A parasitoid wasp of Drosophila employs preemptive and reactive strategies to deplete its host's blood cells.

The wasps Leptopilina heterotoma parasitize and ingest their Drosophila hosts. They produce extracellular vesicles (EVs) in the venom that are packed with proteins, some of which perform immune suppressive functions. EV interactions with blood cells of host larvae are linked to hematopoietic depletion, immune suppression, and parasite success. But how EVs disperse within the host, enter and kill hematopoietic cells is not well understood. Using an antibody marker for L. heterotoma EVs, we show that these parasite-derived structures are readily distributed within the hosts' hemolymphatic system. EVs converge around the tightly clustered cells of the posterior signaling center (PSC) of the larval lymph gland, a small hematopoietic organ in Drosophila. The PSC serves as a source of developmental signals in naïve animals. In wasp-infected animals, the PSC directs the differentiation of lymph gland progenitors into lamellocytes. These lamellocytes are needed to encapsulate the wasp egg and block parasite development. We found that L. heterotoma infection disassembles the PSC and PSC cells disperse into the disintegrating lymph gland lobes. Genetically manipulated PSC-less lymph glands remain non-responsive and largely intact in the face of L. heterotoma infection. We also show that the larval lymph gland progenitors use the endocytic machinery to internalize EVs. Once inside, L. heterotoma EVs damage the Rab7- and LAMP-positive late endocytic and phagolysosomal compartments. Rab5 maintains hematopoietic and immune quiescence as Rab5 knockdown results in hematopoietic over-proliferation and ectopic lamellocyte differentiation. Thus, both aspects of anti-parasite immunity, i.e., (a) phagocytosis of the wasp's immune-suppressive EVs, and (b) progenitor differentiation for wasp egg encapsulation reside in the lymph gland. These results help explain why the lymph gland is specifically and precisely targeted for destruction. The parasite's simultaneous and multipronged approach to block cellular immunity not only eliminates blood cells, but also tactically blocks the genetic programming needed for supplementary hematopoietic differentiation necessary for host success. In addition to its known functions in hematopoiesis, our results highlight a previously unrecognized phagocytic role of the lymph gland in cellular immunity. EV-mediated virulence strategies described for L. heterotoma are likely to be shared by other parasitoid wasps; their understanding can improve the design and development of novel therapeutics and biopesticides as well as help protect biodiversity.

Venom immunotherapy in patients with clonal mast cell disorders: IgG4 correlates with protection.

BACKGROUND: Patients with clonal mast cell disorders (cMCD), systemic mastocytosis (SM) and monoclonal mast cell activation syndrome (MMAS), represent an increased risk for Hymenoptera venom anaphylaxis (HVA). Lifelong venom immunotherapy (VIT) is recommended; however, its efficacy and safety are controversial. Hence, we sought to evaluate the efficacy and safety of VIT in HVA patients with cMCD. METHODS: A retrospective study was conducted among 46 patients with Vespula venom allergy who had experienced severe HVA, 32 cMCD (22 with SM and 10 with MMAS) and 14 controls. There were no differences between cMCD patients and controls in age (58 vs 66) and duration of VIT (47 vs 48 months), respectively. RESULTS: During VIT, 11 (34%) cMCD patients experienced adverse reactions (ARs) (7% in controls), including 1 anaphylaxis. There were 23 re-stings in 17 (53%) patients during VIT. Of episodes, four (17%) presented with anaphylaxis, 14 (60%) presented with local reaction, and five (23%) were asymptomatic. In 11 episodes (48%), the patient did not take epinephrine, of these 8 (73%) presented with local reaction, and 3 (27%) were asymptomatic. Patient-based protection from anaphylaxis was 76% (4/17) in cMCD vs. 100% in controls during VIT. The venom-specific IgG4 concentrations increased during VIT (P < .001) although tryptase and IgE were unaltered. CONCLUSION: Both safety and efficacy of VIT in cMCD patients were slightly reduced than controls. Severe ARs were rare. The elevated IgG4 levels may be a biomarker for efficacy of VIT in cMCD patients, as it correlates with protection from re-stings.

Exclusive Bee Venom Allergy: Risk Factors and Outcome of Immunotherapy.

INTRODUCTION: Venom immunotherapy (VIT) is considered to be the gold-standard treatment for patients with Hymenoptera venom allergy. Data regarding VIT in bee venom (BV) allergic patients are scarce. AIM: The aim of this study was to evaluate the outcome of VIT in patients with exclusive BV allergy and to try to define risk factors for VIT-induced systemic reactions (VIT-ISR) and VIT failure. METHODS: This is a retrospective study including data from all BV allergic patients that were treated by VIT in the Allergy Unit at the Meir Medical Center in the years 1995-2018. RESULTS: Two hundred and forty-seven patients with exclusive BV allergy were included; 206 (83.4%) preferred to undergo rush buildup. Sixty-nine patients (27.9%) had at least 1 reaction during buildup, with the c-kit mutation being the only significant risk factor (100 vs. 28.9%, p = 0.02). Female gender (25.4 vs. 13.3%, p = 0.04), conventional buildup schedule (26.8 vs. 14.1%, p = 0.04), and c-kit mutation (100 vs. 16.8%, p < 0.01) but not tryptase level were found to be significantly more frequent in recurrent reactors. Females (20.3 vs. 9%, p = 0.03), patients with severe systemic reaction to the index sting (24.3 vs. 9.5%, p = 0.004), and c-kit mutation (66 vs. 12%, p = 0.05) but not tryptase level were found to be risk factors for severe systemic reactions. CONCLUSION: Despite the considerably high rate of VIT-ISR in patients with exclusive BV allergy, VIT can be performed safely and efficiently. C-kit mutation, and not basal serum tryptase level, seems to be a preferable biomarker for VIT-ISR in these patients.

Insect Sting Anaphylaxis-Or Mastocytosis-Or Something Else?

Insect sting anaphylaxis and mast cell disorders are intertwined in a specific and unusual way. There may be specific subsets of clonal mast cell disorders that are predisposed to sting anaphylaxis. The clinical characteristics of the sting reactions should raise suspicion of underlying mastocytosis (eg, hypotension without hives especially in a male). A baseline serum tryptase level is helpful in the evaluation of patients with insect sting anaphylaxis because it correlates with important risks for these patients, and they have a high frequency of abnormally elevated baseline levels. Elevated baseline serum tryptase level has been reported to correlate with clonal mast cell disease in patients with insect sting anaphylaxis but may also indicate one of several possible underlying syndromes, including mast cell activation syndrome (MCAS), familial hypertryptasemia, and idiopathic anaphylaxis. There is some overlap in these conditions, so it is important to evaluate the clinical pattern at presentation as well as laboratory markers, and to consider bone marrow biopsy to make a final and specific diagnosis of clonal mast cell disease. The presence of venom-IgE does not prove that the patient's previous sting reactions were IgE-mediated, but even low levels of venom-IgE in patients with mastocytosis predispose to severe sting anaphylaxis. Evaluation of all these possible factors will affect the recommendation for venom immunotherapy.

Mastocytosis and the Fig Wasp (Blastophaga psenes).

BACKGROUND: Mastocytosis involves the abnormal proliferation of mast cells and clinical variability. In the case of anaphylaxis, the triggering antigen, often associated with Hymenoptera allergens, must be identified. The common fig (Ficus carica) requires the fig wasp (Blastophaga psenes) for pollination. OBJECTIVE: We evaluated the ingestion of B. psenes as a trigger of anaphylaxis in patients with mastocytosis. MATERIAL AND METHODS: Skin prick tests (SPTs) and specific immunoglobulin E to the possible involved allergens were carried out in the patient and in 4 controls allergic to Hymenoptera and fig. Given the possibility of hidden allergens, we studied the source (figs of various origins) and possible hypersensitivity to Hymenoptera allergens, including the fig wasp (B. psenes). RESULTS: In all subjects, the SPT resulted in a wheal (larger than with histamine) with the extract of the inferior part of the female fig but not with the male extract (lower pole and stem). Immune detection was made with the stem and inferior part of figs and venom of Polistes and Vespula. Recognition bands were observed at 25 kDa with female fig extracts that were also recognized by the patient with anaphylaxis to Hymenoptera venom. CONCLUSIONS: We cannot exclude the possibility that the ingestion of fig with Blastophaga antigens may have triggered anaphylaxis in our patient.

Biopanning of allergens from wasp sting patients.

OBJECTIVE: Wasp venom is a potentially important natural drug, but it can cause hypersensitivity reactions. The purpose of the present study was to systematically study the epitopes of wasp venom. METHODS: Using a random 12-peptide phage library, we performed antibody-binding epitope panning on ten serum samples from wasp sting victims at 3 h and 4 days after the sting. The panning epitopes were identified by high-throughput sequencing and matched with wasp venom proteins by BLAST. The panned antibody-binding epitopes were verified by ELISA. RESULTS: A total of 35 specific potential wasp venom epitopes in 4 days were identified. Amongst them, twelve peptide epitopes were matched with nine wasp venom proteins, namely, vitellogenin precursor, hexamerin 70b precursor, venom carboxylesterase-6 precursor, MRJP5, major royal jelly protein 8 precursor, venom acid phosphatase Acph-1 precursor, phospholipase A2, venom serine protease 34 precursor, and major royal jelly protein 9 precursor. The changes in serum IgM antibodies induced by wasp venom were confirmed by ELISA based on the 12 peptide epitopes. CONCLUSION: The nine wasp venom proteins are potential allergens, which should be excluded or modified in the potential biomedical applications of wasp venom.

Hymenoptera Anaphylaxis as a Clonal Mast Cell Disorder.

Up to 7% of adult patients with Hymenoptera venom allergy may suffer from a clonal mast cell disease. Patients with clonal mast cell disease and Hymenoptera venom anaphylaxis are commonly males, without skin lesions, and anaphylaxis is characterized by hypotension and syncope in the absence of urticaria and angioedema. A normal value of tryptase does not exclude a mastocytosis. The diagnosis of a mast cell disease leads to several therapeutic consequences concerning the treatment of Hymenoptera venom allergy as matter of fact these patients have to undergo long-life venom immunotherapy, to prevent further, potentially fatal severe reactions.

The high molecular weight dipeptidyl peptidase IV Pol d 3 is a major allergen of Polistes dominula venom.

Hymenoptera venom allergy can cause severe anaphylaxis in untreated patients. Polistes dominula is an important elicitor of venom allergy in Southern Europe as well as in the United States. Due to its increased spreading to more moderate climate zones, Polistes venom allergy is likely to gain importance also in these areas. So far, only few allergens of Polistes dominula venom were identified as basis for component-resolved diagnostics. Therefore, this study aimed to broaden the available panel of important Polistes venom allergens. The 100 kDa allergen Pol d 3 was identified by mass spectrometry and found to be a dipeptidyl peptidase IV. Recombinantly produced Pol d 3 exhibited sIgE-reactivity with approximately 66% of Polistes venom-sensitized patients. Moreover, its clinical relevance was supported by the potent activation of basophils from allergic patients. Cross-reactivity with the dipeptidyl peptidases IV from honeybee and yellow jacket venom suggests the presence of exclusive as well as conserved IgE epitopes. The obtained data suggest a pivotal role of Pol d 3 as sensitizing component of Polistes venom, thus supporting its status as a major allergen of clinical relevance. Therefore, Pol d 3 might become a key element for proper diagnosis of Polistes venom allergy.

Fatal Anaphylaxis to Yellow Jacket Stings in Mastocytosis: Options for Identification and Treatment of At-Risk Patients.

BACKGROUND: Patients with indolent systemic mastocytosis (ISM) are at risk for severe anaphylactic reactions to yellow jacket (YJ) stings while demonstration of sensitization can be challenging because specific IgE (sIgE) levels are regularly below 0.35 kUA/L. The implication of missing YJ allergy is illustrated by a case of fatal anaphylaxis. OBJECTIVE: To explore the natural course of YJ venom allergy and the diagnostic accuracy and therapeutic consequence of YJ venom sIgE in patients with ISM. METHODS: All patients with ISM seen from 1981 to 2015 (n = 243) were evaluated on the number of YJ stings, reaction severity, and sensitivity and specificity of YJ venom sIgE. YJ venom allergic patients without mastocytosis served as control (n = 313). RESULTS: A total of 153 patients with ISM were stung during adult life. The first systemic reaction was more often severe in patients with ISM than in patients without mastocytosis (69.9% vs 22.0%) and reactions recurred in 40 of 41 re-stung patients with ISM. ISM reactors showed lower YJ venom sIgE levels than nonmastocytosis reactors (0.61 vs 4.83 kUA/L; P < .001) and asymptomatic sensitization was exceedingly rare. In ISM the current clinical threshold of 0.35 kUA/L yields a sensitivity and specificity of 77.6% and 87.5%, respectively. The optimal diagnostic accuracy is achieved at 0.17 kUA/L (sensitivity, 83.6%; specificity, 85.0%). CONCLUSIONS: The high rate of severe reactions and the fatal case underscore the importance of adequate diagnostic sensitivity of sIgE in patients with ISM. The sensitivity of sIgE can be ameliorated by lowering the threshold to 0.17 kUA/L, retaining good specificity. We recommend sIgE screening in all patients with ISM and discussing immunotherapy when YJ venom sIgE exceeds 0.17 kUA/L.

Human Allergen-Specific IgG Subclass Antibodies Measured Using ImmunoCAP Technology.

BACKGROUND: Knowledge of human IgG subclass antibody responses to various allergens has been hampered by a lack of reliable standardized assays. The aim here was to develop quantitative immunoassays for human IgG1, IgG2, and IgG3 antibodies using ImmunoCAP® technology and to evaluate their application. METHODS: Enzyme conjugates with isotype-specific monoclonal antibodies and calibrators composed of purified myeloma paraproteins were developed for each assay and used together with other standardized assay reagents for the Phadia® 100 instrument. The calibrators were adjusted to the international reference preparation IRP 67/86. The assays were characterized and used together with other standard ImmunoCAP assays to measure antibodies to various allergens in preliminary studies. RESULTS: The new assays had limits of quantitation of 1.0 (IgG1), 4.6 (IgG2), and 0.04 mgA/L (IgG3), and coefficients of variation of <20%. Only some minor cross-reactivity with IgG2 was observed for the specific IgG1 assay. The specific IgG2 assay showed a bias for the allotype G2m(23) and compensation factors were used to adjust the measured concentrations accordingly. Preliminary studies indicated a strong and stable IgG4 antibody response to ß-lactoglobulin in healthy individuals, a high IgG1 and even higher IgG2 antibody response to house dust mite in sensitized and nonsensitized subjects, and a mixed IgG subclass response to venom allergens in allergic patients with increasing IgG4 antibody levels during venom immunotherapy. CONCLUSIONS: The new research assays are valuable tools for immunological studies, enabling the characterization of antibody profiles using a standardized approach, and facilitating data interpretation and the comparison of results across studies.

[Insect venom allergy ­ the diagnosis can be difficult to make but good treatment is available]. / Insektsgiftallergi - diagnostiken kan vara svår men bra behandling finns.

Bee and wasp stings can cause allergic reactions. Although the local reactions are more frequent, anaphylaxis due to insect stings can be potentially fatal. Rapid recognition of anaphylaxis is therefore critical and reactions should immediately be treated with i.m. adrenaline. Patients having experienced anaphylaxis should be referred to an allergist for diagnostic evaluation and possible venom-immunotherapy (VIT). The clinical history is essential in diagnosis of venom allergy as the test results are not always reliable. Diagnostic testing with venom components might be beneficial in appropriate patients. The analysis of serum tryptase from the acute episode can be crucial. Mastocytosis is associated in about 8 percent of patients with severe anaphylaxis from insect stings and should be considered in the differential diagnosis. VIT is indicated for patients with a history of anaphylaxis and is effective in preventing future anaphylaxis from Hymenoptera stings.

Agreement of skin test with IL-4 production and CD40L expression by T cells upon immunotherapy of subjects with systemic reactions to Hymenoptera stings.

Venom immunotherapy is the only curative intervention for subjects with Hymenoptera venom allergy who suffering systemic reactions upon bee or wasp stings. Venom immunotherapy can restore normal immunity against venom allergens, as well as providing to allergic subjects a lifetime tolerance against venoms. Nevertheless, it is necessary using safety assays to monitoring the development of tolerance in the VIT protocols to avoid fatal anaphylactic reactions. The purpose of this study was to assess the modifications in several markers of tolerance induction in subjects with Hymenoptera venom allergy undergoing immunotherapy. The studies were performed at baseline time and after six month of VIT. Intradermal skin tests, basophil activation tests, specific IgE levels; and the T-cell markers (IL-4 and IFN-γ producing cells; and expression of the surface activation markers CD40L and CTLA-4) were assayed. At six month of immunotherapy all parameters studied had significant alterations. All decreased, except the IFN-γ producing cells. In addition, modifications in intradermal skin test showed a significant correlation with both, CD40L expression on CD4 T lymphocytes (p=0.043) and IL-4 producing T lymphocytes (p=0.012). Neither basophil activation test nor serum levels of sIgE demonstrated any correlation with the immunological parameters studied nor among them. These results suggest that both IL-4 production and CD40L expression could be two good indicators of the beneficial effects of venom immunotherapy which translate into skin tests.

Rush immunotherapy for wasp venom allergy seems safe and effective in patients with mastocytosis.

BACKGROUND: Patients with mastocytosis and wasp venom allergy (WA) may benefit from venom immunotherapy (VIT). However, fatal insect sting reactions have been described in mastocytosis patients despite previous immunotherapy. We investigated the safety and efficacy of (rush) VIT in patients with mastocytosis and WA. OBJECTIVE: To investigate the safety and efficacy of (rush) VIT in patients with mastocytosis and WA. METHODS: We describe nine patients with cutaneous mastocytosis and WA who received VIT. Cutaneous mastocytosis was confirmed by histopathology and systemic mastocytosis was diagnosed according to World Health Organization criteria. VIT was given according to a rush protocol. Given the difference in safety and efficacy of VIT in patients with WA and honeybee venom allergy, we reviewed the literature for VIT with the focus on WA patients with mastocytosis and addressed the difference between patients with cutaneous versus systemic mastocytosis. RESULTS: Nine patients had WA and mastocytosis, of whom six had cutaneous mastocytosis, two combined cutaneous and systemic mastocytosis and one systemic mastocytosis. All patients received rush IT with wasp venom. Most patients had only mild local side effects, with no systemic side effects during the course of VIT. One patient had a systemic reaction upon injection on one occasion, during the updosing phase, with dyspnoea and hypotension, but responded well to treatment. Immunotherapy was continued after temporary dose adjustment without problems. Two patients with a previous anaphylactic reaction were re-stung, without any systemic effects. CONCLUSIONS: VIT is safe in cutaneous mastocytosis patients with WA, while caution has to be made in case of systemic mastocytosis. VIT was effective in the patients who were re-stung.

Anaphylaxis after hymenoptera sting: is it venom allergy, a clonal disorder, or both?

A 47-year-old man presented with loss of consciousness 5 minutes after being stung by a yellow jacket in his backyard. Epinephrine and fluids were required for resuscitation. Allergy evaluation revealed specific IgE to yellow jacket and honeybee, and the patient was started on venom immunotherapy. He had systemic reactions during buildup and a severe anaphylactic episode requiring 3 doses of intramuscular epinephrine at maintenance doses. Immunotherapy was discontinued. Serum tryptase level after 1 such episode was 29 ng/mL, with a baseline level of 25 ng/mL 4 weeks later. The physical examination was unremarkable including no skin lesions of cutaneous mastocytosis. Because of elevated baseline tryptase level, a bone marrow biopsy was performed, which revealed multifocal dense infiltrates of mast cells. A diagnosis of systemic mastocytosis was made. The patient was treated with omalizumab and was able to tolerate immunotherapy and is currently maintained on lifelong immunotherapy. He was restung in the field and has not had anaphylaxis.

Two cases of elevated tryptase in abdominal aortic aneurysm.

INTRODUCTION: From the literature, patients with a history of anaphylaxis to hymenoptera venom and positive specific IgE have shown a correlation between elevated tryptase levels and two clinical situations: systemic mastocytosis and an increased risk of reactions to venom immunotherapy or hymenoptera sting. Other clinical scenarios could explain elevated tryptase levels. MATERIAL AND METHODS: A 67 year old male (P1) and a 77 year old male (P2) were evaluated for previous severe anaphylaxis to hymenoptera sting. They underwent standard diagnostic work-up for hymenoptera venom allergy. Having found elevated tryptase levels, these were followed by a bone marrow biopsy to rule out systemic mastocytosis. RESULTS: P1: specific IgE and skin tests were positive for Vespula species; tryptase 52.8 ng/ml; P2: specific IgE and skin tests were positive for Vespa cabro and tryptase 153 ng/ml. Bone marrow biopsy results were negative for mastocytosis. We carried out magnetic resonance imaging, in P1 to better characterize the severe osteoporosis and in P2 because during physical examination a pulsating mass had been identified in the mesogastrium, and an aneurysm of the abdominal aorta which required surgical intervention in both patients was detected. Eight months after surgery, tryptase levels had diminished significantly (P1: 11.6 ng/ml and P2: 14.5 ng/ml). DISCUSSION: The elevated tryptase levels were correlated to abdominal aneurysm in both patients. In fact, post-surgery tryptase levels dramatically decreased. These two cases demonstrate that high tryptase levels in subjects with a history of hymenoptera venom anaphylaxis can be associated to undiagnosed aneurysmatic disease.