The prevalence of pollinosis caused by cedar pollen has increased by 10% these ten years of 26.5% in the investigation of 2008 in Japan. The pharmacotherapy is a main treatment tool for pollinosis, and the surgical treatment is not acknowledged to the treatment of pollinosis internationally. Moreover, allergen immunotherapy enters a special treatment method, and is an important therapeutic procedure. The allergen immunotherapy is unique for having possibility of curing allergen specific allergic diseases. However the side effect of allergen subcutaneous immunotherapy (SCIT), such as anaphylaxis is kept at a distance in a medical situation in Japan. Then, a sublingual immunotherapy (SLIT) that was safer than it, developed in Europe for pollinosis induced by grass or ragweed, but not in Japan. As a result, the effect of SLIT was proven in the cedar pollinosis in Japan as high level evidence. A whole body immunity induction is thought in the appearance of the effect, and, in addition, it is necessary to be going to be cleared the accurate mechanism of the effect in the future. Moreover, the development of a special SLIT and the import of an overseas product are needed in Japan.
Recent observations have suggested significant worldwide increase in the prevalence of allergic rhinitis and cedar pollinosis. In Japan, Japanese cedar (Cryptometria japonica) and Japanese cypress (Chamaecyparis obtusa) pollens are considered to be the major unique allergens and their extent of dispersal is quite large, travelling more than 100km and thus causing serious pollinosis. Cedar pollinosis is a typical type 1 allergic disease by an adaptive immune response that occurs through the induction of allergen-specific effector T cells from naïve T cells. We examined the number of Japanese cedar pollen specific memory Th cells in the peripheral blood of the patients and found that the cedar pollen specific IL-4-producing Th2 memory cells increased during the pollen season and decreased during the off-season. However, more than 60% of the cedar-specific memory Th2 cells survived up to 8 months after the pollen season. Natural killer T(NKT) cells represent a unique lymphocyte subpopulation and their activity is not restricted to MHC antigens. NKT cells play an important role in innate immunity, however, the participation in development of allergic rhinitis could not be clarified.
To initiate, monitor, and complete effective immunotherapy, biomarkers to predict and visualize the immune responses are needed. First, we need to identify the right candidate for immunotherapy. Secondly, the immune responses induced by immunotherapy should be monitored. For the first objective, analysis of polymorphisms of candidate genes may be helpful, but still be in development. Regarding biomarkers for immune responsese, there are numerous reports that evaluate immunotherapy-induced immune changes such as suppression of effector cells, deviation to Th1 cytokine production, and induction of regulatory T cells. No standardized methods, however, have been established. Among them, a functional assay of blocking IgG activity, the IgE-facilitated allergen binding assay, may be useful. We quantitated induced expression of an activation marker, CD203c, on basophils and found that the assay efficiently predicts sensitivity to particular allergen and severity of the allergen-induced symptoms. In patients who received rush immunotherapy for Japanese cedar pollinosis, reduction in CD203c expression after the therapy was observed, suggesting the utility of the test for monitoring immunotherapy.
Background: Japanese cedar pollen-induced allergic rhinitis in a guinea pig model clearly induced not only sneezing but also biphasic nasal blockage. To date, there have only been a few reports on models of murine allergic rhinitis which clearly show nasal blockage. Therefore, in order to try and develop such a model, we administered multiple dosages of intranasal pollen or purified antigen protein Cry j 1. Methods: B10.S mice were sensitized by intranasal instillations of either pollen extract or Cry j 1 twice a day for 7 days, which was adsorbed on Al(OH)3. Subsequently, once a week, the mice were given multiple intranasal instillation challenges of either the pollen suspension or Cry j 1 and the frequency of sneezing was observed after respective challenges were made. Specific airway resistance (sRaw) was measured as an indicator for nasal blockage. Cry j 1-specific IgE levels were measured using an enzyme-linked immunosorbent assay. Results: The serum Cry j 1-specific IgE level showed clear elevation only in the group sensitized by Cry j 1 + Al(OH)3 and then challenged by Cry j 1. No elevations were seen in the groups sensitized by pollen extract + Al(OH)3 followed by a pollen suspension challenge. There was an immediate increase in sneezing after challenges in all of the sensitized-challenged groups. Nevertheless, no increases in sRaw in any of the groups were detected at any of the time points during the 8 hours following the challenges. Conclusions: Cry j 1 may be more effective than crude antigens for efficient sensitization/challenge in mice. No increase in sRaw occurred, even in mice that possessed high amounts of Cry j 1-specific IgE and that exhibited sneezing.
Background: Recently, some common foods in daily life, especially lactic acid bacteria, have been found to have anti-allergic effects. We previously isolated a halophilic lactic acid bacterium, Tetragenococcus halophilus Th221, from soy sauce moromi, a mixture of koji and salt solution, and showed that it possesses an immunomodulatory activity that promotes T helper type 1 immunity. Methods: To evaluate the anti-allergic effects of Th221, we performed a randomized, double-blind, placebo-controlled study in 45 subjects with perennial allergic rhinitis (PAR) treated by oral administration of Th221 (high dose, 60mg/day, 15 subjects; low dose, 20.4mg/day, 15 subjects) or a placebo (15 subjects) for 8 weeks. Results: There were no significant differences among the groups that ingested Th221 and the placebo group regarding the disease severities, total nasal symptom scores and total nasal sign scores examined by physicians. However, the disease severity examined by physicians significantly improved in the high-dose group at the end of the trial compared with the beginning (p < 0.05). The total score for nasal symptoms of subjects who received a high dose of Th221 also showed a significant improvement at the end of the trial compared with the beginning (p < 0.01). According to the subjects' diaries, significant improvements in sneezing and rhinorrhea were observed during some periods in the high-dose group. The change in serum total immunoglobulin E improved significantly at the end of the trial compared with the beginning in this group (p < 0.05). The safety of Th221 treatment was confirmed by laboratory tests and inspection of the general condition of each subject. Conclusions: Th221 can be expected to safely improve the symptoms of PAR.
Background: Asthma is the most common chronic diseases in school-aged children in Japan. It is important to consider health-related quality of life (QoL) among children with chronic diseases when treatment decisions are made. Methods: A school-based survey was conducted in randomly selected public schools in Tokyo by using a KINDL® questionnaire for evaluating QoL and the international study of asthma and allergy on childhood (ISAAC) questionnaire, which is designed for comparing the asthma prevalence in various countries, from May to June in 2005. We recruited approximately 10% of the total children 6-7-years-old and 13-14-years-old living in Tokyo for sampling. Results: Response rate of this questionnaire was 86% (22,645 children) in the 6-7-year-old group and 64% (12,879 children) in the 13-14-year-old group. Comparing asthmatics with non-asthmatics in the same age, QoL of children with asthma was significantly impaired. The severity of QoL of children with asthma was significantly impaired. QoL of children with exercise-induced asthma (EIA) were more significantly impaired than ones without EIA and showed lower scores in the categories of physical functioning, emotional and school activities than those without EIA. Of note, QoL was more impaired in the EIA-positive group among severe asthmatics, suggesting that QoL of children with even severe asthma could be improved when EIA is appropriately controlled. Conclusions: Existence of EIA among asthmatic children most strongly impairs their QoL. We should be more cautious about the management of EIA.
Background: For in vitro diagnosis of wheat allergy, specific IgE to wheat is known to be a poor predictive marker. Oral food challenge, the gold standard for the diagnosis, is accompanied by a risk of severe induced reactions. Reliable in vitro tests are needed to be developed for safe indication for oral challenge. Objective: We examined the utility of a basophil activation marker, CD203c, for the diagnosis of IgE-mediated wheat allergy. Methods: Fifty-eight children with suspected wheat allergy with positive CAP-FEIA to wheat were enrolled. On 70 occasions, the clinical distinction between patients with wheat allergy (WA) and patients tolerant to wheat (TW) was made by means of an oral food challenge test or recent history of immediate allergic reactions or tolerance after ingestion of wheat. Twelve replicate evaluations were performed in 9 patients over more than a 6-month interval. Thirty two patients on 43 occasions were diagnosed with WA and 27 were confirmed to be TW. One patient had both diagnoses 18 months apart. Peripheral blood was incubated with fractionated wheat extracts, purified native omega-5 gliadin (nOG5) and recombinant omega-5 gliadin (rOG5). Expression of CD203c on basophils was then analyzed by flow cytometry using a commercial kit. Results: All wheat proteins induced concentration-dependent enhancement of CD203c expression in WA, but did not in TW. The analysis of receiver operating characteristics (ROC) showed that nOG5-induced CD203chigh% values provided the best power for discriminating between WA and TW, with a sensitivity of 85.0% and specificity of 77.0% at the cut-off level of 14.4%. AUC for CD203c with nOG5 were significantly higher than that for conventional CAP-FEIA, 0.89 and 0.73, respectively (p < 0.01). Conclusions: Measurement of nOG-induced enhancement of CD203c on basophils is useful for the diagnosis of immediate wheat allergy in children.
Background: Allergic rhinitis is not a fatal disease, but its symptoms deteriorate the quality of life. High morbidity raises a concern about its impact on health care resources. Utility weights, which are required for cost-utility analysis by the level of severity, have not been established to date. This study aims to derive the weights based on a community survey with a time trade-off technique. Methods: Self-administered monthly time trade-off questionnaires were administered to representative samples in the community. Four levels of severity were defined by clinical stratification proposed in the "Practical Guideline for the Management of Allergic Rhinitis in Japan". Results: 146 responses (response rate: 51.0%) were collected. Utility weights by the four levels of severity were found to be 0.96, 0.94, 0.89 and 0.83, from mild to severest symptoms, respectively. These values were found to be statistically independent from the respondent's characteristics such as sex, age, existence of current nasal symptoms or history of allergic rhinitis. Conclusions: The authors consider that the elicited utility weights are reliable. The results of this study could facilitate economic evaluations regarding allergic rhinitis in various contexts, contributing to better management of the disease.
Background: Few cases of cashew nut (CN) allergy have been reported in Japan. We evaluated the clinical features of 4 cases with CN allergy and investigated the allergens involved. Methods: In order to investigate the cross-reactivity between CN and pistachios, we performed ImmunoCAP inhibition tests using sera of 4 cases with positive histories of CN allergy and positive results of specific IgE measurement (ImmunoCAP) and skin prick tests. Furthermore, we analyzed the molecular weights of allergens of CN and pistachios by IgE-immunoblotting. Results: Of the 4 cases (male : female = 1 : 3), there were 3 cases (patient #2-4) and 1 case (patient #1) of anaphylaxis and oral allergy syndrome, respectively. The initial symptom was an oropharyngeal symptom in 3 of the 4 cases, of which 2 cases developed anaphylaxis within 10 minutes after eating only a few pieces of CN. All 4 cases reacted positively to the skin prick test with CN, although 1 case of anaphylaxis tested negatively for CN by ImmunoCAP. Additionally, in 2 cases, IgE-binding to CN and pistachio were inhibited with both pistachios and CN, indicating cross-reactivity between CN and pistachios. IgE-immunoblotting of CN using sera from the 4 cases revealed 2 bands at molecular weights of approximately 33 kd and 42 kd, whereas that of pistachios showed a single band at 36 kd. However, IgE in all 4 sera did not bind to rAna o 2. Conclusions: In CN allergy, a small amount of CN could induce a severe anaphylactic reaction. Moreover, in cases of suspected CN allergy, reactions to not only CN but also pistachio, which could be cross-reactive to CN, should be examined.
Background: In Japan, the number of asthma deaths has been gradually decreasing. However, in the management of asthma, there are still some problems originating from patient-related factors and iatrogenic factors, both of which should be further analyzed. Methods: We investigated clinical and background characteristics of 164 patients with asthma who were admitted to our hospital with acute exacerbations, by reviewing their clinical records. Results: Fifty-two patients had received long-term management (LTM) based on the guidelines (the LTM group), while 112 had not (the non-LTM group). In patients whose asthma severity had been intermittent (step 1), the proportion of severe and near fatal exacerbations was significantly higher in the non-LTM group than in the LTM group. However, even in the LTM-group, 23% of mild persistent (step 2) and 38% of moderately and severely persistent (step 3 & 4) patients had severe or near fatal exacerbations. In these patients, the peak expiratory flow rate significantly improved after discharge, and poor adherence was also significantly higher in the non-LTM group than in the LTM group. A multivariate analysis revealed that the factors associated with poor adherence were: 1) no history of previous admission due to asthma exacerbation; 2) the patient was male; and 3) the patient was young (<60 years). Conclusions: In the LTM group, re-evaluation of the actual severity of asthma and prompt treatment corresponding to the severity of disease should still be encouraged. In the non-LTM group, establishing countermeasures against factors causing poor adherence would be the next step in ensuring strong adherence with LTM.
Background: In studies on allergies in mouse models, IgE production is an essential parameter to be evaluated. Here, we examine the effect of commercially available immunoreaction enhancer solutions and different blocking reagents in enzyme-linked immunosorbent assay (ELISA) for total or antigen-specific murine IgE in order to improve the assays. Methods: Sera from mice immunized with recombinant house dust mite major allergens, Der f 1 and Der p 1, were used for the assays. Total IgE was measured by sandwich ELISA using monoclonal antibodies against murine IgE. Antigen-specific IgE was assayed using allergen-coated plates. Sensitivity or signal intensity in ELISA was compared among conditions differing in the use of enhancer solutions, blocking reagents, or monoclonal antibodies, and incubation time. Results: Use of enhancer solutions improved the sensitivity of ELISA for total IgE by approximately 30-fold of that using a conventional buffer. A blocking reagent caused more unwanted enhancement of the background signal in blank wells in ELISA for total IgE compared with another blocking reagent, however, improved signal intensity in ELISA for antigen-specific ELISA without significant enhancement of the background signal. Optimal assay conditions were determined. Conclusions: Enhancer solutions are effective in improving ELISAs for total and antigen-specific murine IgE. Selection of blocking reagents was important to decrease unwanted enhancement of background signals and was effective in enhancing signals for positive samples. The ELISAs improved in this study are useful for the study of allergies in mouse models.
Background: Pollens from species of the Cupressaceae family are one of the most important causes of respiratory allergies worldwide. Many patients with pollinosis have specific IgE to both allergens from Japanese cedar and Japanese cypress pollen. We set out to identify T cell epitopes in Cha o 2, the second major allergen of Japanese cypress pollen. Methods: T cell lines (TCL) and T cell clones (TCC) specific to Cha o 2 were generated from allergic patients cross-reactive to Japanese cedar and Japanese cypress pollen. T cell epitopes in Cha o 2 were identified by responses of TCL stimulated with overlapping peptides. Abilities of IL-4/IFN-γ production by TCC were evaluated using enzyme immunoassay. Results: Using TCL, 11 dominant and subdominant T cell epitopes were identified in Cha o 2. The subsets of TCC were predominantly of T helper 2-type. A T cell epitope p141-160 in Cha o 2 and corresponding peptide in Cry j 2 showed high homology. Although TCC PC.205.159 responded to stimulation with p141-160 in Cha o 2, it did not respond with corresponding peptide in Cry j 2, therefore, the T cell epitope was unique to Cha o 2. Conclusions: Eleven T cell epitopes that were identified are unique to Cha o 2. Cha o 2 is a putative aeroallergen that can potentially sensitize human T cells. We concluded that generation of T cells specific to Cha o 2 in allergic patients acts as one of the causes of continuous allergic symptoms in April.
Background: Peroxisome proliferator-activated receptor γ(PPAR-γ) has been shown to play an important role in the control of inflammatory responses acting on macrophages, mast cells, T cells, and eosinophils. The present study was aimed at investigating the effects of PPAR-γ agonist on nasal symptoms and eosinophil accumulations in nasal mucosa by using a murine allergic rhinitis model. Furthermore, we examined the expression of PPAR-γ in the nasal mucosa in mice. Methods: BALB/c mice were sensitized and challenged intranasally with ovalbumin. Ciglitazone, a PPAR-γ agonist, was administered orally 6 hours before each nasal challenge. Results: Administration of PPAR-γ agonist significantly decreased the number of nasal rubs, nasal histamine responsiveness, serum IgE, IL-5 production from the spleen, and eosinophilic infiltration in the nasal mucosa. Furthermore, PPAR-γ was expressed in eosinophils and epithelial cells in the nasal mucosa by immunohistochemistry. Conclusions: PPAR-γ was expressed in eosinophils and epithelial cells in the nasal mucosa. Also, the oral administration of ciglitazone is effective in upper airway allergic inflammation in mice.
Background: Although Japanese cedar (Cryptomeria japonica) pollinosis has developed into a health problem, few methods eradicate indoor allergens completely. In a recent study, however, the effectiveness of inactivation with sodium hypochlorite (NaOCl) treatment was revealed. Therefore, the present study aimed to elucidate the ability of chlorine bleach (NaOCl) to reduce the immunogenicity of the major allergenic protein of Japanese cedar (Cry j 1). Methods: Sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, enzyme-linked immunosorbant assay, and skin testing were carried out in 7 individuals. Results: The allergenic protein was undetectable using sodium dodecyl sulfate polyacrylamide gel electrophoresis and silver staining at a sodium hypochlorite/allergenic protein molar ratio of 457. Western blotting with human sera showed the same dose-dependent efficacy. The immunogenicity of the purified protein and cedar pollen was also demonstrated on enzyme-linked immunosorbant assay to be reduced by sodium hypochlorite treatment in a dose- and time-dependent manner. Moreover, sodium hypochlorite-treatment inhibited the skin test response to the protein in all 7 individuals. Conclusions: Hypochlorous acid generated by electrolysis is an effective method for significantly reducing the immunogenicity of Cry j 1.
Background: Pollens from the Salsola spp. are an important source of respiratory allergy in tropical countries. Our aim was to characterize the IgE binding proteins of S. incanescens pollen extract and study its cross-reactivity with S. kali pollen allergens. Methods: Prick tests with S. kali and S. incanescens pollen extracts were performed on eight respiratory allergy patients from Mashhad, Northeast Iran. The antigenic profiles and IgE-binding patterns of S. kali and S. incanescens pollen extracts were compared by SDS-PAGE and Western blotting, using individual sera from the salsola pollen-sensitive patients. Cross-reactivity of proteins in the two weeds was assessed by IgE- immunoblotting inhibition. Results:S. kali and S. incanescens pollen extracts showed similar IgE-binding profiles in Western blotting. The IgE binding components of 39, 45, 66 and 85kDa were detected in both pollen extracts. Furthermore, inhibition of the immunoblots revealed extensive inhibition of IgE binding to proteins and a close relationship between these two weeds allergens. Conclusions:S. incanescens pollen is a potent allergen source with several IgE binding components that shows a close allergenic relationship with S. kali. Our results suggest that in S. incanescens-rich areas, S. kali pollen extracts could be used as a diagnostic reagent for allergic patients to S. incanescens pollen.
Background: Phosphodiesterase 4 selective inhibitor may prevent airway inflammation and remodeling. Objective: The aim of this study was to investigate the effects of KF19514, a phosphodiesterase 4 and 1 dual inhibitor, on chronic airway inflammation and remodeling following chronic exposure to aerosolized antigen in mice. Methods: Ovalbumin (OVA) was administered intraperitoneally to BALB/c mice on days 0 and 14, and the mice were then exposed to aerosolized OVA daily for 4 weeks. Twenty-four hours following the final inhalation, bronchial responsiveness to acetylcholine was measured, and histologic examination and hydroxyproline content of the lung were evaluated. Results: Bronchial responsiveness to acetylcholine, number of inflammatory cells and eosinophils in the lamina propria, thickness of epithelial and subepithelial collagen layers, and hydroxyproline content of the lung increased following chronic exposure to OVA for 7 weeks. KF19514 significantly prevented all of these changes. Conclusions: Phosphodiesterase 4 and 1 inhibitors such as KF19514 may help prevent bronchial hyperresponsiveness and chronic asthma-induced airway remodeling.
Background: Recent studies have suggested that allergic rhinitis is closely related to bronchial asthma, reflecting the "one airway-one disease" hypothesis. It is unclear if the effects of pranlukast, a leukotriene-receptor antagonist, are consistent with this hypothesis. Objective: The goal of the study was to determine if pranlukast has effects on the upper and lower airways through a comparison of the effects of fexofenadine and pranlukast on airway hyperresponsiveness in non-asthmatic patients with cedar pollinosis before the Japanese cedar pollen season and during the peak pollen season. Methods: Patients received fexofenadine hydrochloride plus oral mequitazine (fexofenadine group) or pranlukast hydrate plus oral mequitazine (pranlukast group) as an initial treatment. Subsequent changes in airway responsiveness to acetylcholine were measured. Results: Among patients in whom coughing developed during the peak pollen season, airway responsiveness significantly increased in the fexofenadine group. In the pranlukast group, airway responsiveness did not increase significantly, regardless of the presence or absence of coughing. Conclusions: The results indicate that pranlukast hydrate inhibits airway hyperresponsiveness in non-asthmatic patients with Japanese cedar pollinosis. In turn, this suggests that cysteinyl leukotrienes have a role in increased airway responsiveness.
Background: Although the number of patients requiring hospitalization due to asthma attacks has decreased over the years, there are many who still require hospitalization for tracheal intubation and mechanical ventilation following a severe asthma attack. Therefore, we evaluated the characteristics of patients with asthma who required tracheal intubation and mechanical ventilation in our hospital. Methods: We evaluated 20 patients who had severely exacerbated asthma, requiring tracheal intubation and mechanical ventilation. An evaluation was made based on their smoking history, the number of days from the onset of the asthma attack to admission, the level of asthma control, treatments before presenting to our hospital, the frequency of hospital visits, the reason for tracheal intubation and mechanical ventilation, and outcome. Results: Of the 20 patients with asthma 13 were men and 7 women, with a mean age of 48.7 years. The characteristics of patients who required tracheal intubation and mechanical ventilation were as follows: (1) smokers, (2) not taking or irregularly taking medication, (3) using inhaled short-acting β2agonist (SABA) alone as needed, and (4) not using inhaled corticosteroids (ICS). Conclusions: Our findings suggest that treatment mainly using ICS, in addition to increased awareness of the dangers of asthma among the patients themselves, are important in preventing severe asthma attacks requiring tracheal intubation and mechanical ventilation.
Background: Salmon is one of the most widely consumed seafoods in Japan and many other countries around the world. Due to the confirmed cases of salmon-induced allergy, the food sanitation law in Japan stipulates salmon as one of the specific food items for which labeling is recommended when used as an ingredient of processed foods. However, trout, the landlocked form of anadromous salmon, is not subject to the allergen-labeling requirements, even though both populations belong to a single species. Since no supporting data have been demonstrated to make a clear distinction between these two populations in terms of allergenicity, we comparatively examined their allergenic properties using sera from patients allergic to fish. Methods: Extracts of Oncorhynchus nerka from different habitats were obtained: kokanee (landlocked) and red salmon (anadromous). Control extracts were derived from four other species. This study focused on the (1) IgE-binding capacity of the fish extracts in patients' sera (n = 50), (2) ELISA inhibition test (n = 6), and (3) inhibition immunoblot test (n = 8) between the kokanee and red salmon. Results: The extracts from kokanee and red salmon showed the highest correlation with each other in terms of the IgE-binding capacity, and showed complete (100%) reciprocal cross-inhibition in the ELISA inhibition test. On immunoblotting, there was no marked difference in the staining pattern between the two extracts, and each IgE-binding band gradually disappeared when the patients' sera were preincubated with the counterpart antigen in a dose-dependent manner. Conclusions: These results suggest that kokanee has similar allergenic properties to red salmon.
Background: Acute generalized exanthematous pustulosis (AGEP) is a rare and severe cutaneous reaction usually triggered by drugs. Other causative factors such as viral infections are rarely involved. In this study, we report a case of AGEP caused by a spider bite. Case Summary: A 56-year-old woman was referred to the allergy unit after a spider bite at the left popliteal fossa, while gardening, 5 days earlier. The offending spider was captured and identified by an entomologist as belonging to the Loxosceles rufescens species. No acute reaction was observed; however, after 24 hours, due to the occurrence of typical dermonecrotic skin lesions associated with erythema and edema, Cefuroxime and Clindamycin were administered intramuscularly after medical advice was given. Almost 72 hours after the spider bite, an erythematous and partly edematous eruption appeared locally in the gluteus area bilaterally, which progressively expanded to the trunk, arms and femors. Within 24 hours dozens of small, pinhead sized, non-follicular pustules were present, mainly in the folds. The patient complained of a burning sensation of the skin in addition to pruritus; and simultaneously had a fever of 38-39°C as the eruption expanded. Discussion: A spider bite may represent a possible causative factor of AGEP. A spider's venom contains sphingomyelinase that stimulates the release of IL8 and GM-CSF, which are involved in AGEP pathogenesis. Whether or not the con-current use of antibiotics has an effect in AGEP appearance when combined with a spider's venom, cannot be excluded.