TLR Agonists Modify NK Cell Activation and Increase Its Cytotoxicity in Acute Lymphoblastic Leukemia.

Natural killer (NK) cells play a crucial role in innate immunity, particularly in combating infections and tumors. However, in hematological cancers, NK cells often exhibit impaired functions. Therefore, it is very important to activate its endosomal Toll-like receptors (TLRs) as a potential strategy to restore its antitumor activity. We stimulated NK cells from the peripheral blood mononuclear cells from children with acute lymphoblastic leukemia and NK cells isolated, and the NK cells were stimulated with specific TLR ligands (Poly I:C, Imiquimod, R848, and ODN2006) and we evaluated changes in IFN-γ, CD107a, NKG2D, NKp44 expression, Granzyme B secretion, cytokine/chemokine release, and cytotoxic activity. Results revealed that Poly I:C and Imiquimod enhanced the activation of both immunoregulatory and cytotoxic NK cells, increasing IFN-γ, CD107a, NKG2D, and NKp44 expression. R848 activated immunoregulatory NK cells, while ODN2006 boosted CD107a, NKp44, NKG2D, and IFN-γ secretion in cytotoxic NK cells. R848 also increased the secretion of seven cytokines/chemokines. Importantly, R848 and ODN 2006 significantly improved cytotoxicity against leukemic cells. Overall, TLR stimulation enhances NK cell activation, suggesting TLR8 (R848) and TLR9 (ODN 2006) ligands as promising candidates for antitumor immunotherapy.

Reassessing HIV Detection Strategies: An Analysis of Opportunistic Screening vs. Indicator-Condition-Driven Diagnosis in Valencia, Spain.

Our study assessed the characteristics of people living with HIV (PLWH) detected via opportunistic screening in Valencia (Spain) to determine diagnoses potentially missed under a more restrictive, indicator-condition diagnostic strategy. We conducted a retrospective analysis of electronic health records of 97 PLWH diagnosed between April 2019 and August 2022. The main outcomes reported were patient CD4+ T cell count, known HIV risk factors at diagnosis, and missed opportunities for diagnosis, defined as the failure of a previously untested patient to undergo HIV testing despite attending previous visits to healthcare facilities prior to diagnosis. Successful linkage to care was achieved for 95.9% of diagnosed patients. Half of the PLWH were diagnosed late, while 47.8% did not meet the criteria for indicator-condition-driven HIV diagnosis at the time of their diagnosis. Additionally, 52.2% did not receive HIV testing despite an average of 5.1 ± 6.0 healthcare visits in the 12 months prior to diagnosis. Spaniards had more missed opportunities for diagnosis than foreigners (64% vs. 40%, p = 0.02). Depending solely on an indicator-condition-driven HIV diagnosis approach could result in 47.8% of cases being missed. Including "migrants" as a testing criterion could lower missed diagnoses to 25.3% but might create inequities in prevention access. In conclusion, our findings provide valuable insights to enhance HIV testing, early diagnosis, and linkage to care. While it is crucial to uphold the indicator-condition-driven HIV diagnosis as baseline practice, improving screening strategies will decrease late diagnoses and missed opportunities, thereby effectively contributing to end the epidemic.

Influence of controlled environmental conditions in potential salivary ocular pain biomarkers for enhancing the assessment of ocular pain.

PURPOSE: We endeavored to identify objective salivary biomarkers for pain, a subjective sensation with a biological basis, using molecules already described related to pain. The study aimed to analyze inter-individual differences and intersession variability in salivary potential ocular pain biomarkers on healthy subjects, in samples obtained under the influence of controlled environmental conditions. METHODS: Thirty-four healthy subjects, 20 male, 14 female, median age 35.44 years (range 30-40) were exposed for 30 minutes under standard environmental conditions (T: 22°C, 50% relative humidity) in the Controlled Environmental Research Laboratory (CE-Lab, Vision R&D, Valladolid Spain) in two separate visits (V1, V2) at least 24 hours apart. Saliva was collected after the exposure in each of the visits, and cortisol, α-amylase (sAA), secretory IgA (sIgA), testosterone, and soluble fraction of TNFα receptor II (sTNFαRII) were analyzed by ELISA. Repeatability of inter-subject inter-session measurements was assayed by intraclass correlation coefficient (ICC). RESULTS: There were no significant inter-session differences in testosterone (p = 0.2497), sTNFαRII (p = 0.6451) and sIgA (p = 0.9689) salivary levels. The reproducibility for salivary cortisol, sAA, testosterone, sTNFαRII and sIgA were 0.98 ng/ml, 20.58 U/ml, 21.07 µg/ml, 24.68 pg/ml and 0.19 pg/ml, respectively. Salivary cortisol, sAA, testosterone, sTNFαRII and sIgA yielded the following ICCs: 0.506, 0.569, 0.824, 0.870 and 0.4295, respectively; all these ICCs (except that for cortisol and sIgA) were found to be improved compared to those found previously by our group in a previous study in salivary samples obtained from healthy subjects under non-controlled environmental conditions; Cortisol´s ICC didn´t improve and was in both cases at the limit of acceptability. CONCLUSION: Environmental factors such as temperature and relative humidity affect the reproducibility of measurement of some salivary molecules which have been proposed as potential pain biomarkers. The exposure of subjects to standard controlled environmental conditions before salivary sample obtention would improve the reproducibility of these molecule measures' as potential biomarkers of chronic ocular pain.

Aminopeptidase N/CD13 Crosslinking Promotes the Activation and Membrane Expression of Integrin CD11b/CD18.

The ß2 integrin CD11b/CD18, also known as complement receptor 3 (CR3), and the moonlighting protein aminopeptidase N (CD13), are two myeloid immune receptors with overlapping activities: adhesion, migration, phagocytosis of opsonized particles, and respiratory burst induction. Given their common functions, shared physical location, and the fact that some receptors can activate a selection of integrins, we hypothesized that CD13 could induce CR3 activation through an inside-out signaling mechanism and possibly have an influence on its membrane expression. We revealed that crosslinking CD13 on the surface of human macrophages not only activates CR3 but also influences its membrane expression. Both phenomena are affected by inhibitors of Src, PLCγ, Syk, and actin polymerization. Additionally, after only 10 min at 37 °C, cells with crosslinked CD13 start secreting pro-inflammatory cytokines like interferons type 1 and 2, IL-12p70, and IL-17a. We integrated our data with a bioinformatic analysis to confirm the connection between these receptors and to suggest the signaling cascade linking them. Our findings expand the list of features of CD13 by adding the activation of a different receptor via inside-out signaling. This opens the possibility of studying the joint contribution of CD13 and CR3 in contexts where either receptor has a recognized role, such as the progression of some leukemias.

Flow cytometry: A powerful analytical technique for characterizing the biological function of biotherapeutics and biosimilars.

Biotherapeutics are complex molecules with therapeutic activity produced through biotechnology and/or genetic engineering. These medicines have clinical applications in diagnostic procedures and therapies for many disorders, including cancer, autoimmunity, and chronic degenerative diseases. Most biotherapeutics are expensive and sometimes unaffordable for low-income patients suffering from cancer or chronic illness. Biosimilars emerged in the 2000 s after patents of many innovative biotherapeutic products expired. The Biosimilar market is growing fast and demands reliable technologies for analyzing the physicochemical properties and bioactivity of products. A big challenge for biosimilar development is to prove comparable bioactivity, safety, efficacy, and toxicity profile as the innovator product. Bioactivity assessment can utilize different analytical techniques such as ELISA, flow cytometry, and surface plasmon resonance. Flow cytometry is a versatile analytical tool that can be used for the development of quantitative, reproducible, and accurate protocols suitable for routine evaluation of bioactivity in-vitro. Nevertheless, flow cytometry has been very scarcely used in comparability evaluation between biosimilar versus an originator product. Here, we review potential applications of flow cytometry to carry out functional bioassays of biotherapeutics or biosimilars.

Molecular Basis of Plant Profilins' Cross-Reactivity.

Profilins are ubiquitous allergens with conserved structural elements. Exposure to profilins from different sources leads to IgE-cross-reactivity and the pollen-latex-food syndrome. Monoclonal antibodies (mAbs) that cross-react with plant profilins and block IgE-profilin interactions are relevant for diagnosis, epitope mapping, and specific immunotherapy. We generated IgGs mAbs, 1B4, and 2D10, against latex profilin (anti-rHev b 8) that inhibit the interaction of IgE and IgG4 antibodies from sera of latex- and maize-allergic patients by 90% and 40%, respectively. In this study, we evaluated 1B4 and 2D10 recognition towards different plant profilins, and mAbs recognition of rZea m 12 mutants by ELISAs. Interestingly, 2D10 highly recognized rArt v 4.0101 and rAmb a 8.0101, and to a lesser extent rBet v 2.0101, and rFra e 2.2, while 1B4 showed recognition for rPhl p 12.0101 and rAmb a 8.0101. We demonstrated that residue D130 at the α-helix 3 in profilins, which is part of the Hev b 8 IgE epitope, is essential for the 2D10 recognition. The structural analysis suggests that the profilins containing E130 (rPhl p 12.0101, rFra e 2.2, and rZea m 12.0105) show less binding with 2D10. The distribution of negative charges on the profilins' surfaces at the α-helices 1 and 3 is relevant for the 2D10 recognition, and that may be relevant to explain profilins' IgE cross-reactivity.

Increasing and sustaining blood-borne virus screening in Spain and Portugal throughout the COVID-19 pandemic: a multi-center quality improvement intervention.

Background: Around 57,000 people in Spain and Portugal currently living with HIV or chronic hepatitis C are unaware of their infection. The COVID-19 pandemic severely disrupted screening efforts for these infections. We designed an intervention to increase and sustain opportunistic blood-borne virus (BBV) screening and linkage to care (SLTC) by implementing the TEST model. Methods: The Plan Do Study Act (PDSA) method of quality improvement (QI) was implemented in 8 healthcare organizations (HCOs), including four hospitals, two clusters of community health centers, and two community-based organizations (CBOs). Baseline assessment included a review of BBV SLTC practices, testing volume, and results 12 months before the intervention. Changes in BBV testing rates over time were measured before, during, and after the COVID-19 lockdowns in 2020. A mixed ANOVA model was used to analyze the possible effect on testing volumes among HCOs over the three study periods. Intervention: BBV testing was integrated into normal clinical flow in all HCOs using existing clinical infrastructure and staff. Electronic health record (EHR) systems were modified whenever possible to streamline screening processes, implement systemic institutional policy changes, and promote QI. Results: Two years after the launch of the intervention in screening practices, testing volumes increased by 116%, with formal healthcare settings recording larger increases than CBOs. The start of the COVID-19 lockdowns was accompanied by a global 60% decrease in testing in all HCOs. Screening emergency department patients or using EHR systems to automate screening showed the highest resilience and lowest reduction in testing. HCOs recovered 77% of their testing volume once the lockdowns were lifted, with CBOs making the fullest recovery. Globally, enhanced screening techniques enabled HCOs to diagnose a total of 1,860 individuals over the research period. Conclusions: Implementation of the TEST model enabled HCOs to increase and sustain BBV screening, even during COVID-19 lockdowns. Although improvement in screening was noted in all HCOs, additional work is needed to develop strong patient linkage to care models in challenging times, such as global pandemics.

Crosslinking of membrane CD13 in human neutrophils mediates phagocytosis and production of reactive oxygen species, neutrophil extracellular traps and proinflammatory cytokines.

Aminopeptidase N, or CD13, is a cell membrane ectopeptidase highly expressed in myeloid cells. Through its enzymatic activity, CD13 regulates the activity of several bioactive peptides, such as endorphins and enkephalins, chemotactic peptides like MCP-1 and IL-8, angiotensin III, bradikinin, etc. In recent years, it has been appreciated that independently of its peptidase activity, CD13 can activate signal transduction pathways and mediate effector functions such as phagocytosis and cytokine secretion in monocytes and macrophages. Although neutrophils are known to express CD13 on its membrane, it is currently unknown if CD13 can mediate effector functions in these cells. Here, we show that in human neutrophils CD13 can mediate phagocytosis, which is dependent on a signaling pathway that involves Syk, and PI3-K. Phagocytosis mediated by CD13 is associated with production of reactive oxygen species (ROS). The level of phagocytosis and ROS production mediated by CD13 are similar to those through FcγRIII (CD16b), a widely studied receptor of human neutrophils. Also, CD13 ligation induces the release of neutrophil extracellular traps (NETs) as well as cytokine secretion from neutrophils. These results support the hypothesis that CD13 is a membrane receptor able to activate effector functions in human neutrophils.

Psychological readiness to return to sports practice and risk of recurrence: Case studies.

Returning to sport after the sports injury is a difficult decision because it's multicausal and the fact that a rash decision can result in numerous negative consequences. Given the importance of psychological variables for the correct rehabilitation of the injured athlete and his or her optimal return to sports practice, there seems to be little information on this subject. In this sense, the objective is to determine the relationship between the subjective psychological disposition of the athlete in the process of Return to Play (RTP) with the type of mood profile and his mental health. This is based on the fact that each athlete evaluates his or her recovery differently and has different levels of anxiety, depression, and stress. For this purpose, four athletes participated in the study. Two males and two females from the sports of indoor soccer and soccer, who had just returned to sports after a moderate or severe injury. The average age was 24.25 years. Various measurements were taken after practices and after matches, to assess mood, psychological readiness, anxiety, stress, and depression. The results confirm Morgan's iceberg profile and the influence that subjective psychological perceptions and assessed emotional states have on athletes' incorporation into their sports practice with a guarantee of success.

Phenotypic characterization of patients developing chronic dry eye and pain after refractive surgery: A cross-sectional study.

PURPOSE: To describe the clinical characteristics of patients suffering from chronic dry eye (DE) and pain after refractive surgery (RS). METHODS: Cross-sectional, observational, single-visit study. DE-, pain- and psychological-related symptoms were evaluated with specific questionnaires. DE-related tests evaluated tear osmolarity, conjunctival hyperemia, Meibomian gland dysfunction, tear stability and production, and ocular surface staining. Corneal mechanical sensitivity (Cochet-Bonnet) was measured pre/post topical anesthesia, and symptomatic variation post-anesthesia (anesthetic challenge test) was recorded. When pain was present, it was further categorized as neuropathic or nociceptive based on published criteria. RESULTS: We recruited 104 patients (39.5 ± 9.5 years). Most, 85.6%, had corneal RS as opposed to intraocular RS. Migraines, anxiety, depression (p < 0.0001), and central sensitization syndromes (p = 0.0214) were more frequent post-RS than pre-RS. Persistent DE-symptoms, severe in 86.5% patients, developed in a range of 0-204 months post-RS. Dryness and pain were the two most frequent symptoms. The only DE-related tests showing abnormal values were tear osmolarity (315.2 ± 17.1 mOsm/L; normal ≤308) and tear break-up time (4.1 ± 2.5 s; normal >7). Corneal sensitivity was 55.4 ± 7.0 mm, and decreased (p < 0.0001) after topical anesthesia, 6.0 ± 10.4 mm. However, it remained pathologically elevated, ≥10 mm in 61 (58.7%) patients. The normal symptomatic post-anesthesia improvement was absent in 58 (55.7%) patients. Ocular pain was present in 82 (78.8%) patients, and it was categorized as neuropathic in 66 (80.5%) of them, 63.5% of the entire cohort. CONCLUSIONS: Chronic ocular pain and its neuropathic subtype were diagnosed in 78.8% and 63.5% respectively of patients seeking consultation for persistent symptomatic DE post-RS.

A native IgE in complex with profilin provides insights into allergen recognition and cross-reactivity.

Allergies have become a rising health problem, where plentiful substances can trigger IgE-mediated allergies in humans. While profilins are considered minor allergens, these ubiquitous proteins are primary molecules involved in cross-reactivity and pollen-food allergy syndrome. Here we report the first crystal structures of murine Fab/IgE, with its chains naturally paired, in complex with the allergen profilin from Hevea brasiliensis (Hev b 8). The crystallographic models revealed that the IgE's six complementarity-determining regions (CDRs) interact with the allergen, comprising a rigid paratope-epitope surface of 926 Å2, which includes an extensive network of interactions. Interestingly, we also observed previously unreported flexibility at Fab/IgE's elbow angle, which did not influence the shape of the paratope. The Fab/IgE exhibits a high affinity for Hev b 8, even when using 1 M NaCl in BLI experiments. Finally, based on the encouraging cross-reactivity assays using two mutants of the maize profilin (Zea m 12), this antibody could be a promising tool in IgE engineering for diagnosis and research applications.

Leukocyte Membrane Enzymes Play the Cell Adhesion Game.

For a long time, proteins with enzymatic activity have not been usually considered to carry out other functions different from catalyzing chemical reactions within or outside the cell. Nevertheless, in the last few years several reports have uncovered the participation of numerous enzymes in other processes, placing them in the category of moonlighting proteins. Some moonlighting enzymes have been shown to participate in complex processes such as cell adhesion. Cell adhesion plays a physiological role in multiple processes: it enables cells to establish close contact with one another, allowing communication; it is a key step during cell migration; it is also involved in tightly binding neighboring cells in tissues, etc. Importantly, cell adhesion is also of great importance in pathophysiological scenarios like migration and metastasis establishment of cancer cells. Cell adhesion is strictly regulated through numerous switches: proteins, glycoproteins and other components of the cell membrane. Recently, several cell membrane enzymes have been reported to participate in distinct steps of the cell adhesion process. Here, we review a variety of examples of membrane bound enzymes participating in adhesion of immune cells.

Neutral evolution test of the spike protein of SARS-CoV-2 and its implications in the binding to ACE2.

As the SARS-CoV-2 has spread and the pandemic has dragged on, the virus continued to evolve rapidly resulting in the emergence of new highly transmissible variants that can be of public health concern. The evolutionary mechanisms that drove this rapid diversity are not well understood but neutral evolution should open the first insight. The neutral theory of evolution states that most mutations in the nucleic acid sequences are random and they can be fixed or disappear by purifying selection. Herein, we performed a neutrality test to better understand the selective pressures exerted over SARS-CoV-2 spike protein from homologue proteins of Betacoronavirus, as well as to the spikes from human clinical isolates of the virus. Specifically, Tyr and Asn have higher occurrence rates on the Receptor Binding Domain (RBD) and in the overall sequence of spike proteins of Betacoronavirus, whereas His and Arg have lower occurrence rates. The in vivo evolutionary phenomenon of SARS-CoV-2 shows that Glu, Lys, Phe, and Val have the highest probability of occurrence in the emergent viral particles. Amino acids that have higher occurrence than the expected by the neutral control, are favorable and are fixed in the sequence while the ones that have lower occurrence than expected, influence the stability and/or functionality of the protein. Our results show that most unique mutations either for SARS-CoV-2 or its variants of health concern are under selective pressures, which could be related either to the evasion of the immune system, increasing the virus' fitness or altering protein - protein interactions with host proteins. We explored the consequences of those selected mutations in the structure and protein - protein interaction with the receptor. Altogether all these forces have shaped the spike protein and the continually evolving variants.

A 2-(benzothiazol-2-yl)-phenolato platinum(II) complex as potential photosensitizer for combating bacterial infections in lung cancer chemotherapy†.

Cancer and antibiotic resistance are two global health threats that usually hamper clinical chemotherapeutic efficacy. Particularly for lung cancer, bacterial infections frequently arise thereby complicating the course of cancer treatment. In this sense, three new neutral luminescent cycloplatinated(II) photosensitizers of the type [Pt(dmba)(L)] (dmba = N,N-dimethylbenzylamine-κN,κC; L = 2-(benzo[d]oxazol-2-yl)-phenolato-κN,κO1, 2-(benzo[d]thiazol-2-yl)-phenolato-κN,κO2, and 2-(1-methyl-1H-benzo[d]imidazole-2-yl)phenolato-κN,κO3) have been characterized and developed to potentially eliminate both resistant bacteria and lung cancer cells. The phototherapeutic effects of complex 2 have been evaluated using low doses of blue light irradiation. Complex 2 exerted promising photoactivity against pathogenic Gram-positive bacteria strains of clinical interest, displaying a phototoxic index (PI) of 15 for methicillin-resistant Staphylococcus aureus, one of the major microorganisms predominating lung infections. Likewise, the anticancer activity of 2 was also increased upon light irradiation in human lung A549 cancer cells (PI = 36). Further in vitro experiments with this platinum(II) complex suggest that ROS-generating photodynamic reactions were involved upon light irradiation, thus providing a reasonable mechanism for its dual anticancer and antibacterial activities.

Neutrophils: Many Ways to Die.

Neutrophils or polymorphonuclear leukocytes (PMN) are key participants in the innate immune response for their ability to execute different effector functions. These cells express a vast array of membrane receptors that allow them to recognize and eliminate infectious agents effectively and respond appropriately to microenvironmental stimuli that regulate neutrophil functions, such as activation, migration, generation of reactive oxygen species, formation of neutrophil extracellular traps, and mediator secretion, among others. Currently, it has been realized that activated neutrophils can accomplish their effector functions and simultaneously activate mechanisms of cell death in response to different intracellular or extracellular factors. Although several studies have revealed similarities between the mechanisms of cell death of neutrophils and other cell types, neutrophils have distinctive properties, such as a high production of reactive oxygen species (ROS) and nitrogen species (RNS), that are important for their effector function in infections and pathologies such as cancer, autoimmune diseases, and immunodeficiencies, influencing their cell death mechanisms. The present work offers a synthesis of the conditions and molecules implicated in the regulation and activation of the processes of neutrophil death: apoptosis, autophagy, pyroptosis, necroptosis, NETosis, and necrosis. This information allows to understand the duality encountered by PMNs upon activation. The effector functions are carried out to eliminate invading pathogens, but in several instances, these functions involve activation of signaling cascades that culminate in the death of the neutrophil. This process guarantees the correct elimination of pathogenic agents, damaged or senescent cells, and the timely resolution of the inflammation that is essential for the maintenance of homeostasis in the organism. In addition, they alert the organism when the immunological system is being deregulated, promoting the activation of other cells of the immune system, such as B and T lymphocytes, which produce cytokines that potentiate the microbicide functions.

Organoplatinum(II) Complexes Self-Assemble and Recognize AT-Rich Duplex DNA Sequences.

The specific recognition of AT-rich DNA sequences opens up the door to promising diagnostic and/or therapeutic strategies against gene-related diseases. Here, we demonstrate that amphiphilic PtII complexes of the type [Pt(dmba)(N∧N)]NO3 (dmba = N,N-dimethylbenzylamine-κN, κC; N∧N = dpq (3), dppz (4), and dppn (5)) recognize AT-rich oligonucleotides over other types of DNA, RNA, and model proteins. The crystal structure of 4 shows the presence of significant π-stacking interactions and a distorted coordination sphere of the d8 PtII atom. Complex 5, containing the largest π-conjugated ligand, forms supramolecular assemblies at high concentrations under aqueous environment. However, its aggregation can be promoted in the presence of DNA at concentrations as low as 10 µM in a process that "turns on" its excimer emission around 600 nm. Viscometry, gel electrophoresis, and theoretical calculations demonstrate that 5 binds to minor groove when self-assembled, while the monomers of 3 and 4 intercalate into the DNA. The complexes also inhibit cancer cell growth with low-micromolar IC50 values in 2D tissue culture and suppress tumor growth in 3D tumor spheroids with a multicellular resistance (MCR) index comparable to that of cisplatin.

A Versatile Open-Source Printhead for Low-Cost 3D Microextrusion-Based Bioprinting.

Three-dimensional (3D) bioprinting promises to be essential in tissue engineering for solving the rising demand for organs and tissues. Some bioprinters are commercially available, but their impact on the field of Tissue engineering (TE) is still limited due to their cost or difficulty to tune. Herein, we present a low-cost easy-to-build printhead for microextrusion-based bioprinting (MEBB) that can be installed in many desktop 3D printers to transform them into 3D bioprinters. We can extrude bioinks with precise control of print temperature between 2-60 °C. We validated the versatility of the printhead, by assembling it in three low-cost open-source desktop 3D printers. Multiple units of the printhead can also be easily put together in a single printer carriage for building a multi-material 3D bioprinter. Print resolution was evaluated by creating representative calibration models at different temperatures using natural hydrogels such as gelatin and alginate, and synthetic ones like poloxamer. Using one of the three modified low-cost 3D printers, we successfully printed cell-laden lattice constructs with cell viabilities higher than 90% after 24-h post printing. Controlling temperature and pressure according to the rheological properties of the bioinks was essential in achieving optimal printability and great cell viability. The cost per unit of our device, which can be used with syringes of different volume, is less expensive than any other commercially available product. These data demonstrate an affordable open-source printhead with the potential to become a reliable alternative to commercial bioprinters for any laboratory.

Novel murine mAbs define specific and cross-reactive epitopes on the latex profilin panallergen Hev b 8.

The production of specific antibodies able to recognize allergens from different sources or block interactions between allergens and antibodies mediating allergic reactions is crucial for developing successful tools for diagnostics and therapeutics. Panallergens are highly conserved proteins present in widely different species, implicated in relevant cross-reactions. The panallergen latex profilin (Hev b 8) has been associated with the latex-food-pollen syndrome. We generated five monoclonal IgGs and one IgE from murine hybridomas against recombinant Hev b 8 and evaluated their interaction with this allergen using ELISA and biolayer interferometry (BLI). Affinity purified mAbs exhibited high binding affinities towards rHev b 8, with KD1 values ranging from 10-10 M to 10-11 M. Some of these antibodies also recognized the recombinant profilins from maize and tomato (Zea m 12 and Sola l 1), and the ash tree pollen (Fra e 2). Competition ELISA demonstrated that some mAb pairs could bind simultaneously to rHev b 8. Using BLI, we detected competitive, non-competitive, and partial-competition interactions between pairs of mAbs with rHev b 8, suggesting the existence of at least two non-overlapping epitopes on the surface of this allergen. Three-dimensional models of the Fv of 1B4 and 2D10 IgGs and docking simulations of these Fvs with rHev b 8 revealed these epitopes. Furthermore, these two mAbs inhibited the interaction of polyclonal IgE and IgG4 antibodies from profilin-allergic patients with rHev b 8, indicating that the mAbs and the antibodies present in sera from allergic patients bind to overlapping epitopes on the allergen. These mAbs can be useful tools for immune-localization studies, immunoassay development, or standardization of allergenic products.