Long-term storage stability of PGL-I coated ELISA plates for anti-Mycobacterium leprae IgM detection.

The assessment of ELISA plates coated with phenolic glycolipid-I/PGL-I revealed excellent stability during eight years of storage at room temperature, promoting consistent IgM antibody detection in multibacillary leprosy patients. These stable, standardized plates can significantly contribute to efficient leprosy serology research and support its widespread distribution and use in endemic countries.

Oxidized Melanoma Antigens Promote Activation and Proliferation of Cytotoxic T-Cell Subpopulations.

Increasing evidence suggests the role of reactive oxygen and nitrogen species (RONS) in regulating antitumor immune effects and immunosuppression. RONS modify biomolecules and induce oxidative post-translational modifications (oxPTM) on proteins that can alarm phagocytes. However, it is unclear if and how protein oxidation by technical means could be a strategy to foster antitumor immunity and therapy. To this end, cold gas plasma technology producing various RONS simultaneously to oxidize the two melanoma-associated antigens MART and PMEL is utilized. Cold plasma-oxidized MART (oxMART) and PMEL (oxPMEL) are heavily decorated with oxPTMs as determined by mass spectrometry. Immunization with oxidized MART or PMEL vaccines prior to challenge with viable melanoma cells correlated with significant changes in cytokine secretion and altered T-cell differentiation of tumor-infiltrated leukocytes (TILs). oxMART promoted the activity of cytotoxic central memory T-cells, while oxPMEL led to increased proliferation of cytotoxic effector T-cells. Similar T-cell results are observed after incubating splenocytes of tumor-bearing mice with B16F10 melanoma cells. This study, for the first time, provides evidence of the importance of oxidative modifications of two melanoma-associated antigens in eliciting anticancer immunity.

Germline-specific RNA helicase DDX4 forms cytoplasmic granules in cancer cells and promotes tumor growth.

Cancer cells undergo major epigenetic alterations and transcriptomic changes, including ectopic expression of tissue- and cell-type-specific genes. Here, we show that the germline-specific RNA helicase DDX4 forms germ-granule-like cytoplasmic ribonucleoprotein granules in various human tumors, but not in cultured cancer cells. These cancerous DDX4 complexes contain RNA-binding proteins and splicing regulators, including many known germ granule components. The deletion of DDX4 in cancer cells induces transcriptomic changes and affects the alternative splicing landscape of a number of genes involved in cancer growth and invasiveness, leading to compromised capability of DDX4-null cancer cells to form xenograft tumors in immunocompromised mice. Importantly, the occurrence of DDX4 granules is associated with poor survival in patients with head and neck squamous cell carcinoma and higher histological grade of prostate cancer. Taken together, these results show that the germ-granule-resembling cancerous DDX4 granules control gene expression and promote malignant and invasive properties of cancer cells.

A rare case of food-dependent exercise-induced anaphylaxis caused by potato snacks.

A rare case of food-dependent exercise-induced anaphylaxis caused by potato snacks is reported. Specific food triggers for anaphylaxis were identified by using the skin prick test, antigen analysis, and serum IgE assays. Four potato proteins were considered candidate antigens for food-dependent exercise-induced anaphylaxis.

Potential biomarkers for evaluating the BCG vaccination response based on humoral immunity.

Background: The current prophylactic tuberculosis vaccine Bacille Calmette-Guérin (BCG), was derived in the 1920s, but the humoral immune responses induced by BCG vaccination have not been fully elucidated to date. In this study, our aim was to reveal the profiles of antibody responses induced by BCG vaccination in adults and identify the potential biomarkers for evaluating the BCG vaccination response. Methods: Proteome microarrays were performed to reveal the serum profiles of antibody responses induced by BCG vaccination in adults. ELISA was used to validate the potential biomarkers in validation cohort (79 healthy controls and 58 BCG-vaccinated subjects). Then combined panel was established by logistic regression analysis based on OD values of potential biomarkers. Results: Multiple antigens elicited stronger serum IgG or IgM antibody responses in BCG vaccinated subjects than healthy subjects at 12 weeks post BCG vaccination; among the antigens, Rv0060, Rv2026c and Rv3379c were further verified using 137 serum samples and presented the moderate performance in assessment of the BCG vaccination response by receiver operating characteristic analysis. Furthermore, a combined panel exhibited an improved AUC of 0.923, and the sensitivity and specificity were 77.59 % and 91.14 %, respectively. In addition, the antibody response against Rv0060, Rv2026c and Rv3379c was related to the clinical background to a certain extent. Conclusions: The novel antigens identified in our study could offer better knowledge towards developing a more efficacious vaccine based on humoral immune responses, and they could be potential biomarkers in assessments of BCG vaccination responses.

Novel mRNA adjuvant ImmunER enhances prostate cancer tumor-associated antigen mRNA therapy via augmenting T cell activity.

Prostate cancer (PCa) is characterized as a "cold tumor" with limited immune responses, rendering the tumor resistant to immune checkpoint inhibitors (ICI). Therapeutic messenger RNA (mRNA) vaccines have emerged as a promising strategy to overcome this challenge by enhancing immune reactivity and significantly boosting anti-tumor efficacy. In our study, we synthesized Tetra, an mRNA vaccine mixed with multiple tumor-associated antigens, and ImmunER, an immune-enhancing adjuvant, aiming to induce potent anti-tumor immunity. ImmunER exhibited the capacity to promote dendritic cells (DCs) maturation, enhance DCs migration, and improve antigen presentation at both cellular and animal levels. Moreover, Tetra, in combination with ImmunER, induced a transformation of bone marrow-derived dendritic cells (BMDCs) to cDC1-CCL22 and up-regulated the JAK-STAT1 pathway, promoting the release of IL-12, TNF-α, and other cytokines. This cascade led to enhanced proliferation and activation of T cells, resulting in effective killing of tumor cells. In vivo experiments further revealed that Tetra + ImmunER increased CD8+T cell infiltration and activation in RM-1-PSMA tumor tissues. In summary, our findings underscore the promising potential of the integrated Tetra and ImmunER mRNA-LNP therapy for robust anti-tumor immunity in PCa.

Boosting antigen-specific T cell activation with lipid-stabilized protein nanoaggregates.

Vaccines based on protein antigens have numerous advantages over inactivated pathogens, including easier manufacturing and improved safety. However, purified antigens are weakly immunogenic, as they lack the spatial organization and the associated 'danger signals' of the pathogen. Formulating vaccines as nanoparticles enhances the recognition by antigen presenting cells, boosting the cell-mediated immune response. This study describes a nano-precipitation method to obtain stable protein nanoaggregates with uniform size distribution without using covalent cross-linkers. Nanoaggregates were formed via microfluidic mixing of ovalbumin (OVA) and lipids in the presence of high methanol concentrations. A purification protocol was set up to separate the nanoaggregates from OVA and liposomes, obtained as byproducts of the mixing. The nanoaggregates were characterized in terms of morphology, ζ-potential and protein content, and their interaction with immune cells was assessed in vitro. Antigen-specific T cell activation was over 6-fold higher for nanoaggregates compared to OVA, due in part to the enhanced uptake by immune cells. Lastly, a two-dose immunization with nanoaggregates in mice induced a significant increase in OVA-specific CD8+ T splenocytes compared to soluble OVA. Overall, this work presents for the first time the microfluidic production of lipid-stabilized protein nanoaggregates and provides a proof-of-concept of their potential for vaccination.

Advances in CAR-T-cell therapy in T-cell malignancies.

Significant advances have been made in chimeric antigen receptor T (CAR-T)-cell therapy for the treatment of recurrent or refractory B-cell hematologic malignancies. However, CAR-T-cell therapy has not yet achieved comparable success in the management of aggressive T-cell malignancies. This article reviews the challenges of CAR-T-cell therapy in treating T-cell malignancies and summarizes the progress of preclinical and clinical studies in this area. We present an analysis of clinical trials of CAR-T-cell therapies for the treatment of T-cell malignancies grouped by target antigen classification. Moreover, this review focuses on the major challenges encountered by CAR-T-cell therapies, including the nonspecific killing due to T-cell target antigen sharing and contamination with cell products during preparation. This review discusses strategies to overcome these challenges, presenting novel therapeutic approaches that could enhance the efficacy and applicability of CAR-T-cell therapy in the treatment of T-cell malignancies. These ideas and strategies provide important information for future studies to promote the further development and application of CAR-T-cell therapy in this field.

IGH Complementarity Determining Region-3-Cytomegalovirus Protein Chemical Complementarity Linked to Better Overall Survival Probabilities for Glioblastoma.

Cytomegalovirus (CMV) has long been thought to have an association with glioblastoma multiforme (GBM), although the exact role of CMV and any subsequent implications for treatment have yet to be fully understood. This study addressed whether IGH complementarity determining region-3 (CDR3)-CMV protein chemical complementarity, with IGH CDR3s representing both tumor resident and blood-sourced IGH recombinations, was associated with overall survival (OS) distinctions. IGH recombination sequencing reads were obtained from (a) the Clinical Proteomic Tumor Analysis Consortium, tumor RNAseq files; and (b) the cancer genome atlas, blood exome-derived files. The Adaptive Match web tool was used to calculate chemical complementarity scores (CSs) based on hydrophobic interactions, and those scores were used to group GBM cases and assess survival probabilities. We found a higher OS probability for cases whose hydrophobic IGH CDR3-CMV protein chemical complementarity scores (Hydro CSs) were in the upper 50th percentile for several CMV proteins, including UL99 and UL123, as well as for CSs based on known B cell epitopes representing these proteins. We also identified multiple immune signature genes, including CD79A and TNFRSF17, for which higher RNA expression was associated with higher Hydro CSs. Results were consistent with the idea that stronger immunoglobulin responses to CMV are associated with better OS probabilities for GBM.

PRAMEF12, a novel cancer/testis gene, regulates proliferation and apoptosis to promote progression of glioma.

Aim: To evaluate whether PRAMEF12 can serve as a diagnostic biomarker for glioma. Methods: We examined PRAMEF12 expression in multiple normal and glioma tissues. The diagnostic value of PRAMEF12 was evaluated using receiver operating characteristic curve analysis. The effect of PRAMEF12 ablation on proliferation, cell cycle and apoptosis was investigated. Database analyses were utilized for functional enrichment analysis. Results: PRAMEF12 expression in normal tissue was restricted to the human testis. PRAMEF12 displayed significant diagnostic value in glioma. PRAMEF12 knockdown inhibited cell proliferation, induced apoptosis and resulted in induction of S-phase cell cycle arrest. Pathway enrichment analysis indicated that PRAMEF12 may participate in cancer. Conclusion: PRAMEF12, a novel cancer/testis gene, may be a potential new diagnostic biomarker for glioma.

[Box: see text].

Antibody responses after sequential vaccination with PCV13 and PPSV23 in patients with moderate to severe plaque psoriasis under immunosuppressive therapy.

A crucial step in lowering the risk of invasive pneumococcal illness in high-risk populations, such as individuals with plaque psoriasis, is pneumococcal vaccination. The serologic response to the sequential vaccination with Prevenar 13 (PCV13) and Pneumovax 23 (PPSV23) in psoriasis patients under immunosuppressive therapy is still poorly characterized despite national recommendations suggesting vaccination for immunocompromised patients. In this prospective study, we investigated the serological response in 57 patients under active systemic treatment for moderate to severe plaque psoriasis who underwent sequential vaccination with PCV13 followed by PPSV23. Our analysis focused on global and serotype-specific anti-pneumococcal antibody responses over a 7-month period post-vaccination. Our findings reveal a robust serological response in patients with plaque psoriasis under systemic therapy. When comparing our results with a cohort of kidney transplant recipients who completed a similar sequential vaccination protocol, psoriasis patients showed higher antibody concentrations. In psoriasis patients, the mean levels of all global antibody classes tested (IgG, IgG2, IgA, IgM) increased more than 4-fold (P < 0.0001) and serotype-specific antibodies more than 1.9-fold (P < 0.01). In addition to providing strong evidence of the safety and effectiveness of sequential pneumococcal vaccination in individuals with plaque psoriasis, our work sheds light on the complex interactions that exist between immunosuppressive treatment, vaccination schedule, and antibody responses in various risk groups. IMPORTANCE: To protect against severe courses of infection with Streptococcus pneumoniae, the national guidelines recommend sequential vaccination for these patients. However, there are only studies on the efficacy of a single administration of these vaccines in this particular risk group. The immunological responses to the vaccine were correlated with clinical patient data. In summary, our study shows for the first time that sequential vaccination is immunogenic in patients with moderate to severe plaque psoriasis.

Exploring T Cell Receptor Repertoires in Myocardial Diseases.

Mounting experimental and clinical evidence has revealed that adaptive immune mechanisms targeting myocardial antigens are triggered by different forms of cardiac injury and impact disease progression. B and T lymphocytes recognize specific antigens via unique adaptive immune receptors generated through a somatic rearrangement process that generates a potential repertoire of 1019 unique receptors. While the adaptive immune receptor repertoire diversity provides the basis for immunologic specificity, making sense of it can be a challenging task. In the present review, we discuss key aspects underlying the generation of TCRs (T cell receptors) and emerging tools for their study in the context of myocardial diseases. Moreover, we outline how exploring TCR repertoires could lead to a deeper understanding of myocardial pathophysiological principles and potentially serve as diagnostic tools.

Stepping forward: T-cell redirecting bispecific antibodies in cancer therapy.

T cell-redirecting bispecific antibodies are specifically designed to bind to tumor-associated antigens, thereby engaging with CD3 on the T cell receptor. This linkage between tumor cells and T cells actively triggers T cell activation and initiates targeted killing of the identified tumor cells. These antibodies have emerged as one of the most promising avenues within tumor immunotherapy. However, despite success in treating hematological malignancies, significant advancements in solid tumors have yet to be explored. In this review, we aim to address the critical challenges associated with T cell-redirecting bispecific antibodies and explore novel strategies to overcome these obstacles, with the ultimate goal of expanding the application of this therapy to include solid tumors.

Engineering nanomedicines for immunogenic eradication of cancer cells: Recent trends and synergistic approaches.

Resistance to cancer immunotherapy is mainly attributed to poor tumor immunogenicity as well as the immunosuppressive tumor microenvironment (TME) leading to failure of immune response. Numerous therapeutic strategies including chemotherapy, radiotherapy, photodynamic, photothermal, magnetic, chemodynamic, sonodynamic and oncolytic therapy, have been developed to induce immunogenic cell death (ICD) of cancer cells and thereby elicit immunogenicity and boost the antitumor immune response. However, many challenges hamper the clinical application of ICD inducers resulting in modest immunogenic response. Here, we outline the current state of using nanomedicines for boosting ICD of cancer cells. Moreover, synergistic approaches used in combination with ICD inducing nanomedicines for remodeling the TME via targeting immune checkpoints, phagocytosis, macrophage polarization, tumor hypoxia, autophagy and stromal modulation to enhance immunogenicity of dying cancer cells were analyzed. We further highlight the emerging trends of using nanomaterials for triggering amplified ICD-mediated antitumor immune responses. Endoplasmic reticulum localized ICD, focused ultrasound hyperthermia, cell membrane camouflaged nanomedicines, amplified reactive oxygen species (ROS) generation, metallo-immunotherapy, ion modulators and engineered bacteria are among the most innovative approaches. Various challenges, merits and demerits of ICD inducer nanomedicines were also discussed with shedding light on the future role of this technology in improving the outcomes of cancer immunotherapy.

Variable Surface Antigens of Plasmodium falciparum: Protein Families with Divergent Roles.

Malaria caused by Plasmodium falciparum (Pf) is an illness that contributes significantly to the global health burden. Pf makes significant alterations to the host cell to meet its metabolic demands and escape the immune response of the host. These include the export of a large number of parasite proteins to the infected Red Blood Cells (iRBC). Variable Surface Antigens (VSAs), which are highly polymorphic protein families with important roles in immune evasion, form an important component of the exported proteins. A total of five protein families constitute the VSAs, viz. PfEMP1 (Pf erythrocyte membrane protein 1), RIFIN (repetitive interspersed family), STEVOR (sub-telomeric open reading frame), SURFIN (surface-associated interspersed gene family), and PfMC-2TM (Pf Maurer's cleft two transmembrane). With orthologues present in various simian-infecting species, VSAs take up a variety of domain topologies and organizational structures while exhibiting differential expressions throughout the parasite life cycle. Their expression varies across clinical isolates and laboratory strains, which suggests their crucial role in host cell survival and defense. Members of VSAs are reported to contribute significantly to disease pathogenesis through immune evasion processes like cytoadherence, iRBC sequestration in the host vasculature, rosetting, reduced erythrocyte deformability, and direct immunosuppression. In this study, we have gathered information on various aspects of VSAs, like their orthologues, domain architecture, surface topology, functions and interactions, and three-dimensional structures, while emphasizing discoveries in the field. Considering the vast repertoire of Plasmodial VSAs with new emergent functions, a lot remains unknown about these families and, hence, malaria biology.

Gut microbiota as a sensor of autoimmune response and treatment for rheumatoid arthritis.

Rheumatoid arthritis (RA) is considered a multifactorial condition where interaction between the genetic and environmental factors lead to immune dysregulation causing autoreactivity. While among the various genetic factors, HLA-DR4 and DQ8, have been reported to be the strongest risk factors, the role of various environmental factors has been unclear. Though events initiating autoreactivity remain unknown, a mucosal origin of RA has gained attention based on the recent observations with the gut dysbiosis in patients. However, causality of gut dysbiosis has been difficult to prove in humans. Mouse models, especially mice expressing RA-susceptible and -resistant HLA class II genes have helped unravel the complex interactions between genetic factors and gut microbiome. This review describes the interactions between HLA genes and gut dysbiosis in sex-biased preclinical autoreactivity and discusses the potential use of endogenous commensals as indicators of treatment efficacy as well as therapeutic tool to suppress pro-inflammatory response in rheumatoid arthritis.

Mobile calculator application for estimating human erythrocyte antigen frequency in Korea.

OBJECTIVES: This study aimed to establish a comprehensive human erythrocyte antigen (HEA) frequency data set for Koreans. It also sought to develop a mobile app that facilitates the calculation of the frequencies of specific antigen-negative red blood cell units and the average number of units required for antigen typing. METHODS: Human erythrocyte antigen frequencies were compiled from large-scale blood donor data and 5 previous papers. Based on the collected data, we developed a mobile calculator app for HEA frequency and evaluated its usability. RESULTS: Human erythrocyte antigen frequency data for 20 blood group systems, including the ABO, Rh, MNS, Duffy, Kidd, and Diego systems, were established. The app was designed to enable users to select the desired phenotype from a drop-down menu and display the calculated frequency at the bottom. The number of units required for antigen typing to find 1 compatible red blood cell unit was also displayed. Five users participated in app evaluation and rated the functionality and information categories highly. In quizzes prompting users to calculate frequencies using the app, all participants provided correct answers, confirming the app's user-friendly functionality. CONCLUSIONS: This app, which encompasses comprehensive HEA frequency data, is expected to find multiple uses in transfusion medicine, including optimizing blood bank workflow and defining rare blood groups in Korea.

Long-term changes in the phenotype and cytokine production of monocytes in COVID-19 recovered and vaccinated individuals.

Monocytes play a crucial role in the immune response against pathogens. Here, we sought to determine COVID-19 and the vaccine Gam-COVID-Vac induce long-term changes in the phenotype and cytokine production of circulating monocytes. Monocytes were purified from peripheral blood mononuclear cells of healthy donors who had not had COVID-19 or vaccination, who had received two doses of Gam-COVID-Vac, and who had mild/moderate COVID-19 in the last 6 months and evaluated by flow cytometry. To investigate the effect of SARS-CoV-2 proteins, monocytes were cultured for 2 days with or without stimulation with recombinant SARS-CoV-2 S1 and N peptides. Monocytes obtained from vaccinated and recovered individuals showed increased basal expression of HLA-DR, CD63, CXCR2, and TLR7. We also observed an increased frequency of CD63+ classical monocytes in both groups, as well as an increased frequency of HLA-DR+ non-classical monocytes in the COVID-19-recovered group compared to the control group. Monocytes from vaccinated and recovered donors produced higher basal levels of IL-6, IL-1ß, and TNF-α cytokines. Ex vivo stimulation with SARS-CoV-2 antigens induced increased expression of HLA-DR and TLR7 on monocytes obtained from the control group. The challenge with SARS-CoV-2 antigens had no effect on the production of IL-6, IL-1ß, and TNF-α cytokines by monocytes. The acquired data offer compelling evidence of enduring alterations in both the phenotype and functional status of circulating monocytes subsequent to vaccination with Gam-COVID-Vac and mild/moderate COVID-19 infection. At least some of these changes appear to be a consequence of exposure to SARS-CoV-2 S1 and N antigens.

Biomimetic Multilayered Lipid Nanovesicles for Potent Protein Vaccination.

Lipid vesicles are widely used for drug and gene delivery, but their structural instability reduces in vivo efficacy and requires specialized handling. To address these limitations, strategies like lipid cross-linking and polymer-lipid conjugation are suggested to enhance stability and biological efficacy. However, the in vivo metabolism of these altered lipids remains unclear, necessitating further studies. A new stabilization technique without chemical modification is urgently needed. Here, a bio-mimetic approach for fabricating robust multilamellar lipid vesicles to enhance in vivo delivery and stabilization of protein antigens is presented. This method leverages 1-O-acylceramide, a natural skin lipid, to facilitate the self-assembly of lipid nanovesicles. Incorporating 1-O-acylceramide, anchoring lipid bilayers akin to its role in the stratum corneum, provides excellent stability under environmental stresses, including freeze-thaw cycles. Encapsulating ovalbumin as a model antigen and the adjuvant monophosphoryl lipid A demonstrates the vesicle's potential as a nanovaccine platform. In vitro studies show enhanced immune responses with both unilamellar and multilamellar vesicles, but in vivo analyses highlight the superior efficiency of multilamellar vesicles in inducing higher antibody and cytokine levels. This work suggests ceramide-induced multilamellar lipid vesicles as an effective nanovaccine platform for enhanced antigen delivery and stability.

Expression and field evaluation of new Mycobacterium bovis antigens.

Bovine tuberculosis (bTB) represents a threat to livestock production. Mycobacterium bovis is the main causative agent of bTB and a pathogen capable of infecting wildlife and humans. Eradication programs based on surveillance in slaughterhouses with mandatory testing and culling of reactive cattle have failed to eradicate bTB in many regions worldwide. Therefore, developing effective tools to control this disease is crucial. Using a computational tool, we identified proteins in the M. bovis proteome that carry predictive binding peptides to BoLADRB3.2 and selected Mb0309, Mb1090, Mb1810 and Mb3810 from all the identified proteins. The expression of these proteins in a baculovirus-insect cell expression system was successful only for Mb0309 and Mb3810. In parallel, we expressed the ESAT-6 family proteins EsxG and EsxH in this system. Among the recombinant proteins, Mb0309 and EsxG exhibited moderate performance in distinguishing between cattle that test positive and negative to bTB using the official test, the intradermal tuberculin test (IDT), when used to stimulate interferon-gamma production in blood samples from cattle. However, when combined as a protein cocktail, Mb0309 and EsxG were reactive in 50 % of positive cattle. Further assessments in cattle that evade the IDT (false negative) and cattle infected with Mycobacterium avium paratuberculosis are necessary to determine the potential utility of this cocktail as an additional tool to assist the accurate diagnosis of bTB.