG group. However, no epigenetic changes were observed in the IFA group. Conclusions: Our data provide new insights into the mechanisms of Freund’s adjuvants and the immunomodulatory effects of CFA could involve the features of TI.

Wheat streak mosaic disease (WSMD) is the most important viral disease affecting wheat production in the US Great Plains. WSMD is caused by a single or co-infection of wheat curl mite (WCM)-transmitted wheat streak mosaic virus (WSMV), Triticum mosaic virus (TriMV), and/or High Plains wheat mosaic virus (HPWMoV). Nebraska, one of the US Great Plains states, significantly contributes to the national small grain production and utilizes cereal crops as forage and cover crops. In this study, a state-wide sampling was conducted during the 2023-2025 growing seasons across Nebraska to examine the infection dynamics of the causal agents of WSMD in winter and spring cereal crops. A total of 1624 symptomatic leaf samples were assayed with multiplex RT-PCR. In 2024, co-infection of wheat with WSMV and TriMV was more common, whereas WSMV single infections dominated in 2023. In contrast, WSMV single infections were predominant in field oats compared to WSMV-TriMV coinfection. We found a high incidence of WSMV and TriMV as single and co-infections in other cereal and forage crops: rye (80%), triticale (93%), and barley (72%). HPWMoV was found only in a few wheat samples as co-infections with WSMV or WSMV+TriMV in 2023. The P1 and CP sequences of WSMV and TriMV from different cereal hosts showed minimal sequence diversity, indicating that these viruses circulate freely among diverse cereal hosts. Our data suggest that various commercial, forage, and cover cereal crops are infected with mite-transmitted viruses in field conditions and may serve as a green bridge for the continuum of WSMD in wheat.

Yellow dwarf viruses (YDV), comprising strains of barley yellow dwarf virus (BYDV) and cereal yellow dwarf virus (CYDV), cause economically important diseases in cereal crops in the United States. Although YDV are widespread in Nebraska, information on species distribution and their temporal variability is limited. A statewide survey was conducted during the 2023 and 2024 growing seasons to characterize YDV strain diversity and associated aphid vectors in cereal crops. Total RNA was extracted from symptomatic leaf samples collected from commercial and experimental fields, and viral detection was performed using a multiplex one-step reverse transcription polymerase chain reaction (RT-PCR). Aphid abundance was assessed separately using Berlese funnel from the field-collected plant materials, and species were determined morphologically. In 2023, 68.1% (644/946) of the tested samples were positive for at least one YDV strain, whereas in 2024, only 25.5% (144/564) of the samples tested positive. BYDV-SGV was the predominant strain in 2023, particularly in oats and wheat, whereas SGV+MAV and RPV+SGV strains were the most common coinfections. In contrast, CYDV-RPV and BYDV-MAV predominated in wheat and barley in 2024, whereas PAV was most frequently detected in oats. The bird cherry-oat aphid (Rhopalosiphum padi) was the most abundant aphid species across all crops and locations. These results demonstrate substantial year-to-year variation in the composition of YDV strains in Nebraska cereal crops and confirm the utility of multiplex RT-PCR for field diagnosis. Furthermore, the observed patterns of virus prevalence and aphid abundance emphasize the epidemiological relevance of these findings and support the role of aphid vectors in shaping strain distribution and composition.

Background. Inbred mouse models of autoimmune myocarditis are routinely used to investigate the immune mechanisms underlying dilated cardiomyopathy. However, their translational relevance is limited because observations made in a single inbred strain may not reflect those of outbred human populations. This limitation can be overcome by using Diversity Outbred (DO) mice, whose genetic variability is comparable to that of humans. Methods. To investigate the utility of DO mice, we characterized their immune cell distributions and induced myocarditis by immunization with porcine cardiac myosin (PCM) emulsified in complete Freund’s adjuvant. Antigen-specific T cell and antibody responses were evaluated using lymphocytes and serum samples, respectively, and hearts were examined histologically for inflammatory changes. Results. First, we noted no significant variations in the majority of immune cell populations, which include T cells and B cells. However, NK cells, double positive for CD49b and NK1.1, were lacking in both sexes. While we noted sex differences in the expression of major histocompatibility complex class II molecules in antigen-presenting cells, expression of costimulatory molecules was similar in both sexes. Second, upon immunization, we demonstrated that the PCM was immunogenic, and the PCM-reactive T cell responses were generated in both males and females, as measured by a proliferation assay. Third, cytokine analysis revealed marginal detection of Th1 (IFN-γ) and Th17 (IL-17 and IL-22) cytokines, mainly with three doses of immunization. Fourth, determination of PCM-reactive antibody responses revealed significant amounts of IgG1 and IgG2b isotypes. Finally, histological analysis revealed varying degrees of myocarditis in individual mice of both sexes. Conclusions. Our data suggest that mild autoimmune myocarditis can be induced in DO mice. However, to capture the heterogeneity in disease susceptibility, large sample cohorts are required.