Bivalent foot-and-mouth disease virus mRNA vaccine induces well-balanced humoral and cellular immune responses.

Dual TSHR/IGF-1Rα electroporation induces progressive orbital remodeling and retinochoroidal alterations in a murine model of thyroid eye disease.

In clinical practice, thyroid function tests (TFTs) usually comprise thyroid-stimulating hormone (TSH) and free thyroxine (FT4), with free triiodothyronine (FT3) measured less frequently. We report a 76-year-old woman with autoimmune thyroiditis presenting with raised TSH and incongruously elevated FT4. The discordance between biochemical findings and clinical status prompted further investigations for assay interference. TFTs were assessed using four different immunoassay platforms and equilibrium dialysis. Additional studies included heterophilic antibody blocking and polyethylene glycol (PEG) precipitation. PEG precipitation demonstrated a high-molecular-weight interferent, consistent with thyroid hormone autoantibodies. FT4 measured by two-step immunoassays (Abbott Alinity and Beckman DxI) aligned with FT4 reported by equilibrium dialysis, whereas one-step assays (Roche Cobas and Siemens Atellica) reported spuriously elevated FT4. One-step immunoassays are particularly susceptible to interference from thyroid hormone autoantibodies, which may yield misleading results. Recognition of potential TFT assay interference and the use of alternative methodologies are essential for accurate diagnosis and appropriate patient management.

Resistin signaling via NF-κB pathway plays a key role in obesity-induced and -aggravated asthma.

In humans and mice, the T-cell receptor (TCR) of each effector/memory T-cell clone recognizes one to several cognate peptide-MHC complexes (pMHC). Limited knowledge of TCR repertoire specificities restricts our capacity to rationally interpret this information, both diagnostically and in preclinical research. In this study, we (i) developed and validated a cost-efficient wet lab and computational pipeline to identify mouse TCRs specific to particular peptides, (ii) produced a dataset of helper T cell (Th) TCR beta chain CDR3s specific to B16 melanoma neoantigens in the I-Ab pMHCII context, available in VDJdb, and (iii) applied this dataset to track tumor-specific T-cell responses to the CTLA4 blocking immunotherapy in the orthotopic B16 melanoma model. We showed that B16-specific TCR motifs expanded in both Th and regulatory T cell (Treg) repertoires of tumor-bearing mice, with stronger expansion in the Th subset of mice treated with the CTLA4 blocking antibody. The response was stochastic across individual mice with respect to the specific TCR motifs and target peptides involved-each mouse displayed a unique response pattern, likely reflecting the natural diversity of molecular pathways underlying antitumor immunity. We also showed that CTLA4 blockade promotes prominent Th clonal expansion and induces general, nontumor-specific Th-to-Treg plasticity. Altogether, we provide a universal pipeline for the investigation of mouse T-cell responses at the antigen-specific level, facilitating the development and validation of immunotherapeutic and vaccination approaches.

Hybrid BSA-PEO nanofibers as a stable biointerface for electrochemical cortisol immunosensing.

Sigma-2 receptor modulators alter low-density lipoprotein receptor-mediated lipid uptake in retinal pigment epithelial cells.

Classical swine fever (CSF), caused by the classical swine fever virus (CSFV), is an acute, febrile, and highly contagious disease that has led to significant economic losses in the global swine industry. Although the attenuated lapinized CSF vaccine (C-strain) has effectively controlled CSF outbreaks in China since the 1950s, it remains challenging to serologically differentiate infected from vaccinated animals (DIVA). Currently, the application of E2 subunit vaccines allows for DIVA by detecting antibodies against the Erns protein. Therefore, this study aimed to develop a blocking ELISA for CSFV Erns antibody detection using porcine monoclonal antibodies (mAbs) derived from single B cell technology. Peripheral blood mononuclear cells (PBMCs) were isolated from immunized pigs, and single CD21+IgM−Erns-His tag+ B cells were sorted via flow cytometry. Using one-step PCR, full-length genes of porcine IgG heavy and light chains were amplified separately, yielding 11 porcine mAbs against the CSFV Erns protein. Among these, three mAbs (E0S3, E0S5, and E0S10) exhibited broad reactivity, while two (E0S1, E0S4) showed no cross-reaction with bovine viral diarrhea virus (BVDV). Using mAb E0S4 as the blocking antibody, a blocking ELISA was established and optimized. The assay demonstrated a detection limit of 1:128, no cross-reactivity with other swine viruses or BVDV, and intra- and inter-assay coefficients of variation below 10%. ROC curve analysis determined an optimal cut-off value of 48.4%, with high sensitivity and specificity. In conclusion, the developed blocking ELISA provides a reliable tool for high-throughput serological surveillance, facilitating the DIVA strategy and contributing to CSF eradication programs.

Sublingual allergen immunotherapy (SLIT) is an effective treatment for immunoglobulin (Ig)E-mediated allergies. Its success is associated with allergen-specific (s)IgG, which blocks IgE-mediated mechanisms. Preventive effects of SLIT in children before allergy-symptom onset remain largely unexplored. A randomized trial was conducted between October 06, 2017 and December 15, 2022, which included house dust mite (HDM)-sensitized preschool children (aged 3-5 years) showing no allergy symptoms. They were randomized (2:2 blocks) to HDM-SLIT (300 index of reactivity/day, Staloral) or placebo solution for 2 years. Children receiving > 4 months of treatment were included in the analysis. Primary objective of the study was to compare the groups for change in major HDM allergen-Der p 1-sIgG levels from baseline to end of treatment (EOT). Secondary objectives were to compare longitudinal changes (at 4 and 12 months, and EOT) in the (i) levels of HDM-sIgG subclasses and -sIgE using ImmunoCAP/-ISAC and/or ELISA (ii) Der p-reactivity in skin and basophils using skin prick test and basophil activation test (BAT), (iii) sensitization status defined as sIgE-positivity in skin and/or serum, and (iv) Der p-sIgE-blocking activity using inhibition-BAT. Eighteen children received HDM-SLIT (mean age = 4.26 ± 0.37 years, eight females) and 15 placebo (mean age = 4.16 ± 0.59 years, nine females). HDM-SLIT increased Der p 1-sIgG levels between baseline and EOT versus placebo (effect size = 3.11 [95% CL 1.36-4.82], p = 0.001). Additional changes induced by HDM-SLIT were: increased levels of HDM-sIgG1 and -sIgG4, no long-term increase in HDM-sIgE levels, reduced Der p-reactivity in skin and basophils (p values ≤ 0.05), and blunted development of new sensitizations. Sera from HDM-SLIT treated individuals displayed blocking activity on basophils (p values ≤ 0.05). Preventive application of HDM-SLIT in allergy-prone children induces immunomodulatory effects early during treatment (elevated sIgG levels with blocking activity and reduced effector cell responses), thereby revealing potential to interfere with allergy development. EudraCT#2016-002844-18.

Lymphocyte activation relies on the integration of signals from multiple receptors at cell-cell contacts, but the spatiotemporal regulation of this process is unclear. Here, we show that programmed cell death protein 1 (PD-1) and T cell receptor (TCR) signals are integrated at nanoscale microvillar close contacts formed during T cell interactions with their targets. PD-1 signaling begins as these contacts form and selectively limits the duration, but not the amplitude, of TCR signaling. PD-1 suppresses TCR activity directly, by locally recruiting Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP2), and indirectly, by reducing cell-spreading, close-contact formation, and TCR engagement. A PD-1 blocking antibody induced inhibitory signaling when Fc receptor engagement trapped PD-1 at close contacts. Engineering the antibody to prevent PD-1 trapping eliminated these agonistic effects and improved blockade efficacy. These findings identify microvillar contacts as crucial hubs of initial signal integration and provide a framework for optimizing checkpoint immunotherapies.

Natural and in vitro-generated regulatory T-cell subsets differentially modulate phenotype and drug response in K562 leukemic cells.

Lifileucel is a tumor-derived autologous T cell therapy approved for unresectable or metastatic melanoma in adults. This article discusses 10 clinical studies on lifileucel for the treatment of advanced melanoma. The phase II study data available evaluated the safety and efficacy of lifileucel in a global, multicenter study involving adult patients with unresectable or metastatic melanoma who had previously received systemic therapies, including a PD-1 blocking antibody and, if applicable, BRAF and MEK inhibitors. Among 73 patients treated with the recommended dose, the objective response rate was 31.5%, with 4.1% achieving a complete response and 27.4% a partial response. Of the responders, 56.5%, 47.8%, and 43.5% maintained their responses without progression or death at 6, 9, and 12 months, respectively. The most common adverse events seen in clinical studies include thrombocytopenia, anemia, and febrile neutropenia. Additional studies are underway to provide further data on the use of lifileucel as a safe and effective intravenous treatment option for unresectable or metastatic melanoma. This article reviews the published data encompassing the preliminary development, pharmacology, efficacy, and safety of lifileucel.

Post-stroke inflammation contributes to poor outcomes in both clinical and experimental studies. Interleukin-6 (IL-6) is a key inflammatory mediator in ischemic stroke, and higher circulating IL-6 levels are associated with greater stroke severity and worse clinical outcomes. Targeting IL-6 signaling therefore represents a potential therapeutic strategy. We tested whether inhibition of IL-6 signaling with the IL-6 receptor (IL-6R) blocking antibody tocilizumab (TCZ) improves recovery after experimental stroke. Aged mice (18–20 months) underwent 60 minutes of middle cerebral artery occlusion. TCZ (20 mg/kg) was administered 5 hours after ischemia onset, and behavioral outcomes were assessed weekly for 5 weeks. Delayed TCZ treatment improved long-term functional recovery in aged male mice but not in aged females. To explore this difference, we measured circulating soluble IL-6R (sIL-6R) levels in mice and patients with ischemic stroke. Females exhibited significantly higher post-stroke sIL-6R levels. Increasing the TCZ dose to 100 mg/kg restored efficacy in aged female mice and improved long-term outcomes. These findings support a role for IL-6R pathway modulation in improving recovery after experimental stroke and suggest that therapeutic response may differ by sex and target availability, potentially related to differences in circulating sIL-6R after ischemic injury.

Neurocysticercosis (NCC), a parasitic brain infection caused by Taenia solium larvae, remains a leading cause of preventable epilepsy globally. Although calcified brain lesions were formerly considered as the quiescent end stage of NCC, they may act as epileptogenic foci. It has been suggested that parasitic antigens within calcified lesions may act as potential triggers of inflammation and subsequent seizure activity. In this study, we developed and optimized immunohistochemistry (IHC) assays employing anti-Taenia solium monoclonal antibodies (mAbs) to detect residual cyst antigens in calcified lesions in a porcine NCC model and assessed antigen persistence for up to 12 months after successful antiparasitic treatment. Six mAbs raised against T. solium whole cyst (TsW5, TsW8, and TsW12), vesicular fluid (TsV3 and TsV4), and excretory/secretory products (TsE1) were used for IHC assay development and tested in brain sections containing viable brain cysts from NCC pigs and uninfected tissue from controls to optimize assay conditions, blocking, primary and secondary antibody dilutions. Optimized assays were subsequently performed in selected calcified granulomas (n = 20) obtained from NCC-infected pigs sacrificed at 4, 8, and 12 months after antiparasitic treatment to identify residual cyst antigens as well as their localization and area of reactivity. We observed residual cyst antigens in 65-80% of calcified granulomas, with TsW8 and TsV3 showing the highest percentages of immunoreactivity. Antigen localization followed two patterns, one with antigens entirely located within the calcified lesions (TsW5, TsW8, TsW12, and Tsv4) and another with antigens located outside the cyst in the perilesional brain tissue (TsV3 and TsE1). Antigen detection and the extent of reactivity declined progressively after antiparasitic treatment but persisted at detectable immunoreactive areas in calcified granulomas up to month 12 months after treatment. T. solium antigens remain detectable in calcified granulomas and in the perilesional tissue for up to 12 months after antiparasitic treatment in the pig model.

Vitamin B12 deficiency is a worldwide public health issue, with its prevalence high in the developing countries. It is a micronutrient synthesized by microorganisms and obtained through diet and its deficiency can lead to megaloblastic anaemia and neurological deficits. The elderly are more prone to it. Pernicious anaemia is a prevalent cause of megaloblastic anaemia, which is associated with autoimmune destruction of parietal cells and intrinsic factor deficiency by APCA and AIFA antibodies. These antibodies can cause mucosal damage and hinder vitamin B12 absorption. The present prospective observational study included 166 patients with severe vitamin B12 deficiency (defined by Vitamin B12 levels ≤ 150 pg/mL) who were treated at a tertiary care centre in western Rajasthan. Expression of AIFA and APCA were assessed and their association with different parameters were evaluated. The present study didnot find any significant association between APCA and AIFA impressions, indicating that the absence or presence of one antibody did not strongly predict the presence or absence of the other antibody. This suggests that these autoantibodies may have distinct roles or mechanisms in PA and further research is needed to understand their relationship.

Emerging Therapies for Anaphylaxis.

Subarachnoid hemorrhage (SAH) often results in severe neurological impairment. While spliced X-box binding protein 1 (XBP1s) has been implicated in brain injury, its specific role and mechanism in SAH-induced mice remain unclear. A murine SAH model was established to explore the function of XBP1 knockdown. Neurological function was evaluated through behavioral tests, brain edema measurement, and hematoxylin and eosin staining. Blood-brain barrier (BBB) integrity and neuronal pyroptosis were assessed using Evans blue extravasation, western blotting, immunofluorescence and LDH release assay. The direct interaction of IL-6 by XBP1s was confirmed using chromatin immunoprecipitation and luciferase reporter assays. Finally, the functional central to our hypothesis was verified by administering exogenous IL-6 or IL-6 blocking antibody to SAH mice. We found that XBP1s and p-IRE1 were significantly up-regulated after SAH. Knockdown of XBP1 ameliorated neurological deficits, preserved BBB integrity (as indicated by increased ZO-1/Occludin and reduced Evans blue leakage), and suppressed neuronal pyroptosis, evidenced by decreased levels of pyroptosis-related proteins and LDH release. Mechanistically, XBP1s was identified as a direct transcriptional enhancer of IL-6. In the mouse SAH model, the protective effects of XBP1 knockdown on the BBB and against pyroptosis were effectively abolished by the exogenous administration of IL-6 and were comparable to administration of the IL-6 blocking antibody. The IRE1-XBP1s signaling axis played an important role in SAH-induced neuronal damage and pyroptosis by directly up-regulating IL-6 expression. Inhibition of this axis represents a mechanistically grounded and promising strategy for SAH intervention.

BACKGROUNDCoccidioidomycosis ranges from self-limiting uncomplicated Valley fever (UVF) in most cases to life-threatening disseminated coccidioidomycosis (DCM) in rare individuals. A few patterns of immunologic deficits allowing for dissemination have been identified, although the specific defects in most individuals with DCM remain undefined. We hypothesized that chronic antigen exposure in DCM engenders a state of T cell exhaustion.METHODSFrom a cohort of over 300 individuals with confirmed diagnoses of coccidioidomycosis, circulating T cell phenotypes were characterized via flow cytometry and Coccidioides-specific T cell responses were measured by activation-induced marker (AIM) assay.RESULTSMale sex was significantly associated with disseminated disease (OR 2.5, 95% CI 1.5–4.0). A majority (52%) of individuals showed Coccidioides-specific T cell responses in our AIM assay. We noted a significant difference in patients sampled in the first year of diagnosis, where only 8% of patients with DCM had T cell responses during this time, as compared with 44% of individuals with UVF (P = 0.04). Among DCM patients with detectable AIM responses, CD4+ T cells demonstrated an exhausted phenotype with elevated PD-1 expression compared with UVF individuals. In vitro PD-1 blockade augmented IFN-γ production in most tested individuals with DCM.CONCLUSIONThese findings suggest that dissemination may occur in some individuals during a period of impaired antigen-specific T cell activity. Importantly, these responses can be augmented in vitro by PD-1–blocking antibodies, supporting further study of immune checkpoint therapy as an adjunct to antifungal treatment in disseminated coccidioidomycosis.FUNDINGNational Institute of Allergy and Infectious Diseases grants U19 AI166059 and R21 AI149654 and University of California Office of the President grant VFR-19-633386.

The systemic administration of therapeutic agents, particularly large, charged molecules such as antibodies, has limited efficacy in treating central nervous system (CNS) disorders. In addition, the slow progression of neurodegenerative diseases makes repeated intrathecal injections unfeasible. Alzheimer’s disease is characterized by the accumulation of Aβ amyloid plaques. Microglia contribute to the clearance of Aβ, but are inhibited by the expression of CD33. Therefore, antibody blocking of CD33 may enhance the phagocytosis of Aβ by microglial cells, slowing AD progression. Here, we use cells as “targeted cellular micropharmacies” that are retained in the CNS to deliver therapeutic proteins directly into the brain. To achieve this, we genetically engineered CD4 T-cells to express: (1) a chimeric antigen receptor against GD2 to retain the cells in the brain, (2) ectopic FoxP3 to reduce inflammation, (3) secreted IL-2 to promote cell longevity, and (4) secreted anti-CD33 scFv antibody. Our proof-of-concept demonstrates that therapeutic antibodies can be delivered to the brain for at least 8 weeks to treat neurological disorders. Other agents could be similarly delivered into the brain by this platform.

The manuscript demonstrates that the activity of cytokine blocking antibodies can be enhanced by introducing a highly sialylated IgG Fc-domain.