Results of phase 2 randomized multi-center study to evaluate the safety and efficacy of infusion of memory T cells as adoptive therapy in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pneumonia and/or lymphopenia (RELEASE NCT04578210).

BACKGROUND AIMS: There are currently no effective anti-viral treatments for coronavirus disease 2019 (COVID-19)-hospitalized patients with hypoxemia. Lymphopenia is a biomarker of disease severity usually present in patients who are hospitalized. Approaches to increasing lymphocytes exerting an anti-viral effect must be considered to treat these patients. Following our phase 1 study, we performed a phase 2 randomized multicenter clinical trial in which we evaluated the efficacy of the infusion of allogeneic off-the-shelf CD45RA- memory T cells containing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cells from convalescent donors plus the standard of care (SoC) versus just the SoC treatment. METHODS: Eighty-four patients were enrolled in three Spanish centers. The patients were randomized into the infusion of 1 × 106/kg CD45RA- memory T cells or the SoC. We selected four unvaccinated donors based on the expression of interferon gamma SARS-CoV-2-specific response within the CD45RA- memory T cells and the most frequent human leukocyte antigen typing in the Spanish population. RESULTS: We analyzed data from 81 patients. The primary outcome for recovery, defined as the proportion of participants in each group with normalization of fever, oxygen saturation sustained for at least 24 hours and lymphopenia recovery through day 14 or at discharge, was met for the experimental arm. We also observed faster lymphocyte recovery in the experimental group. We did not observe any treatment-related adverse events. CONCLUSIONS: Adoptive cell therapy with off-the-shelf CD45RA- memory T cells containing SAR-CoV-2-specific T cells is safe, effective and accelerates lymphocyte recovery of patients with COVID-19 pneumonia and/or lymphopenia. TRIAL REGISTRATION: NCT04578210.

CLA+ memory T cells in atopic dermatitis: CLA+ T cells and atopic dermatitis.

Circulating skin-homing cutaneous lymphocyte-associated antigen (CLA)+ T cells constitute a small subset of human memory T cells involved in several aspects of atopic dermatitis: Staphylococcus aureus related mechanisms, the abnormal Th2 immune response, biomarkers, clinical aspects of the patients, pruritus, and the mechanism of action of targeted therapies. Superantigens, IL-13, IL-31, pruritus, CCL17 and early effects on dupilumab-treated patients have in common that they are associated with the CLA+ T cell mechanisms in atopic dermatitis patients. The function of CLA+ T cells corresponds with the role of T cells belonging to the skin-associated lymphoid tissue and could be a reason why they reflect different mechanisms of atopic dermatitis and many other T cell mediated skin diseases. The goal of this review is to gather all this translational information of atopic dermatitis pathology.

Untargeted serum metabolomic analysis reveals a role for purinergic signaling in FPIES.

BACKGROUND: Food protein-induced enterocolitis syndrome (FPIES) is a non-IgE-mediated food allergy with a typical onset in infancy. Its symptoms are distinct from those of IgE-mediated food allergies and include severe repetitive vomiting, lethargy, and pallor. FPIES reactions are associated with TH17 cytokines and a systemic innate immune activation; however, the link between immune activation and symptoms is poorly understood. OBJECTIVE: Our aim was to use an untargeted metabolomics approach to identify novel pathways associated with FPIES reactions. METHODS: Serum samples were obtained before, during, and after oral food challenge (OFC) (10 subjects with FPIES and 10 asymptomatic subjects), and they were analyzed by untargeted metabolomics. Two-way ANOVA with false discovery rate adjustment was used for analysis of metabolites. Stomach and duodenal biopsy specimens from non-FPIES donors were stimulated with adenosine in vitro and serotonin measured by immunoassay. RESULTS: The levels of a total of 34 metabolites, including inosine and urate of the purine signaling pathway, were increased during OFCs performed on the patients with symptomatic FPIES compared with the levels found for asymptomatic subjects. Expression of the purine receptors P2RX7 and P2RY10 and the ectonucleotidase CD73 in peripheral blood was significantly reduced after OFC of the patients with FPIES. The level of the serotonin metabolite 5-hydroxyindoleacetate was significantly elevated after reaction. Adenosine stimulation of gastric and duodenal biopsy specimens from FPIES-free donors induced a significant release of serotonin, suggesting a link between purinergic pathway activation and serotonin release. CONCLUSIONS: Activation of the purinergic pathway during FPIES reactions provides a possible mechanism connecting inflammation and vomiting by triggering serotonin release from gastric and duodenal mucosa.

Allergen recognition by specific effector Th2 cells enables IL-2-dependent activation of regulatory T-cell responses in humans.

Type 2 allergen-specific T cells are essential for the induction and maintenance of allergies to foods, and Tregs specific for these allergens are assumed to be involved in their resolution. However, it has not been convincingly demonstrated whether allergen-specific Treg responses are responsible for the generation of oral tolerance in humans. We observed that sustained food allergen exposure in the form of oral immunotherapy resulted in increased frequency of Tregs only in individuals with lasting clinical tolerance. We sought to identify regulatory components of the CD4+ T-cell response to food allergens by studying their functional activation over time in vitro and in vivo. Two subsets of Tregs expressing CD137 or CD25/OX40 were identified with a delayed kinetics of activation compared with clonally enriched pathogenic effector Th2 cells. Treg activation was dependent on IL-2 derived from effector T cells. In vivo exposure to peanut in the form of an oral food challenge of allergic subjects induced a delayed and persistent activation of Tregs after initiation of the allergen-specific Th2 response. The novel finding of our work is that a sustained wave of Treg activation is induced by the release of IL-2 from Th2 effector cells, with the implication that therapeutic administration of IL-2 could improve current OIT approaches.

New applications of advanced instrumental techniques for the characterization of food allergenic proteins.

Current approaches based on electrophoretic, chromatographic or immunochemical principles have allowed characterizing multiple allergens, mapping their epitopes, studying their mechanisms of action, developing detection and diagnostic methods and therapeutic strategies for the food and pharmaceutical industry. However, some of the common structural features related to the allergenic potential of food proteins remain unknown, or the pathological mechanism of food allergy is not yet fully understood. In addition, it is also necessary to evaluate new allergens from novel protein sources that may pose a new risk for consumers. Technological development has allowed the expansion of advanced technologies for which their whole potential has not been entirely exploited and could provide novel contributions to still unexplored molecular traits underlying both the structure of food allergens and the mechanisms through which they sensitize or elicit adverse responses in human subjects, as well as improving analytical techniques for their detection. This review presents cutting-edge instrumental techniques recently applied when studying structural and functional aspects of proteins, mechanism of action and interaction between biomolecules. We also exemplify their role in the food allergy research and discuss their new possible applications in several areas of the food allergy field.

Acute FPIES reactions are associated with an IL-17 inflammatory signature.

BACKGROUND: Food protein-induced enterocolitis syndrome (FPIES) is a non-IgE-mediated food allergy characterized by profuse vomiting within hours of ingestion of the causative food. We have previously reported that FPIES is associated with systemic innate immune activation in the absence of a detectable antigen-specific antibody or T-cell response. The mechanism of specific food recognition by the immune system remains unclear. OBJECTIVE: Our aim was to identify immune mechanisms underlying FPIES reactions by proteomic and flow cytometric analysis of peripheral blood. METHODS: Children with a history of FPIES underwent supervised oral food challenge. Blood samples were taken at baseline, at symptom onset, and 4 hours after symptom onset. We analyzed samples from 23 children (11 reactors and 12 outgrown). A total of 184 protein markers were analyzed by proximity ligation assay and verified by multiplex immunoassay. Analysis of cell subset activation was performed by mass cytometry and spectral cytometry. RESULTS: Symptomatic FPIES challenge results were associated with significant elevation of levels of cytokines and chemokines, including IL-17 family markers (IL-17A, IL-22, IL-17C, and CCL20) and T-cell activation (IL-2), and innate inflammatory markers (IL-8, oncostatin M, leukemia inhibitory factor, TNF-α, IL-10, and IL-6). The level of the mucosal damage marker regenerating family member 1 alpha (REG1A) was also significantly increased. These biomarkers were not increased in asymptomatic challenges or IgE-mediated allergy. The level of phospho-STAT3 was significantly elevated in myeloid and T cells after challenge in individuals with symptoms. Mass cytometry indicated preferential activation of nonconventional T-cell populations, including γδ T cells and CD3+CD4-CD8-CD161+ cells; however, the potential sources of IL-17 in PBMCs were primarily CD4+ TH17 cells. CONCLUSIONS: These results demonstrate a unique IL-17 signature and activation of innate lymphocytes in FPIES.

Egg yolk augments type 2 immunity by activating innate cells.

PURPOSE: Egg yolk (EY) may play a role during the sensitizing phase of egg allergy by exerting intestinal type 2-biasing effects. We aimed to identify the mechanism and role of EY in the induction of allergy to egg white (EW). METHODS: BALB/c mice were exposed intragastrically to EW, EY, or the mixture of EW:EY. In addition in vitro experiments were conducted with intestinal epithelial cells (IECs), dendritic cells (DCs), and T cells from naïve mice. Inflammatory and type 2 responses were evaluated. RESULTS: Administration of EW upregulated duodenal expression of factors that influence epithelial barrier integrity and function, such as Muc2 and Cldn2, type 2-promoting epithelial cytokines Il33 and Il25, DC genes Irf4 and Tnfsf4, and Th2-cytokines Il14 and Il13. EW:EY further increased the expression of Il25 and Tslp in the duodenum, Il33 and Tslp in the jejunum, and the proportion of lamina propria group 2 innate immune cells (ILC2s) over EW alone. Moreover, it distinctively enhanced the expression of Irf4 and Cd1d1 in the Peyer's patches (PPs), and of Il6, Il33, Gata3, and Il13, both in PPs and mesenteric lymph nodes. In co-cultures of DCs and T cells, EW:EY induced a higher expression of Gata3, Il4, and Il13, secretion of IL-13 and expansion of CD4+ T cells expressing ST2, the IL-33 receptor, than EW or EY added individually. CONCLUSION: Co-administration of EY may promote sensitization to EW through activation of innate immune cells, such as IECs, DCs and ILC2s, that are central to the progress of allergies.

Immunomodulating peptides for food allergy prevention and treatment.

Among the most promising strategies currently assayed against IgE-mediated allergic diseases stands the possibility of using immunomodulating peptides to induce oral tolerance toward offending food allergens or even to prevent allergic sensitization. This review focuses on the beneficial effects of food derived immunomodulating peptides on food allergy, which can be directly exerted in the intestinal tract or once being absorbed through the intestinal epithelial barrier to interact with immune cells. Food peptides influence intestinal homeostasis by maintaining and reinforcing barrier function or affecting intestinal cell-signalling to nearby immune cells and mucus secretion. In addition, they can stimulate cells of the innate and adaptive immune system while supressing inflammatory responses. Peptides represent an attractive alternative to whole allergens to enhance the safety and efficacy of immunotherapy treatments. The conclusions drawn from curative and preventive experiments in murine models are promising, although there is a need for more pre-clinical studies to further explore the immunomodulating strategy and its mechanisms and for a deeper knowledge of the peptide sequence and structural requirements that determine the immunoregulatory function.

Anaphylaxis induced by a drug containing lysozyme and papain: influence of papain on the IgE response.

BACKGROUND: This paper reports the case of an egg-allergic pediatric patient who, once desensitized to egg following a successful rush oral immunotherapy protocol, could also tolerate Lizipaina®, a drug containing lysozyme (LYS) and papain, which had previously caused him a severe allergic reaction. Because the LYS amount that elicited the anaphylactic reaction (5 mg) was much lower than that tolerated during a double-blind placebo-controlled food challenge (corresponding to approximately 60 mg of LYS), the possibility that the presence of papain could increase the allergenic potential of LYS was investigated. METHODS: Lizipaina, LYS and LYS hydrolyzed with papain were analyzed by SDS-PAGE under reducing and nonreducing conditions, and Western blotting of sera from egg-allergic patients was performed in order to detect IgE-binding fragments. Finally, sequence identification of the IgE-reactive bands was carried out by MALDI-TOF/TOF. RESULTS: The SDS-PAGE pattern of LYS treated with papain under nonreducing conditions showed the presence of intact LYS that partially disappeared following reduction with ß-mercaptoethanol, releasing IgE-reactive fragments as determined by Western blotting. MALDI-TOF/TOF revealed that papain degraded LYS, giving rise to three IgE-binding fragments: LYS (22-129), LYS (34-96) and LYS (62-128) that likely remained linked through the disulfide bonds present in the LYS molecule. CONCLUSION: The combined administration of LYS with proteolytic enzymes such as papain may have developed a severe allergic reaction in the patient studied, underlining the importance of considering all the components and their interactions when drugs are to be consumed by allergic persons.

Anti-correlated feature selection prevents false discovery of subpopulations in scRNAseq.

While sub-clustering cell-populations has become popular in single cell-omics, negative controls for this process are lacking. Popular feature-selection/clustering algorithms fail the null-dataset problem, allowing erroneous subdivisions of homogenous clusters until nearly each cell is called its own cluster. Using real and synthetic datasets, we find that anti-correlated gene selection reduces or eliminates erroneous subdivisions, increases marker-gene selection efficacy, and efficiently scales to millions of cells.

Effects of unfolding treatment assisted glycation on the IgE/IgG binding capacity and antioxidant activity of ovomucoid.

Ovomucoid is the immune-dominant allergen in the egg white of hens. Due to its structure based on nine disulfide bonds as well as its resistance to heat and enzymatic hydrolysis, the allergenicity of this food protein is difficult to decrease by technological processes. We sought to reduce its allergenicity through the Maillard reaction. The unfolding of ovomucoid with L-cysteine-mediated reduction was used to increase accessibility to conformational and linear epitopes by modifying the secondary and tertiary structures of the allergen. Glycation with different saccharides revealed the beneficial effect of maltose glycation on the IgG-binding capacity reduction. By determining the better glycation conditions of unfolded ovomucoid, we produced ovomucoid with reduced IgE binding capacity due to the glycation sites (K17, K112, K129, and K164) on epitopes. Moreover, after simulated infant and adult gastrointestinal digestion, the unfolded plus glycated ovomucoid showed higher ABTS˙+ scavenging activity, O2˙- scavenging activity, ˙OH scavenging activity, Fe2+ chelating activity, and a FRAP value; in particular, for ˙OH scavenging activity, there was a sharp increase of more than 100%.

Molecular Approaches for Food Protein Allergenicity Assessment and the Diagnosis and Treatment of Food Allergies.

Food allergy, an adverse immune reaction triggered by commonly innocuous food proteins, is a health problem that affects millions of people worldwide (around 10% of the global population), and the most recent reports suggest its increasing progression [...].

Intestinal factors promoting the development of RORγt+ cells and oral tolerance.

The gastrointestinal tract has to harmonize the two seemingly opposite functions of fulfilling nutritional needs and avoiding the entry of pathogens, toxins and agents that can cause physical damage. This balance requires a constant adjustment of absorptive and defending functions by sensing environmental changes or noxious substances and initiating adaptive or protective mechanisms against them through a complex network of receptors integrated with the central nervous system that communicate with cells of the innate and adaptive immune system. Effective homeostatic processes at barrier sites take the responsibility for oral tolerance, which protects from adverse reactions to food that cause allergic diseases. During a very specific time interval in early life, the establishment of a stable microbiota in the large intestine is sufficient to prevent pathological events in adulthood towards a much larger bacterial community and provide tolerance towards diverse food antigens encountered later in life. The beneficial effects of the microbiome are mainly exerted by innate and adaptive cells that express the transcription factor RORγt, in whose generation, mediated by different bacterial metabolites, retinoic acid signalling plays a predominant role. In addition, recent investigations indicate that food antigens also contribute, analogously to microbial-derived signals, to educating innate immune cells and instructing the development and function of RORγt+ cells in the small intestine, complementing and expanding the tolerogenic effect of the microbiome in the colon. This review addresses the mechanisms through which microbiota-produced metabolites and dietary antigens maintain intestinal homeostasis, highlighting the complementarity and redundancy between their functions.

JAK1 inhibition with abrocitinib decreases allergen-specific basophil and T-cell activation in pediatric peanut allergy.

Background: JAK1 is a signaling molecule downstream of cytokine receptors, including IL-4 receptor α. Abrocitinib is an oral JAK1 inhibitor; it is a safe and effective US Food and Drug Administration-approved treatment for adults with moderate-to-severe atopic dermatitis. Objective: Our objective was to investigate the effect of abrocitinib on basophil activation and T-cell activation in patients with peanut allergy to determine the potential for use of JAK1 inhibitors as a monotherapy or an adjuvant to peanut oral immunotherapy. Methods: Basophil activation in whole blood was measured by detection of CD63 expression using flow cytometry. Activation of CD4+ effector and regulatory T cells was determined by the upregulation of CD154 and CD137, respectively, on anti-CD3/CD28- or peanut-stimulated PBMCs. For the quantification of peanut-induced cytokines, PBMCs were stimulated with peanut for 5 days before harvesting supernatant. Results: Abrocitinib decreased the allergen-specific activation of basophils in response to peanut. We showed suppression of effector T-cell activation when stimulated by CD3/CD28 beads in the presence of 10 ng of abrocitinib, whereas activation of regulatory T-cell populations was preserved in the presence of abrocitinib. Abrocitinib induced statistically significant dose-dependent inhibition in IL-5, IL-13, IL-10, IL-9, and TNF-α in the presence of peanut stimulation. Conclusion: These results support our hypothesis that JAK1 inhibition decreases basophil activation and TH2 cytokine signaling, reducing in vitro allergic responses in subjects with peanut allergy. Abrocitinib may be an effective adjunctive immune modulator in conjunction with peanut oral immunotherapy or as a monotherapy for individuals with food allergy.

Egg yolk lipids induce sensitization to egg white proteins in a mouse model without adjuvant and exacerbate Th2 responses to egg white in cells from allergic patients.

This study evaluates the influence of egg lipid fractions in the induction of allergic sensitization to egg white (EW) proteins, using a mouse model of orally adjuvant-free induced allergy. Egg triglycerides (TG) and phospholipids (PL), and to a higher extent the whole egg lipid fraction (EL), induced allergy to EW proteins characterized by increased EW-specific IgG1. EL also increased EW-specific IgE. The administration to mice of a mixture of EW and EL increased the intestinal expression of Il33, Il25, and Tslp, the secretion of IL-33 and IL-6, the expansion of group 2 innate lymphoid cells, the regulation of Gata3, Il4 and Il13, dendritic cell (DC) activation and expression of DC molecules that drive Th2 differentiation. TG promoted the absorption of proteins through the intestinal epithelium, enhancing local Th2 responses, while PL favoured the delivery of antigens to the Peyer's Patches. This differential modulation of the site of absorption of egg proteins determined the different behaviour of TG and PL. Egg yolk lipids also induced activation of Th2-inducing innate responses on intestinal human cells in vitro and enhanced adaptive Th2 functions through the activation of DCs in egg-allergic subjects.

Maintenance of Potent Cellular and Humoral Immune Responses in Long-Term Hemodialysis Patients after 1273-mRNA SARS-CoV-2 Vaccination.

Continuous evaluation of the coronavirus disease 2019 (COVID-19) vaccine effectiveness in hemodialysis (HD) patients is critical in this immunocompromised patient group with higher mortality rates due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The response towards vaccination in HD patients has been studied weeks after their first and second SARS-CoV-2 vaccination dose administration, but no further studies have been developed in a long-term manner, especially including both the humoral and cellular immune response. Longitudinal studies that monitor the immune response to COVID-19 vaccination in individuals undergoing HD are therefore necessary to prioritize vaccination strategies and minimize the pathogenic effects of SARS-CoV-2 in this high-risk group of patients. We followed up HD patients and healthy volunteers (HV) and monitored their humoral and cellular immune response three months after the second (V2+3M) and after the third vaccination dose (V3+3M), taking into consideration previous COVID-19 infections. Our cellular immunity results show that, while HD patients and HV individuals secrete comparable levels of IFN-γ and IL-2 in ex vivo stimulated whole blood at V2+3M in both naïve and COVID-19-recovered individuals, HD patients secrete higher levels of IFN-γ and IL-2 than HV at V3+3M. This is mainly due to a decay in the cellular immune response in HV individuals after the third dose. In contrast, our humoral immunity results show similar IgG binding antibody units (BAU) between HD patients and HV individuals at V3+3M, independently of their previous infection status. Overall, our results indicate that HD patients maintain strong cellular and humoral immune responses after repeated 1273-mRNA SARS-CoV-2 vaccinations over time. The data also highlights significant differences between cellular and humoral immunity after SARS-CoV-2 vaccination, which emphasizes the importance of monitoring both arms of the immune response in the immunocompromised population.

A Mutation-driven oncofetal regression fuels phenotypic plasticity in colorectal cancer.

Targeting cancer stem cells (CSCs) is crucial for effective cancer treatment 1 . However, the molecular mechanisms underlying resistance to LGR5 + CSCs depletion in colorectal cancer (CRC) 2,3 remain largely elusive. Here, we unveil the existence of a primitive cell state dubbed the oncofetal (OnF) state, which works in tandem with the LGR5 + stem cells (SCs) to fuel tumor evolution in CRC. OnF cells emerge early during intestinal tumorigenesis and exhibit features of lineage plasticity. Normally suppressed by the Retinoid X Receptor (RXR) in mature SCs, the OnF program is triggered by genetic deletion of the gatekeeper APC. We demonstrate that diminished RXR activity unlocks an epigenetic circuity governed by the cooperative action of YAP and AP1, leading to OnF reprogramming. This high-plasticity state is inherently resistant to conventional chemotherapies and its adoption by LGR5 + CSCs enables them to enter a drug-tolerant state. Furthermore, through phenotypic tracing and ablation experiments, we uncover a functional redundancy between the OnF and stem cell (SC) states and show that targeting both cellular states is essential for sustained tumor regression in vivo . Collectively, these findings establish a mechanistic foundation for developing effective combination therapies with enduring impact on CRC treatment.

Control of Listeria monocytogenes growth and virulence in a traditional soft cheese model system based on lactic acid bacteria and a whey protein hydrolysate with antimicrobial activity.

"Torta del Casar" is a Spanish soft-ripened cheese made with sheep's raw milk and subjected to a short ripening process, which favors the growth of pathogenic microorganisms including Listeria monocytogenes. The development of strategies to control pathogens and minimize health risks associated with the presence of L. monocytogenes in these products is of great interest. In this regard, the anti-Listeria activity of a whey protein hydrolysate (ProH) alone or combined with six lactic acid bacteria strains isolated from cheese was evaluated in this study as a biocontrol strategy using a "Torta del Casar" cheese-based medium. The most active combinations of lactic acid bacteria assayed induced a reduction higher than two logarithmic units in the growth of L. monocytogenes (serotype 4b) compared to their respective control when they were co-inoculated in "Torta del Casar" cheese-based medium at 7 °C for 7 days. In addition, the observed downregulation of some key virulence genes of L. monocytogenes suggests that the strain Lactiplantibacillus plantarum B2 alone and combined with the strain Lactiplantibacillus spp. B4 are good candidates to be used as biocontrol agents against L. monocytogenes growth in traditional soft cheeses based on raw milk during their storage at refrigeration temperatures.