Institute of Immunology and Physiology

<italic>Cannabis sativa</italic> L. is a prolific, but not exclusive, producer of a diverse group of isoprenylated resorcinyl polyketides collectively known as phytocannabinoids.

Currently, testing for immunoglobulin E (IgE) sensitization is the cornerstone of diagnostic evaluation in suspected allergic conditions. This review provides a thorough and updated critical appraisal of the most frequently used diagnostic tests, both in vivo and in vitro. It discusses skin tests, challenges, and serological and cellular in vitro tests, and provides an overview of indications, advantages and disadvantages of each in conditions such as respiratory, food, venom, drug, and occupational allergy. Skin prick testing remains the first line approach in most instances; the added value of serum specific IgE to whole allergen extracts or components, as well as the role of basophil activation tests, is evaluated. Unproven, non-validated, diagnostic tests are also discussed. Throughout the review, the reader must bear in mind the relevance of differentiating between sensitization and allergy; the latter entails not only allergic sensitization, but also clinically relevant symptoms triggered by the culprit allergen.

Immunoglobulin E (IgE)-mediated allergy is the most common hypersensitivity disease affecting more than 30% of the population. Exposure to even minute quantities of allergens can lead to the production of IgE antibodies in atopic individuals. This is termed allergic sensitization, which occurs mainly in early childhood. Allergen-specific IgE then binds to the high (FcεRI) and low-affinity receptors (FcεRII, also called CD23) for IgE on effector cells and antigen-presenting cells. Subsequent and repeated allergen exposure increases allergen-specific IgE levels and, by receptor cross-linking, triggers immediate release of inflammatory mediators from mast cells and basophils whereas IgE-facilitated allergen presentation perpetuates T cell-mediated allergic inflammation. Due to engagement of receptors which are highly selective for IgE, even tiny amounts of allergens can induce massive inflammation. Naturally occurring allergen-specific IgG and IgA antibodies usually recognize different epitopes on allergens compared with IgE and do not efficiently interfere with allergen-induced inflammation. However, IgG and IgA antibodies to these important IgE epitopes can be induced by allergen-specific immunotherapy or by passive immunization. These will lead to competition with IgE for binding with the allergen and prevent allergic responses. Similarly, anti-IgE treatment does the same by preventing IgE from binding to its receptor on mast cells and basophils. Here, we review the complex interplay of allergen-specific IgE, IgG and IgA and the corresponding cell receptors in allergic diseases and its relevance for diagnosis, treatment and prevention of allergy.

Recent advances have greatly improved our understanding of the molecular mechanisms behind atherosclerosis pathogenesis. However, there is still a need to systematize this data from a general pathology perspective, particularly with regard to atherogenesis patterns in the context of both canonical and non-classical inflammation types. In this review, we analyze various typical phenomena and outcomes of cellular pro-inflammatory stress in atherosclerosis, as well as the role of endothelial dysfunction in local and systemic manifestations of low-grade inflammation. We also present the features of immune mechanisms in the development of productive inflammation in stable and unstable plaques, along with their similarities and differences compared to canonical inflammation. There are numerous factors that act as inducers of the inflammatory process in atherosclerosis, including vascular endothelium aging, metabolic dysfunctions, autoimmune, and in some cases, infectious damage factors. Life-critical complications of atherosclerosis, such as cardiogenic shock and severe strokes, are associated with the development of acute systemic hyperinflammation. Additionally, critical atherosclerotic ischemia of the lower extremities induces paracoagulation and the development of chronic systemic inflammation. Conversely, sepsis, other critical conditions, and severe systemic chronic diseases contribute to atherogenesis. In summary, atherosclerosis can be characterized as an independent form of inflammation, sharing similarities but also having fundamental differences from low-grade inflammation and various variants of canonical inflammation (classic vasculitis).

The review aims to consolidate research findings on the molecular mechanisms and virulence and pathogenicity characteristics of coronavirus disease (COVID-19) causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and their relevance to four typical stages in the development of acute viral infection. These four stages are invasion; primary blockade of antiviral innate immunity; engagement of the virus's protection mechanisms against the factors of adaptive immunity; and acute, long-term complications of COVID-19. The invasion stage entails the recognition of the spike protein (S) of SARS-CoV-2 target cell receptors, namely, the main receptor (angiotensin-converting enzyme 2, ACE2), its coreceptors, and potential alternative receptors. The presence of a diverse repertoire of receptors allows SARS-CoV-2 to infect various types of cells, including those not expressing ACE2. During the second stage, the majority of the polyfunctional structural, non-structural, and extra proteins SARS-CoV-2 synthesizes in infected cells are involved in the primary blockage of antiviral innate immunity. A high degree of redundancy and systemic action characterizing these pathogenic factors allows SARS-CoV-2 to overcome antiviral mechanisms at the initial stages of invasion. The third stage includes passive and active protection of the virus from factors of adaptive immunity, overcoming of the barrier function at the focus of inflammation, and generalization of SARS-CoV-2 in the body. The fourth stage is associated with the deployment of variants of acute and long-term complications of COVID-19. SARS-CoV-2's ability to induce autoimmune and autoinflammatory pathways of tissue invasion and development of both immunosuppressive and hyperergic mechanisms of systemic inflammation is critical at this stage of infection.

Since the discovery of Stimulator of Interferon Genes (STING) as an important pivot for cytosolic DNA sensation and interferon (IFN) induction, intensive efforts have been endeavored to clarify the molecular mechanism of its activation, its physiological function as a ubiquitously expressed protein, and to explore its potential as a therapeutic target in a wide range of immune-related diseases. With its orthodox ligand 2'3'-cyclic GMP-AMP (2'3'-cGAMP) and the upstream sensor 2'3'-cGAMP synthase (cGAS) to be found, STING acquires its central functionality in the best-studied signaling cascade, namely the cGAS-STING-IFN pathway. However, recently updated research through structural research, genetic screening, and biochemical assay greatly extends the current knowledge of STING biology. A second ligand pocket was recently discovered in the transmembrane domain for a synthetic agonist. On its downstream outputs, accumulating studies sketch primordial and multifaceted roles of STING beyond its cytokine-inducing function, such as autophagy, cell death, metabolic modulation, endoplasmic reticulum (ER) stress, and RNA virus restriction. Furthermore, with the expansion of the STING interactome, the details of STING trafficking also get clearer. After retrospecting the brief history of viral interference and the milestone events since the discovery of STING, we present a vivid panorama of STING biology taking into account the details of the biochemical assay and structural information, especially its versatile outputs and functions beyond IFN induction. We also summarize the roles of STING in the pathogenesis of various diseases and highlight the development of small-molecular compounds targeting STING for disease treatment in combination with the latest research. Finally, we discuss the open questions imperative to answer.

The Janus kinase-signal transducer and transcription activator pathway (JAK-STAT) serves as a cornerstone in cellular signaling, regulating physiological and pathological processes such as inflammation and stress. Dysregulation in this pathway can lead to severe immunodeficiencies and malignancies, and its role extends to neurotransduction and pro-inflammatory signaling mechanisms. Although JAK inhibitors (Jakinibs) have successfully treated immunological and inflammatory disorders, their application has generally been limited to diseases with similar pathogenic features. Despite the modest expression of JAK-STAT in the CNS, it is crucial for functions in the cortex, hippocampus, and cerebellum, making it relevant in conditions like Parkinson's disease and other neuroinflammatory disorders. Furthermore, the influence of the pathway on serotonin receptors and phospholipase C has implications for stress and mood disorders. This review expands the understanding of JAK-STAT, moving beyond traditional immunological contexts to explore its role in stress-related disorders and CNS function. Recent findings, such as the effectiveness of Jakinibs in chronic conditions such as rheumatoid arthritis, expand their therapeutic applicability. Advances in isoform-specific inhibitors, including filgotinib and upadacitinib, promise greater specificity with fewer off-target effects. Combination therapies, involving Jakinibs and monoclonal antibodies, aiming to enhance therapeutic specificity and efficacy also give great hope. Overall, this review bridges the gap between basic science and clinical application, elucidating the complex influence of the JAK-STAT pathway on human health and guiding future interventions.

SARS-CoV-2 mutations in MHC-I epitopes identified by deep viral sequencing evade CTL responses through decreased peptide-MHC-I binding.

Background: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) induced Coronavirus Disease 2019 (COVID-19) has posed a global threat to public health. The immune system is crucial in defending and eliminating the virus and infected cells. However, immune dysregulation may result in the rapid progression of COVID-19. Here, we evaluated the subsets, phenotypic and functional characteristics of natural killer (NK) and T cells in patients with COVID-19 and their associations with disease severity. Methods: Demographic and clinical data of COVID-19 patients enrolled in Wuhan Union Hospital from February 25 to February 27, 2020, were collected and analyzed. The phenotypic and functional characteristics of NK cells and T cells subsets in circulating blood and serum levels of cytokines were analyzed via flow cytometry. Then the LASSO logistic regression model was employed to predict risk factors for the severity of COVID-19. Results: The counts and percentages of NK cells, CD4+ T cells, CD8+ T cells and NKT cells were significantly reduced in patients with severe symptoms. The cytotoxic CD3-CD56dimCD16+ cell population significantly decreased, while the CD3-CD56dimCD16- part significantly increased in severe COVID-19 patients. More importantly, elevated expression of regulatory molecules, such as CD244 and programmed death-1 (PD-1), on NK cells and T cells, as well as decreased serum cytotoxic effector molecules including perforin and granzyme A, were detected in patients with COVID-19. The serum IL-6, IL-10 and TNF-α were significantly increased in severe patients. Moreover, the CD3-CD56dimCD16- cells were screened out as an influential factor in severe cases by LASSO logistic regression. Conclusions: The functional exhaustion and other subset alteration of NK and T cells may contribute to the progression and improve the prognosis of COVID-19. Surveillance of lymphocyte subsets may in the future enable early screening for signs of critical illness and understanding the pathogenesis of this disease.

ubiquitination assay. In addition, Tim-AIII inhibited cell proliferation, induced cell death, led to ROS and iron accumulation, MDA production, GSH depletion, as well as GPX4 ubiquitination and degradation, were markedly abrogated when HSP90 was knockdown by HSP90-shRNA transfection. Importantly, Tim-AIII also showed a strong capacity of preventing tumor growth by promoting ferroptosis in a subcutaneous xenograft tumor model, whether C57BL/6J or BALB/c-nu/nu nude mice. Together, HSP90 was identified as a new target of Tim-AIII. Tim-AIII, by binding and forming a complex with HSP90, further targeted and degraded GPX4, ultimately induced ferroptosis in NSCLC. These findings provided solid evidence that Tim-AIII can serve as a potential candidate for NSCLC treatment.

Alterations in the composition of the commensal microbiota (dysbiosis) seem to be a pathogenic component of functional gastrointestinal disorders, mainly irritable bowel syndrome (IBS), and might participate in the secretomotor and sensory alterations observed in these patients.We determined if a state antibiotics-induced intestinal dysbiosis is able to modify colonic pain-related and motor responses and characterized the neuro-immune mechanisms implicated in mice. A 2-week antibiotics treatment induced a colonic dysbiosis (increments in Bacteroides spp, Clostridium coccoides and Lactobacillus spp and reduction in Bifidobacterium spp). Bacterial adherence was not affected. Dysbiosis was associated with increased levels of secretory-IgA, up-regulation of the antimicrobial lectin RegIIIγ, and toll-like receptors (TLR) 4 and 7 and down-regulation of the antimicrobial-peptide Resistin-Like Molecule-β and TLR5. Dysbiotic mice showed less goblet cells, without changes in the thickness of the mucus layer. Neither macroscopical nor microscopical signs of inflammation were observed. In dysbiotic mice, expression of the cannabinoid receptor 2 was up-regulated, while the cannabinoid 1 and the mu-opioid receptors were down-regulated. In antibiotic-treated mice, visceral pain-related responses elicited by intraperitoneal acetic acid or intracolonic capsaicin were significantly attenuated. Colonic contractility was enhanced during dysbiosis. Intestinal dysbiosis induce changes in the innate intestinal immune system and modulate the expression of pain-related sensory systems, an effect associated with a reduction in visceral pain-related responses. Commensal microbiota modulates gut neuro-immune sensory systems, leading to functional changes, at least as it relates to viscerosensitivity. Similar mechanisms might explain the beneficial effects of antibiotics or certain probiotics in the treatment of IBS.

Precision allergy molecular diagnostic applications (PAMD@) is increasingly entering routine care. Currently, more than 130 allergenic molecules from more than 50 allergy sources are commercially available for in vitro specific immunoglobulin E (sIgE) testing. Since the last publication of this consensus document, a great deal of new information has become available regarding this topic, with over 100 publications in the last year alone. It thus seems quite reasonable to publish an update. It is imperative that clinicians and immunologists specifically trained in allergology keep abreast of the new and rapidly evolving evidence available for PAMD@. PAMD@ may initially appear complex to interpret; however, with increasing experience, the information gained provides relevant information for the allergist. This is especially true for food allergy, Hymenoptera allergy, and for the selection of allergen immunotherapy. Nevertheless, all sIgE tests, including PAMD@, should be evaluated within the framework of a patient's clinical history, because allergen sensitization does not necessarily imply clinical relevant allergies.

activity of PEGylated liposomes is highly dependent on PEG motif abundance. The process of "stealth" coverage formation is a very important parameter for efficient transfection assays and further fate determination of the PEG layer after tissue penetration. In this review, we discuss the latest methods of PEGylated liposome preparation.

Today, in vivo allergy diagnosis and allergen-specific immunotherapy (AIT) are still based on allergen extracts obtained from natural allergen sources. Several studies analyzing the composition of natural allergen extracts have shown severe problems regarding their quality such as the presence of undefined nonallergenic materials, contaminants as well as high variabilities regarding contents and biological activity of individual allergens. Despite the increasing availability of sophisticated analytical technologies, these problems cannot be overcome because they are inherent to allergen sources and methods of extract production. For in vitro allergy diagnosis problems related to natural allergen extracts have been largely overcome by the implementation of recombinant allergen molecules that are defined regarding purity and biological activity. However, no such advances have been made for allergen preparations to be used in vivo for diagnosis and therapy. No clinical studies have been performed for allergen extracts available for in vivo allergy diagnosis that document safety, sensitivity, and specificity of the products. Only for very few therapeutic allergen extracts state-of-the-art clinical studies have been performed that provide evidence for safety and efficacy. In this article, we discuss problems related to the inconsistent quality of products based on natural allergen extracts and share our observations that most of the products available for in vivo diagnosis and AIT do not meet the international standards for medicinal products. We argue that a replacement of natural allergen extracts by defined recombinantly produced allergen molecules and/or mixtures thereof may be the only way to guarantee the supply of clinicians with state-of-the-art medicinal products for in vivo diagnosis and treatment of allergic patients in the future.

Allergen-specific immunotherapy (AIT) is an allergen-specific form of treatment for patients suffering from immunoglobulin E (IgE)-associated allergy; the most common and important immunologically mediated hypersensitivity disease. AIT is based on the administration of the disease-causing allergen with the goal to induce a protective immunity consisting of allergen-specific blocking IgG antibodies and alterations of the cellular immune response so that the patient can tolerate allergen contact. Major advantages of AIT over all other existing treatments for allergy are that AIT induces a long-lasting protection and prevents the progression of disease to severe manifestations. AIT is cost effective because it uses the patient´s own immune system for protection and potentially can be used as a preventive treatment. However, broad application of AIT is limited by mainly technical issues such as the quality of allergen preparations and the risk of inducing side effects which results in extremely cumbersome treatment schedules reducing patient´s compliance. In this article we review progress in AIT made from its beginning and provide an overview of the state of the art, the needs for further development, and possible technical solutions available through molecular allergology. Finally, we consider visions for AIT development towards prophylactic application.

BACKGROUND: In all societies, the burden and cost of allergic and chronic respiratory diseases are increasing rapidly. Most economies are struggling to deliver modern health care effectively. There is a need to support the transformation of the health care system into integrated care with organizational health literacy. MAIN BODY: As an example for chronic disease care, MASK (Mobile Airways Sentinel NetworK), a new project of the ARIA (Allergic Rhinitis and its Impact on Asthma) initiative, and POLLAR (Impact of Air POLLution on Asthma and Rhinitis, EIT Health), in collaboration with professional and patient organizations in the field of allergy and airway diseases, are proposing real-life ICPs centred around the patient with rhinitis, and using mHealth to monitor environmental exposure. Three aspects of care pathways are being developed: (i) Patient participation, health literacy and self-care through technology-assisted "patient activation", (ii) Implementation of care pathways by pharmacists and (iii) Next-generation guidelines assessing the recommendations of GRADE guidelines in rhinitis and asthma using real-world evidence (RWE) obtained through mobile technology. The EU and global political agendas are of great importance in supporting the digital transformation of health and care, and MASK has been recognized by DG Santé as a Good Practice in the field of digitally-enabled, integrated, person-centred care. CONCLUSION: In 20 years, ARIA has considerably evolved from the first multimorbidity guideline in respiratory diseases to the digital transformation of health and care with a strong political involvement.

BACKGROUND: First vaccines for prevention of Coronavirus disease 2019 (COVID-19) are becoming available but there is a huge and unmet need for specific forms of treatment. In this study we aimed to evaluate the anti-SARS-CoV-2 effect of siRNA both in vitro and in vivo. METHODS: To identify the most effective molecule out of a panel of 15 in silico designed siRNAs, an in vitro screening system based on vectors expressing SARS-CoV-2 genes fused with the firefly luciferase reporter gene and SARS-CoV-2-infected cells was used. The most potent siRNA, siR-7, was modified by Locked nucleic acids (LNAs) to obtain siR-7-EM with increased stability and was formulated with the peptide dendrimer KK-46 for enhancing cellular uptake to allow topical application by inhalation of the final formulation - siR-7-EM/KK-46. Using the Syrian Hamster model for SARS-CoV-2 infection the antiviral capacity of siR-7-EM/KK-46 complex was evaluated. RESULTS: We identified the siRNA, siR-7, targeting SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) as the most efficient siRNA inhibiting viral replication in vitro. Moreover, we showed that LNA-modification and complexation with the designed peptide dendrimer enhanced the antiviral capacity of siR-7 in vitro. We demonstrated significant reduction of virus titer and lung inflammation in animals exposed to inhalation of siR-7-EM/KK-46 in vivo. CONCLUSIONS: Thus, we developed a therapeutic strategy for COVID-19 based on inhalation of a modified siRNA-peptide dendrimer formulation. The developed medication is intended for inhalation treatment of COVID-19 patients.

Pro-inflammatory stress is inherent in any cells that are subject to damage or threat of damage. It is defined by a number of universal components, including oxidative stress, cellular response to DNA damage, unfolded protein response to mitochondrial and endoplasmic reticulum stress, changes in autophagy, inflammasome formation, non-coding RNA response, formation of an inducible network of signaling pathways, and epigenetic changes. The presence of an inducible receptor and secretory phenotype in many cells is the cause of tissue pro-inflammatory stress. The key phenomenon determining the occurrence of a classical inflammatory focus is the microvascular inflammatory response (exudation, leukocyte migration to the alteration zone). This same reaction at the systemic level leads to the development of life-critical systemic inflammation. From this standpoint, we can characterize the common mechanisms of pathologies that differ in their clinical appearance. The division of inflammation into alternative variants has deep evolutionary roots. Evolutionary aspects of inflammation are also described in the review. The aim of the review is to provide theoretical arguments for the need for an up-to-date theory of the relationship between key human pathological processes based on the integrative role of the molecular mechanisms of cellular and tissue pro-inflammatory stress.

BACKGROUND: BM32 is a grass pollen allergy vaccine based on recombinant fusion proteins consisting of nonallergenic peptides from the IgE-binding sites of the 4 major grass pollen allergens and the hepatitis B preS protein. OBJECTIVE: We sought to study the safety and clinical efficacy of immunotherapy (allergen immunotherapy) with BM32 in patients with grass pollen-induced rhinitis and controlled asthma. METHODS: A double-blind, placebo-controlled, multicenter allergen immunotherapy field study was conducted for 2 grass pollen seasons. After a baseline season, subjects (n = 181) were randomized and received 3 preseasonal injections of either placebo (n = 58) or a low dose (80 μg, n = 60) or high dose (160 μg, n = 63) of BM32 in year 1, respectively, followed by a booster injection in autumn. In the second year, all actively treated subjects received 3 preseasonal injections of the BM32 low dose, and placebo-treated subjects continued with placebo. Clinical efficacy was assessed by using combined symptom medication scores, visual analog scales, Rhinoconjunctivitis Quality of Life Questionnaires, and asthma symptom scores. Adverse events were graded according to the European Academy of Allergy and Clinical Immunology. Allergen-specific antibodies were determined by using ELISA, ImmunoCAP, and ImmunoCAP ISAC. RESULTS: Although statistical significance regarding the primary end point was not reached, BM32-treated subjects, when compared with placebo-treated subjects, showed an improvement regarding symptom medication, visual analog scale, Rhinoconjunctivitis Quality of Life Questionnaire, and asthma symptom scores in both treatment years. This was accompanied by an induction of allergen-specific IgG without induction of allergen-specific IgE and a reduction in the seasonally induced increase in allergen-specific IgE levels in year 2. In the first year, more grade 2 reactions were observed in the active (n = 6) versus placebo (n = 1) groups, whereas there was almost no difference in the second year. CONCLUSIONS: Injections of BM32 induced allergen-specific IgG, improved clinical symptoms of seasonal grass pollen allergy, and were well tolerated.

Tetraspanins interact with a wide variety of transmembrane and intracellular proteins called molecular partners, and modulate their function. In this article, we describe a new partner of tetraspanin web, membrane metalloprotease CD 10, which is selectively associated with CD 9. By constructing chimeras between tetraspanins CD 9 and CD 82 (the latter does not interact with CD 10) or by using site‐directed mutagenesis, we determined that a portion of the large extracellular loop from the CCG motif to transmembrane domain 4, as well as the C ‐terminal tail of CD 9, are involved in the interaction with CD 10. The stable expression of wild‐type CD 9 in K 562 CD 10‐positive cells enhanced the level of CD 10 released with exosomes five‐fold. In contrast, the expression of chimeric CD 9, which contained the cytoplasmic C ‐terminal domain from CD 82, had little effect on CD 10 release. Short hairpin RNA knockdown of CD 9 expression in Nalm‐6 pre‐B cells resulted in a two‐fold reduction in the amount of endogenous CD 10 released with microvesicles. The peptidase activity of CD 10 measured either on cells or on exosomes correlated with the level of CD 10 expression, and was not significantly modulated by CD 9 expression as such. Our data suggest that the interaction of CD 10 with tetraspanin CD 9 can play an important role in the redistribution of peptidase activity from the cell surface to outer microenvironments. In bone marrow, where CD 10 presumably contributes to the maturation of pre‐ B cells and migration of B cells to the blood circulation, release of CD 10 peptidase activity with exosomes may effectively regulate extracellular matrix microenvironments. Structured digital abstract CD9 physically interacts with CD179b , Tetraspanin-9 , CD63 , CD81 , HLA class II , Uncharacterized protein C14orf166B , Tetraspanin-14 , HLA class I , Galactokinase , Protein disulfide-isomerase A6 , CD98 , CD19 , CD316 , CD146 , CD92 , Annexin A6 , CD156c , CD71 , CD10 , CD29 , CD315 , DAAM1 and CD49f by anti bait coimmunoprecipitation ( View interaction ) CD10 physically interacts with CD9 by anti bait coimmunoprecipitation (View Interaction: 1 , 2 , 3 , 4 ) CD10 physically interacts with CD179b , Protein FAM207A , CD9 , CD81 , Uncharacterized protein C22orf13 , HLA class II , <jats:ext-link xmlns:xlink="http://www.w3.org