Preparation and Neutralization Efficacy of Novel Jellyfish Antivenoms against Cyanea nozakii Toxins.

Jellyfish stings are a common issue globally, particularly in coastal areas in the summer. Victims can suffer pain, itching, swelling, shock, and even death. Usually, hot water, vinegar, or alumen is used to treat the normal symptoms of a jellyfish sting. However, a specific antivenom may be an effective treatment to deal with severe jellyfish stings. Cyanea nozakii often reach a diameter of 60 cm and are responsible for hundreds of thousands of stings per year in coastal Chinese waters. However, there has been no specific C. nozakii antivenom until now, and so the development of this antivenom is very important. Herein, we collected C. nozakii antisera from tentacle extract venom immunized rabbits and purified the immunoglobulin (IgG) fraction antivenom (AntiCnTXs). Subsequently, two complete procedures to produce a refined F(ab')2 type of antivenom (F(ab')2-AntiCnTXs) and Fab type of antivenom (Fab-AntiCnTXs) by multiple optimizations and purification were established. The neutralization efficacy of these three types of antivenoms was compared and analyzed in vitro and in vivo, and the results showed that all types of antibodies displayed some neutralization effect on the lethality of C. nozakii venom toxins, with the neutralization efficacy as follows: F(ab')2-AntiCnTXs ≥ AntiCnTXs > Fab-AntiCnTXs. This study describes the preparation of novel C. nozakii jellyfish antivenom preparations towards the goal of developing a new, effective treatment for jellyfish stings.

Mutation in KARS: A novel mechanism for severe anaphylaxis.

BACKGROUND: Anaphylaxis is a severe allergic reaction that can be lethal if not treated adequately. The underlying molecular mechanisms responsible for the severity are mostly unknown. OBJECTIVE: This study is based on a clinical case of a patient with extremely severe anaphylaxis to paper wasp venom. This patient has a mutation in the KARS gene, which encodes lysyl-tRNA synthetase (LysRS), a moonlight protein with a canonical function in protein synthesis and a noncanonical function in antigen dependent-FcεRI activation in mast cells. In this study, the objective was to characterize the mutation at the molecular level. METHODS: Analysis of the KARS mutation was carried out using biochemical and functional approaches, cell transfection, Western blot, confocal microscopy, cell degranulation, prostaglandin D2 secretion, and proteases gene transcription. Structural analysis using molecular dynamics simulations and well-tempered metadynamics was also performed. RESULTS: The mutation found, P542R (proline was replaced by arginine at aminoacid 542), affects the location of the protein as we show in biochemical and structural analyses. The mutation resembles active LysRS and causes a constitutive activation of the microphthalmia transcription factor, which is involved in critical mast cell functions such as synthesis of mediators and granule biogenesis. Moreover, the structural analysis provides insights into how LysRS works in mast cell activation. CONCLUSIONS: A link between the aberrant LysRS-P542R function and mast cell-exacerbated activation with increase in proinflammatory mediator release after antigen-IgE-dependent response could be established.

Precision Medicine in Hymenoptera Venom Allergy: Diagnostics, Biomarkers, and Therapy of Different Endotypes and Phenotypes.

Allergic reactions to stings of Hymenoptera species may be severe and are potentially fatal deviations of the immunological response observed in healthy individuals. However, venom-specific immunotherapy (VIT) is an immunomodulatory approach able to cure venom allergy in the majority of affected patients. An appropriate therapeutic intervention and the efficacy of VIT not only depend on a conclusive diagnosis, but might also be influenced by the patient-specific manifestation of the disease. As with other diseases, it should be borne in mind that there are different endotypes and phenotypes of venom allergy, each of which require a patient-tailored disease management and treatment scheme. Reviewed here are different endotypes of sting reactions such as IgE-mediated allergy, asymptomatic sensitization or a simultaneous presence of venom allergy and mast cell disorders including particular considerations for diagnosis and therapy. Additionally, phenotypical manifestations of venom allergy, as e.g. differences in age of onset and disease severity, multiple sensitization or patients unsusceptible to therapy, are described. Moreover, biomarkers and diagnostic strategies that might reflect the immunological status of the patient and their value for therapeutic guidance are discussed. Taken together, the increasing knowledge of different disease manifestations in venom hypersensitivity and the growing availability of diagnostic tools open new options for the classification of venom allergy and, hence, for personalized medical approaches and precision medicine in Hymenoptera venom allergy.

Jellyfish anaphylaxis: A wide spectrum of sensitization routes.

Background: Recent studies demonstrated that, in the past few years, the number of jellyfish species is increasing worldwide; this increase can be explained by environmental and climatic reasons. Contacts with jellyfish can cause acute and chronic effects, including allergic reactions. Although anaphylaxis caused by jellyfish is a rare event, repetitive stings during bathing as well as marine sports and job activities represent important risk factors that can increase the probability of sensitization. Recently, it was also pointed out the possibility of anaphylaxis caused by jellyfish ingestion. In these cases, the sensitization could also be related to previous stings. In cases in which there is no history of jellyfish contact or ingestion, it has been hypothesized that there is a sensitization to an unknown cross-reactive antigen. Objective: The purpose of this work was to collect and review published studies and cases of anaphylaxis associated with jellyfish. Methods: We performed a medical literature data base search, which included English language articles published until September 2019, by using the key words "jellyfish" associated with "anaphylaxis" or "anaphylactic shock." Results: The results of our research showed that dangerous reactions can be caused both by contact and ingestion. Moreover, the latest changes in food habits, life style, and globalization could lead to a more frequent exposure to jellyfish both by contact and ingestion, and, consequently, to a higher probability of sensitization. Conclusion: Prospective studies and well-structured research are needed to better understand all the potential immunologic elements of jellyfish, to clarify its role in sensitization, and to avoid possible dangerous allergic reactions caused by cross-reactivity.

A Lutzomyia longipalpis Salivary Protein Induces Cross-Reactive Antibodies to Pemphigus Autoantigen Desmoglein 1.

Fogo selvagem (FS) is a blistering skin disease caused by pathogenic IgG4 autoantibodies to desmoglein 1 (DSG1). Preclinical FS and leishmaniasis are endemic to certain regions of Brazil and exhibit nonpathogenic anti-DSG1 antibodies. Recurring bites from Lutzomyia longipalpis, the sand fly vector of leishmaniasis, immunize individuals with L. longipalpis salivary antigens LJM17 and LJM11. We measured the antibody responses to LJM17, LJM11, and DSG1 in normal settlers and patients with FS from an endemic focus of FS and nonendemic control populations. We also immunized mice with these antigens and assessed the IgG response. Healthy individuals and patients with FS from endemic areas had significantly higher values of IgG4 anti-LJM17 antibodies than nonendemic controls (P < 0.001 for both). The levels of IgG anti-DSG1 and IgG4 anti-LJM17 and anti-LJM11 antibodies correlated positively in normal settlers and patients with FS. Mice immunized with recombinant LJM17 produced IgG1 antibodies (human IgG4 homolog) that strongly cross-reacted with recombinant DSG1; these IgG1 antibodies were inhibited by LJM17, LJM11, and DSG1 in a dose-dependent manner. However, they did not bind human or mouse epidermis by indirect immunofluorescence. Lastly, we identified short-sequence homologies of surface-exposed residues within the human DSG1 ectodomain and LJM17. Inoculation by LJM17 from L. longipalpis-elicited DSG1-cross-reactive IgG4 antibodies may lead to FS in genetically predisposed individuals.

Paratrygon aiereba irradiated anti-mucus serum reduce edematogenic activity induced in experimental model.

Accidents by freshwater stingrays are common in northern Brazil, there is no specific therapy for high morbidity and local tissue destruction. The irradiation of venoms and toxins by ionizing radiation has been used to produce appropriate immunogens for the production of antisera. We planned to study the efficacy of stinging mucus irradiation in the production of antisera, with serum neutralization assays of edematogenic activity and quantification of cytokines performed in animal models of immunization with native and irradiated mucus of Paratrygon aiereba, a large freshwater stingray. Antiserum potency and its cross-reactivity with mucus from other freshwater stingrays were detected by ELISA. Immunization models demonstrated the ability to stimulate a strong humoral response with elevated levels of serum IgG detectable by ELISA, and both native and irradiated mucus were immunogenic and capable of recognizing mucus proteins from other freshwater neotropical stingrays. Mucus P. aiereba causes cellular and humoral adaptive immune responses in cells of immunized mice producing antibodies and cytokines such as TNF-α, IL-6 and IL-17. Rabbit antisera immunized with mucus from P. aiereba irradiated at 2 kGy showed a significant reduction of mucus-induced edematogenic activity in mice. Our data suggest that the use of antisera against freshwater stingray mucus show the possibility of specific therapy for these accidents.

Emergency Treatment of Anaphylaxis in Japanese Beekeepers.

Objectives: Honeybee stings often lead to anaphylactic shock. We surveyed Japanese beekeepers to examine whether adrenaline auto-injectors are properly used after honeybee stings.Methods: We contacted representatives of the Japanese Beekeeping Association in all 47 prefectures for assistance distributing allergist-developed questionnaires. Representatives in 33 prefectures distributed questionnaires to their members and we received valid responses from 826 beekeepers.Results: Adrenaline auto-injectors had been prescribed to only 46 of the 826 participants (5.6%) to prevent systemic reaction (SR) to honeybee stings. Of the 33 beekeepers who experienced a honeybee sting after adrenaline auto-injector prescription, 16 (48.5%) developed SRs; 9 of these 16 (56.3%) were treated with an adrenaline auto-injector.Conclusions: Japanese beekeeping organizations should consider encouraging medical institutions to prescribe adrenaline auto-injectors. Furthermore, physicians and other health care workers should better educate beekeepers and others who have been prescribed an adrenaline auto-injector in order to improve compliance and raise awareness of the risk posed by SRs.

Characterization of IgE-binding proteins in the salivary glands of Simulium nigrogilvum (Diptera: Simuliidae).

Simulium dermatitis is an IgE-mediated skin reaction in animals and humans caused by the bites of black flies. Although Simulium nigrogilvum has been incriminated as the main human-biting black fly species in Thailand, information on its salivary allergens is lacking. Salivary gland extract of S. nigrogilvum females was subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis, and the separated components were applied onto nitrocellulose membranes for immunoblotting, which was performed by probing the protein blots with sera from 17 individuals who were allergic to the bites of S. nigrogilvum. IgE-reactive protein bands were characterized further by liquid chromatography-mass spectrometry (LC-MS/MS) analysis. Nine protein bands (79, 42, 32, 25, 24, 22, 15, 13, and 11 kDa) were recognized in the serum of the subjects. Four of the nine protein bands (32, 24, 15, and 11 kDa) showed IgE reactivity in all (100%) of the tested sera, and they were identified as salivary secreted antigen 5-related protein, salivary serine protease, erythema protein, and hypothetical secreted protein, respectively. Three other proteins, salivary serine protease (25 kDa), salivary D7 secreted protein (22 kDa), and hypothetical protein (13 kDa), reacted with > 50% of the sera. The relevance of the identified protein bands as allergens needs to be confirmed by using pure recombinant proteins, either in the in vivo skin prick test or in vitro detection of the specific IgE in the serum samples of allergic subjects. This will be useful for the rational design of component-resolved diagnosis and allergen immunotherapy for the allergy mediated by the bites of black flies.

In vitro exposure to Hymenoptera venom and constituents activates discrete ionotropic pathways in mast cells.

Calcium entry is central to the functional processes in mast cells and basophils that contribute to the induction and maintenance of inflammatory responses. Mast cells and basophils express an array of calcium channels, which mediate responses to diverse stimuli triggered by small bioactive molecules, physicochemical stimuli and immunological inputs including antigens and direct immune cell interactions. These cells are also highly responsive to certain venoms (such as Hymenoptera envenomations), which cause histamine secretion, cytokine release and an array of pro-inflammatory functional responses. There are gaps in our understanding of the coupling of venom exposure to specific signaling pathways such as activation of calcium channels. In the present study, we performed a current survey of a model mast cell line selected for its pleiotropic responsiveness to multiple pro-inflammatory inputs. As a heterogenous stimulus, Hymenoptera venom activates multiple classes of conductance at the population level but tend to lead to the measurement of only one type of conductance per cell, despite the cell co-expressing multiple channel types. The data show that ICRAC, IARC, and TRPV-like currents are present in the model mast cell populations and respond to venom exposure. We further assessed individual venom components, specifically secretagogues and arachidonic acid, and identified the conductances associated with these stimuli in mast cells. Single-cell calcium assays and immunofluorescence analysis show that there is heterogeneity of channel expression across the cell population, but this heterogeneity does not explain the apparent selectivity for specific channels in response to exposure to venom as a composite stimulus.

Changing microRNA Expression during Three-Month Wasp Venom Immunotherapy.

MicroRNAs are small non-coding molecules playing a significant regulatory role in several allergic diseases. However their role in tolerance induction remains unclear. The aim of this study was to determine the expression of selected microRNAs during the first three months of wasp venom immunotherapy (VIT). 5 adult patients with a history of severe systemic reactions after stinging by wasps and confirmed sensitization were included. Venous blood samples were collected before VIT, 24 hours after completing its initial phase and after 3 months of the maintenance therapy. A control group was comprised of 5 healthy individuals with no history of allergy. In the blood samples expression of 96 microRNAs was determined with the use of microfluidic cards. In a statistical analysis the expression was compared between the study groups as well as between the pre- and post-VIT samples. Significant differences were found between the patients with wasp venom allergy and the healthy controls in the expression of miR-601 and miR-1201 upregulated in allergic patients at every time point (p = 0.04; p = 0.015, respectively). During VIT profile of microRNA was changing with lower expression of 6 microRNAs (including miR-182, miR-342, miR-375) and higher of 11 microRNAs (including let-7d, miR-34b, miR-143). To conclude, VIT has led to some changes in the expression of microRNA associated with Th2-type inflammation and tolerance induction.

The kiss of (cell) death: can venom-induced immune response contribute to dermal necrosis following arthropod envenomations?

Introduction: Snakes, insects, arachnids and myriapods have been linked to necrosis following envenomation. However, the pathways involved in arthropod venom-induced necrosis remain a highly controversial topic among toxinologists, clinicians and the public. On the one hand, clinicians report on alleged envenomations based on symptoms and the victims' information. On the other hand, toxinologists and zoologists argue that symptoms are incompatible with the known venom activity of target species. This review draws from the literature on arthropod envenomations, snakebite, and inflammatory processes to suggest that envenomation by a range of organisms might trigger an intense inflammatory cascade that ultimately lead to necrosis. If confirmed, these processes would have important implications for the treatment of venom-induced necrosis. Objectives: To describe two inflammatory pathways of regulated necrosis, tumour necrosis factor (necroptosis) and Neutrophil Extracellular Traps (NETosis); to discuss existing knowledge about snake venom and arachnid-induced necrosis demonstrating the involvement of tumour necrosis factor and neutrophils in the development of tissue necrosis following envenomation and to contribute to the understanding of venom-induced necrosis by arthropods and provide clinicians with an insight into little known inflammatory processes which may occur post envenomation. Methods: ISI Web of Science databases were searched using the terms "spider bite necrosis", "arthropod envenomation necrosis", "venom necrosis", "venom immune response", "loxoscelism", "arachnidism", "necroptosis venom", "necroptosis dermatitis", "tumour necrosis factor TNF venom", "scorpionism", "scolopendrism", "centipede necrosis", "NETosis venom", "NETosis necrosis". Searches produced 1737 non-duplicate citations of which 74 were considered relevant to this manuscript. Non-peer-reviewed sources or absence of voucher material identifying the organism were excluded. What is necrosis? Necrosis is the breakdown of cell membrane integrity followed by inflowing extracellular fluid, organelle swelling and the release of proteolytic enzymes into the cytosol. Necrosis was historically considered an unregulated process; however, recent studies demonstrate that necrosis can also be a programmed event resulting from a controlled immune response (necroptosis). Tumour necrosis factor and the necroptosis pathway: Tumour necrosis factor is a pro-inflammatory cytokine involved in regulating immune response, inflammation and cell death/survival. The pro-inflammatory cytokine TNF-α participates in the development of necrosis after envenomation by vipers. Treatment with TNF-α-antibodies may significantly reduce the manifestation of necrosis. Neutrophil Extracellular Traps and the NETosis pathway: The process by which neutrophils discharge a mesh of DNA strands in the extracellular matrix to entangle ("trap") pathogens, preventing them from disseminating. Neutrophil Extracellular Traps have been recently described as important in venom-induced necrosis. Trapped venom accumulates at the bite site, resulting in significant localized necrosis. Arthropod venom driving necrosis: Insects, myriapods and arachnids can induce necrosis following envenomation. So far, the processes involved have only been investigated in two arachnids: Loxosceles spp. (recluse spiders) and Hemiscorpius lepturus (scorpion). Loxosceles venom contains phospholipases D which hydrolyse sphingomyelin, resulting in lysis of muscle fibers. Subsequently liberated ceramides act as intermediaries that regulate TNF-α and recruit neutrophils. Experiments show that immune-deficient mice injected with Loxosceles venom experience less venom-induced inflammatory response and survive longer than control mice. Necrosis following Hemiscorpius lepturus stings correlates with elevated concentrations of TNF-α. These observations suggest that necrosis may be indirectly triggered or worsened by pathways of regulated necrosis in addition to necrotic venom compounds. Conclusions: Envenomation often induce an intense inflammatory cascade, which under certain circumstances may produce necrotic lesions independently from direct venom activity. This could explain the inconsistent and circumstantial occurrence of necrosis following envenomation by a range of organisms. Future research should focus on identifying pathways to regulated necrosis following envenomation and determining more efficient ways to manage inflammation. We suggest that clinicians should consider the victim's immune response as an integral part of the envenomation syndrome.

AGEP from a dog bite treated with Ibuprofen.

BACKGROUND: Acute Generalized Exanthematous Pustulosis (AGEP) is a rare dermatologic reaction characterized by an erythematous rash with pustular erosions, fever and leukocytosis. Although most often secondary to antibiotic use, AGEP has also been associated with many drugs. A thorough literature search showed only four previously documented cases of ibuprofen-associated AGEP, and one case of dog bite-associated AGEP. CASE REPORT: We present the case of a 46 year old Caucasian female who developed AGEP after self-treating with ibuprofen for a dog bite. CONCLUSION: In the clinical setting this rash is often dramatic and illuminating the causative agent can be a diagnostic challenge. Our case represents a rare cause of AGEP and an important finding for current practitioners.

Eosinophil-Related Disease and the Skin.

Eosinophils are bone marrow-derived cells that infiltrate skin and mucous membrane in a broad spectrum of primary and reactive inflammatory diseases and malignancies. The eosinophil has potent proinflammatory activities, particularly, through the effects of its toxic granule proteins. In addition, eosinophils have prothrombotic and profibrotic activities. Eosinophil participation in the pathogenesis of certain diseases without identifiable intact eosinophil infiltration may not be recognized because eosinophil degranulation is poorly visualized on hematoxylin-and-eosin-stained histopathology sections. Eosinophil-related pathophysiology can involve virtually every component of skin. Commonly recognized dermatoses associated with eosinophils are arthropod bite and sting reactions and drug eruptions, "bugs and drugs." Skin involvement is common in eosinophil-related systemic diseases including the hypereosinophilic syndromes. Eosinophil-related pathophysiology may play a key role in numerous disorders that, therefore, may benefit from therapies targeted to reduce or eliminate eosinophils.

Surfing as a risk factor for sensitization to poly(γ-glutamic acid) in fermented soybeans, natto, allergy.

BACKGROUND: Poly(γ-glutamic acid) (PGA) is an allergen in natto, fermented soybeans, which causes late-onset anaphylaxis. We hypothesized that jellyfish stings sensitize adults to PGA because a surfer had allergies to both natto and jellyfish, whose sting contains PGA. The aim of the study was to identify behavioral factors, such as marine sports, associated with PGA sensitization. METHODS: Outpatients diagnosed with food allergies based on relevant clinical history, positive skin test and/or food challenge test answered a questionnaire during a regular visit in 2016. RESULTS: Questionnaire data from 140 outpatients were analyzed. These patients were divided into two groups: natto allergy group (13 patients, M:F = 10:3, mean age 40.6 years) and non-natto allergy group (127 patients, M:F = 46:81, mean age 44.5 years). All patients with natto allergy had positive results in skin prick test and basophil activation test with PGA. Of these, 92.3% had a marine sport hobby, especially surfing (84.6%). PGA sensitization was independently associated with marine sports (odds ratio, 278.0, 95 percent confidence interval, 36.9-6315.9, p < 0.001) adjusted for male sex and sea bathing, but not with male sex or sea bathing. In addition, although there was no significant difference in the experience of marine sports between natto and non-natto allergy groups, the natto allergy group participated significantly more frequently in marine sports than the non-natto allergy group (p < 0.001). There was no significant difference between natto consumption amount and PGA sensitization. CONCLUSIONS: Surfing is a risk factor for PGA sensitization in those with allergy to natto.

Skin Test Reactivity to Hymenoptera Venom after Venom Immunotherapy Correlates Inversely with the IgG/IgE Ratio.

BACKGROUND: Skin test reactivity to hymenoptera venom and venom-specific IgE are important for diagnosing venom allergy and deciding on the appropriate allergen for venom immunotherapy (VIT). Longitudinal data on skin test reactivity during VIT and their correlation with venom-specific immunoglobulin (Ig)E and IgG are scarce. METHODS: We retrospectively analyzed shifts in skin test reactivity and serum levels of venom-specific IgE and IgG in patients allergic to hymenoptera venom before the initiation of VIT with ultrarush therapy and after ≥3 years of VIT. RESULTS: Fifty-four patients received ultrarush desensitization and subsequent VIT with wasp venom, 26 with honeybee venom, and 8 with both wasp and honeybee venom. Hymenoptera-specific skin test reactivity decreased during VIT in most patients, and became negative in 8% of the wasp-allergic patients and in 25% of the honeybee-allergic patients. Serum levels of venom-specific IgE positively correlated to skin test reactivity before VIT, but did not change significantly during VIT. IgG serum levels and the IgG/IgE ratio increased during VIT in most patients. A high IgG/IgE ratio correlated with low skin test reactivity after ≥3 years of VIT. CONCLUSIONS: The correlation between a high venom-specific IgG/IgE ratio and low skin test reactivity after VIT may be interesting for future investigations that assess its role as a potential marker for VIT efficacy.

Laboratory Animal Bite Anaphylaxis: A National Survey: Part 1: Case Series and Review of the Literature.

OBJECTIVE: This study documents previously unreported cases of laboratory animal bite anaphylaxis in animal laboratory facilities in the United States. METHODS: An online survey was e-mailed to designated institutional officials at laboratory animal facilities identified by the National Institutes of Health Office of Laboratory Animal Welfare. RESULTS: One hundred ninety eight organizations responded and 15 organizations indicated that workers had experienced anaphylaxis following an animal bite. Case report forms were completed by nine of these institutions for 14 cases, 13 for rodent bites, and one involving a needlestick from a horse. In half of the cases involving rodents, there was no prior history of animal allergy. All workers had uncomplicated recoveries. Treatment, testing, and work restrictions varied across cases. CONCLUSIONS: While uncommon, anaphylaxis from laboratory animal bites occurs more frequently than suggested by the literature.