Abstract
Aims:
To investigate the immunoglobulin (Ig) G response after being fed upon by Cimex lectularius L.
Methods and Results:
Participants were fed upon by 3 male C lectularius insects weekly for a month. Blood was obtained before the feeding and at the last feeding, which was used for immunoblots against bed bug salivary gland extract, with antihuman IgG secondary antibodies. No consistent IgG changes developed in 11 humans serially fed upon by C lectularius. Two participants had new IgG responses to proteins at molecular weights of approximately 12 to 13 kDa, and 1 had an IgG response to a protein at approximately 40 kDa. At the last study visit, more intense IgG bands to proteins at molecular weights of 12 to 13 kDa had developed in 55% of participants (6/11) and at molecular weights of ≈30 kDa, ≈40 kDa, and ≈70 kDa in 45% (5/11) compared with the first study visit. Nitrophorin and apyrase were the most common C lectularius proteins identified with liquid chromatography–tandem mass spectrometry in both crushed bed bug salivary gland extract and post–bed bug feeding extract.
Conclusions:
Human participants did not have consistent IgG responses to crushed C lectularius salivary gland extract.
Keywords: antibody, bed bug, Cimex lectularius, cimicosis, IgG, immunoglobulin, saliva
Introduction
Bed bugs (Cimex lectularius L.) are hematophagous ectoparasites of humans that tend to live in close proximity to their hosts.1 In established infestations the number of C lectularius insects can be in the thousands.2 If a blood meal is available, C lectularius will feed every few days.3 Therefore, a heavily infested person could potentially endure hundreds of bed bug feedings per day, the clinical consequences of which are poorly understood.3 Humans generate immune responses to bed bug saliva, but the pathophysiology of cimicosis, the dermal reaction to bed bug feedings, is uncharacterized.4,5 After a human is fed upon by C lectularius for the first time, a skin reaction develops after about 1 week.6–8 With repeated feedings, the development of a skin reaction can be almost immediate.6,8 Not all humans have a rash after being fed up on by a bed bug, however, and the percentage of anergic people is unknown.9
Rather than being an intrinsic response to the insect exoskeleton, cimicosis is caused by C lectularius saliva.4,5 Bed bugs that had their salivary glands surgically removed were not able to feed and did not cause a skin reaction in sensitized persons.4 Additionally, applying isolated bed bug saliva to the skin of a sensitized person causes cimicosis.5 Previous investigations of bed bug salivary gland homogenate have identified an apyrase, which inhibits platelet and neutrophil aggregation.10–12 Also present in bed bug saliva are a factor X activation inhibitor and nitrophorin, which carries unstable nitric oxide and promotes vasodilatation and inhibits platelet aggregation.13–16 The objectives of the current study were to characterize the immunoglobulin (Ig) G responses to bed bug salivary antigens in humans who were serially fed upon by C lectularius.
Materials and Methods
Study Participants
The study was approved by the University Hospitals Institutional Review Board, and the methods have been previously published.8 All participants provided written informed consent before participating in the study. Inclusion criteria were healthy volunteers older than 18 years. Exclusion criteria included pregnancy; current breastfeeding; hospital admission for allergic reaction during the past 10 years; current use of antihistamines or corticosteroids; cancer diagnosis within the past 5 years; drinking an average of 2 or more alcoholic drinks per day or planning to drink 3 or more alcoholic drinks during any 8-hour period while enrolled in the study; any known hepatic or renal disease; any immunodeficiency; history of asthma requiring treatment by a physician within the previous 6 months; history of angioedema or anaphylaxis in the past 2 years; or not speaking English.
Feeding Protocol
Human participants were enrolled to be serially fed upon by bed bugs. Participants were fed on by bed bugs at day 0 (study visit 1), days 9 to 11 (study visit 2), days 19 to 21 (study visit 3), and days 28 to 32 (study visit 4). Participants were asked to record in a journal the onset and duration of any rash and pruritus associated with a bed bug feeding. Pruritus was recorded on a Likert scale (1, not itchy, to 10, intolerable itch).
At each study visit, 3 male C lectularius (Ridge strain) insects fed on each study participant’s supinated forearm. Male bed bugs were chosen to decrease the risk of a participant inadvertently acquiring a bed bug infestation by participating in the study—C lectularius eggs and first instars are small and could be accidently overlooked. Each bed bug did not feed on more than 1 human participant, and all insects were visually confirmed to have fed. The insects were kept inside 50-mL test tubes, over which sheer fabric was adhered. This allowed the insects to feed through the fabric but not escape. Blood samples were obtained before the first bed bug feeding and at the time of the last bed bug feeding.
C lectularius
Ridge strain bed bugs were used the experiments and were originally captured from an apartment in New Haven, Connecticut, in 2009. The insects then were maintained by feeding on a study investigator until they were used in the experiments.
C lectularius Salivary Gland Salivary glands were surgically removed from 275 C lectularius. The salivary glands were crushed, combined, and placed in 3.5 mL of sterile, distilled water. The samples were stored at −80°C.
Immunoblots
Crushed C lectularius salivary gland was mixed with diluted 4× loading buffer (LI-COR), denatured, and separated by SDS-PAGE on 4% to 15% Mini-PROTEAN TGX gels (Bio-Rad) with diluted 10× Tris/glycine/SDS buffer (Bio-Rad) at 5 μg of protein per well. The separated proteins were transferred to PVDF transfer membranes (Thermo Scientific) through a wet transfer process with transfer buffer (20% methanol, 10% Tris-glycine). The membranes were then blocked with Odyssey Blocking Buffer (LI-COR) for 1 hour and incubated overnight with participant serum diluted 1:10 in TBST (Tris-buffered saline [Bio-Rad] plus 0.1% Tween 20 [Fisher Scientific]). Fluorescence-based Western blotting was then performed on the membranes by probing bands with an IRDye 800CW–labeled goat antihuman IgG secondary antibody (LI-COR) diluted 1:7,000 in TBST. The membranes were then dry scanned on an Odyssey Classic Imaging system (LI-COR).
HBSS-Bed Bug Feeding
Mixed populations of C lectularius L. (Ridge strain) were placed in 50-mL conical test tubes with the opening secured by sheer fabric. Parafilm was placed over the sheer fabric and the test tube inverted into a petri dish containing sterile Hanks balanced salt solution (HBSS) warmed to approximately 37°C. Bed bugs were then allowed to feed on the warmed HBSS. Prefeed (control) and postfeed HBSS was collected and frozen at −80°C.
Liquid Chromatography With Tandem Mass Spectrometry
Sample Preparation
For liquid chromatography with tandem mass spectrometry (LC-MS/MS), crushed bed bug salivary glands were lysed with 2% SDS with pulse sonication. Samples were then cleaned of detergent by using a previously published filter-aided sample preparation protocol with a 10-kDa molecular weight–cutoff filter (Millipore) and buffer exchanged with 8M urea in 50mM Tris, pH 8.0 to a final volume of 50 μL. Proteins were reduced on the filter with 10mM dithiothreitol (8M urea, 50mM Tris, pH 8.0) for 1 hour at 37°C, followed by alkylation with 25mM iodoacetamide (8M Urea, 50mM Tris, pH 8.0) for 30 minutes in the dark. The 8M urea was then adjusted to 4M with 50mM Tris, pH 8.0, and samples were concentrated to a final volume of 50 μL. The sample was concentrated and buffer exchanged with 100mM Tris pH 8.0 using a 10-kDa molecular weight–cutoff filter (Millipore). The samples were reduced with 10mM dithiothreitol for 1 hour at 37°C, followed by alkylation with 25mM iodoacetamide for 30 minutes in the dark.
Digestion and LC-MS/MS Analysis
Protein concentration was measured using a Bradford assay following the manufacturer’s standard protocol (Bio-Rad). Five μg of each sample was digested using 0.3 μg lysyl endopeptidase for 1 hour at 37°C, followed by the addition of 0.3 μg trypsin and incubation overnight at 37°C. Samples were analyzed by LC-MS/MS using a LTQ-Orbitrap Elite mass spectrometer (Thermo Scientific) equipped with a nanoACQUITY ultra-high pressure liquid chromatography system (Waters). Additional LC-MS/MS methods are provided in the Supplemental Methods. “The LC-MS/MS raw files were processed and searched using Mascot (Matrix Science London, version 2.1) and searched against the Uniprot database ([560,537] sequences; [201,466,755] residues). Mascot search settings were as [follows]: trypsin enzyme specificity; mass accuracy window for precursor ion, 8 ppm; mass accuracy window for fragment ions, 0.8 Da; carbamidomethylation of cysteines as fixed modifications; oxidation of methionine as variable modification; and one missed cleavage. Searched files were then imported into Scaffold (Proteome Software Inc., version 4.6.2) for peptide validation and quantification by spectral count. Peptide identifications were accepted if there [was] >95% probability by the Peptide Prophet algorithm. Spectral counts were reported using total spectrum count for each peptide identified.”17
Results
Of 21 participants enrolled in the study, 18 completed the first bed bug feeding and 11 completed the fourth feeding. Six participants provided journal data after the first bed bug feeding and 5 provided data about the fourth feeding. Only 1 participant (#7) reported a history of being fed upon by bed bugs, which occurred more than 1 year previously.
Immunoblots of Crushed Bed Bug Salivary Gland
Immunoblots for 11 participants are shown in the Figure. No consistent changes in the IgG responses were seen in all participants between study visits 1 and 4 (Tables 1 and 2). Participants showed IgG responses to proteins with molecular weights of approximately 12-13, 30, 40, and 70 kDa at both study visits 1 and 4; only participant 6 had IgG responses to a protein with a molecular weight of ≈60 kDa in both study visits 1 and 4 immunoblots. Three participants (27%) had no appreciable changes in their immunoblots between study visits 1 and 4 (Table 1).
Figure.
Immunoblots Probed With Antihuman Immunoglobulin G Using Serum From Study Visits 1 and 4 and Crushed Bed Bug Salivary Gland Antigen. For each participant, blots are shown from study visit 1 (left) and study visit 4 (right). Each blot is shown with standard molecular weight markers. A, Participants 1 and 2. B, Participants 3 and 4. C, Participants 5 and 6. D, Participants 7 and 8. E, Participants 9 and 10. F, Participant 11. Blue arrows indicate changes in the protein bands.
Table 1.
Summary of Clinical Findings and Antihuman Immunoglobulin G Immunoblot Data
| Visit 1 | Visit 4 | |||||
|---|---|---|---|---|---|---|
| Ppt | Age, y; Sex | Time to First Dermal Reaction, d | Maximum Pruritusa | Time to First Dermal Reaction, d | Maximum Pruritusa | Substantial Changes in Immunoblots Between Study Visits 1 and 4 |
| 1 | 30 F | 4 | 3 | 2 | 3 | None |
| 2 | 49 M | No rash to day 30 | 1 | 2 | 1 | None |
| 3 | 18 F | 7 | 2 | 2 | 3 | None |
| 4 | 19 F | No rash to day 7, then ND | 1 until day 7, then ND | No rash to day 12, then ND | 1 until day 12, then ND | New bands at ≈12 and 13 kDa, and possibly at ≈40 kDa, with darker bands at ≈30 and ≈70 kDa |
| 5 | 19 M | ND | ND | ND | ND | New band at ≈13 kDa, and darker bands at ≈12, ≈30, ≈40, and ≈70 kDa |
| 6 | 46 F | 12 | 2 | 2 | 4 | New band at ≈30 kDa |
| 7b | 21 M | 9 | 5 | 1 | 8 | New bands at ≈13, ≈30, and ≈40 kDa; darker band at ≈70 kDa |
| 8 | 57 M | No rash to day 30 | 1 | 1 | ND | None |
| 9 | 27 F | ND | ND | ND | ND | Darker bands at ≈12, ≈13, ≈30, ≈40, and ≈70 kDa |
| 10 | 49 M | ND | ND | ND | ND | Darker band at ≈12 kDa |
| 11 | 21 F | 10 | 6 | ND | ND | Darker bands ≈12, ≈13, ≈30, ≈40, and ≈70 kDa |
Abbreviations: F, female; M, male; ND, no data; Ppt, participant.
Scale 1 to 10 (1, no itch; 10, worst possible itch).
Reported previous bed bug exposure >1 year before study enrollment.
Table 2.
Immunoblot Results for 4 Protein Bands Identified in Participants
| Size of Immunoblot Band (Corresponding Possible Cimex lectularius L. Salivary Gland Proteins) | Present at First Visit | New Band Developed at Study Visit 4 | New or Darker Band Developed at Study Visit 4 |
|---|---|---|---|
| ≈12-13 kDa (lysozyme, odorant binding protein, and dinucleotide phosphodiesterase) | Ppts 1, 2, 3, 4, 5, 6, 9, 11 | Ppts 7, 10 | Ppts 4, 5, 7, 9, 10, 11 |
| ≈30 kDa (nitrophorin) | Ppts 1, 2, 3, 4, 5, 6, 8, 9, 10, 11 | Ppts 4, 5, 7, 9, 11 | |
| ≈40 kDa (apyrase and inositol phosphatase) | Ppts 1, 2, 3, 5, 6, 9, 11 | Ppt 4 | Ppts 4, 5, 7, 9, 11 |
| ≈70 kDa (esterase) | Ppts 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 | Ppts 4, 5, 7, 9, 11 |
Abbreviation: Ppt, participant.
At study visit 1, the frequency of IgG responses to proteins of the following molecular weights were: ≈12-13 kDa, 73% (n=8); ≈30 kDa, 91% (n=10); ≈40 kDa, 64% (n=7); and ≈70 kDa, 100% (n=11) (Table 1). Only 3 participants showed development of novel immunoblot bands between study visits 1 and 4: participants 7 and 10 at ≈12-13 kDa and participant 4 at ≈40 kDa. The following participants had new or more intense bands at study visit 4 compared with study visit 1: ≈12-13 kDa, 55% (n=6); ≈30 kDa, 45% (n=5); ≈40 kDa, 45% (n=5); and ≈70 kDa, 45% (n=5) (Table 2).
Participants With Higher Reported Pruritic Cimicosis After Study Visit 1
At study visit 1, participants 7 and 11 reported their maximum pruritus to be 5 and 6 (out of 10), respectively, compared with a mean (SD) maximum pruritus level of 1.8 (0.8) for 5 other participants (Table 1). In participants 7 and 11, the onset of their study visit 1 post–bed bug feeding rash was on day 9 and 10, respectively. At study visit 1, participant 11 had IgG responses to proteins at molecular weights ≈12-13, ≈30, and ≈40 kDa (Table 2). Both participants 7 and 11 had IgG responses to a protein at ≈70 kDa at study visit 1 (Table 2).
Participants With Higher Reported Pruritic Cimicosis After Study Visit 4
From the study visit 4 bed bug feeding, participants 6 and 7 had the highest levels of pruritus (4 and 8 out of 10, respectively) among the 5 participants reporting pruritus. Both participants 6 and 7 had new IgG responses between study visits 1 and 4, with molecular weights of ≈30 kDa for both participants and molecular weights of ≈13 kDa and ≈40 kDa for participant 7.
Participants With More Rapid Cimicosis After Study Visit 4
Participants 7 and 8 reported a post–bed bug feeding rash within 1 day after study visit 4. The change in their immunoblots between study visits 1 and 4 showed that participant 7 had new IgG responses to proteins with molecular weights of ≈13, ≈30, and ≈40 kDa, and a more intense IgG response to a protein at ≈70 kDa, whereas participant 8 had no substantial changes. Four participants reported a rash on day 2 after the 4th bed bug feeding, among whom no changes between the study visits 1 and 4 immunoblots were seen, except for participant 6 with development of a new IgG response to a protein with a molecular weight of ≈30 kDa.
LC-MS/MS of Crushed Bed Bug Salivary Gland and From Pre– and Post–Feeding Bed Bug Groups
Nine proteins were identified by LC-MS/MS in crushed bed bug salivary glands, of which 2 were most closely aligned with C lectularius: nitrophorin and apyrase (Table 3). The other 7 proteins most commonly aligned to Sus scrofa, Scylla serrata, Acropora formosa, Homo sapiens, Pan troglodytes, Sepia officinalis, and Drosophila melanogaster (Supplemental Table 1). No C lectularius proteins were found in the pre–bed bug feeding HBSS (negative control), and both nitrophorin and apyrase were found in the post–bed bug feeding HBSS (Table 4). Other proteins found in the pre– and post–bed bug feeding HBSS are listed in Supplemental Table 2.
Table 3.
Proteins Identified in Crushed Cimex lectularius L. Salivary Gland Extract by Liquid Chromatography With Tandem Mass Spectrometry
Table 4.
Cimex lectularius L. Proteins Identified in HBSS by Liquid Chromatography With Tandem Mass Spectrometry Before and After Populations of C lectularius Fed on the HBSS
| Peptide Count |
|||||
|---|---|---|---|---|---|
| Identified Proteins | Accession Number | Alternate ID | Molecular Weight | Control | Saliva HBSS |
| Nitrophorin Cim l NP OS=Cimex lectularius OX=79782 PE=1 SV=1 | O76745 | Not available | 0 | 13 | |
| Apyrase OS=Cimex lectularius OX=79782 GN=APY PE=1 SV=1 | sp|O96559|APY_CIMLE | APY | 41 kDa | 0 | 12 |
Abbreviation: HBSS, Hanks balanced salt solution.
Discussion
IgG1 and IgG4 antibodies against mosquito saliva–specific antigens are increased in persons with a positive mosquito bite test and who have severe local, but not systemic, reactions.18 IgG antibodies develop in humans after being fed upon by other hematophagous arthropods including fleas and ticks.19,20 Little is known, however, about the specific human humoral immune response to bed bug saliva, feces, egg casings, or shed skin. A previously reported patient with cimicosis had no detectable IgG antibody responses to C lectularius apyrase, but they did have IgE antibodies that recognized a ≈32 kDa protein that could represent nitrophorin.21 In another study, 30% of participants (9/30) who had reported a history of being fed upon by bed bugs had a positive IgE response against recombinant C lectularius nitrophorin.22 Another study of 3 participants serially fed upon by bed bugs (2 of whom had dermal reactions to the feedings) reported that they had IgG antibodies to a ≈37 kDa protein on immunoblots, and 1 of the participants had a positive IgG response to a protein with a molecular weight of 75 kDa.23 Serum of patients with papular urticaria but without confirmed exposure to bed begs also showed antibodies that recognized proteins of molecular weights 10 and 66 kDa on immunoblots probed with antihuman IgG.24 These proteins were in the same range as the ≈12-13 kDa and ≈70 kDa bands that were recognized by serum immunoglobulins in participants in our study.
Forty-six proteins have previously been identified in the C lectularius salivary gland.25 The C lectularius salivary gland transcriptome showed proteins of ≈10-15 kDa representing lysozyme, odorant binding protein, and dinucleotide phosphodiesterase, of ≈30 kDa with nitrophorin being the most likely protein, of ≈40 kDa likely representing apyrase and inositol phosphatase, and of ≈70 kDa with esterase being the most likely protein.26 Of the transcripts associated with secreted products, the most common were odorant binding protein family (42%), nitrophorin family (37%), esterase (4%), immunity lysozyme (4%), serine protease (3%), “other” (3%), apyrase (2%), antigen 5 family (2%), diadenosine phosphatase (1%), serpins (1%), and mucinlike (<1%).26
Apyrases are found in hematophagous insects, including C lectularius, and are involved in inhibiting platelet aggregation by the hydrolysis of ATP and ADP to AMP and inorganic phosphate.11,27 The apyrase of C lectularius has an approximate molecular weight of 40 kDa,26 and 4 participants in our study (36%) had more intense IgG bands recognizing a ≈40 kDa protein develop between study visits 1 and 4. Antibodies that recognized a ≈40 kDa protein newly developed between study visits 1 and 4 in 1 participant (9%).
Although all 11 participants had IgG bands at ≈70 kDa at study visit 1, 5 (42%) had more intense bands at study visit 4 than visit 1, which suggests that an increased IgG response was occurring. The band at ≈70 kDa recognized by IgG from participant serum may represent an esterase protein.26 Most participants (8, 73%) had IgG recognition of proteins at ≈12-13 kDa at study visit 1, which could represent baseline recognition of an odorant binding protein.26 At study visit 4, 2 participants (18%) had new bands and 6 (55%) had more intense bands at ≈12-13 kDa; 1 participant never had bands at ≈12-13 kDa. Almost all participants (10, 91%) had antibodies to a protein of ≈30 kDa at study visit 1, which may represent nitrophorin, but none had new IgG antibodies at ≈30 kDa during the study, and 5 (45%) had more intense bands at study visit 4.
The current study builds on previous work examining the humoral immune response to C lectularius.21–24 Our data come from a larger number of participants and combine IgG responses with clinical outcomes and LC-MS/MS data. A previous report showed no significant changes in serum levels of interleukin (IL)-1β, IL-4, IL-5, IL-6, IL-10, IL-17A, tumor necrosis factor α, or interferon before and after C lectularius feedings.8 Additionally, there were no changes in total IgG1, IgG2, IgG4, or IgE levels. However, a significant decrease was seen in IgG3 levels between the first and the fourth bed bug feedings.8
Limitations
The study had several limitations. Data regarding dermal reactions were incomplete and missing from some participants, which limits our ability to correlate IgG responses to bed bug salivary gland antigen recognition with clinical outcomes. We only used laboratory-raised male C lectularius, and it is unknown whether the humoral immune response would have varied depending on insect life stage or sex, or if non–laboratory-raised bed bugs were used. Only IgG responses to bed bug salivary gland antigens were measured, although IgE responses against bed bug salivary gland antigens may be better for approximating allergic responses in humans to bed bugs. We attempted to examine IgE responses in our participants, but we identified technical issues with the equipment available in the laboratory, and other financial limitations prevented us from performing these experiments. The LC-MS/MS analysis was limited to the protein sequences available in Uniprot at the time of analysis.
The C lectularius salivary gland transcriptome has been published, and the expected salivary proteins have been reported.25,26 The lack of commercially available immunoglobulins that recognize known bed bug salivary gland antigens limited our ability to confirm the identity of the specific protein bands identified on our immunoblots.
Conclusions
No universally recognized IgG protein band developed in all participants against bed bug salivary gland proteins after repeated feeding by bed bugs. However, 64% of participants had IgG antibodies to a protein at ≈40 kDa at study visit 1, which may represent a baseline immune response to apyrase. Nitrophorin and apyrase were the only 2 proteins recognized in the crushed bed bug salivary gland and in the post–bed bug feeding HBSS. Further research on the human IgE immune response to bed bug salivary gland antigens would provide additional insight into the human immune response to bed bug salivary antigens.
Supplementary Material
Acknowledgements
We thank the Dahms Clinical Research Unit (DCRU) for its assistance in obtaining, processing, and storing clinical specimens from our study participants. The DCRU and the CTSC Bioanalyte Core Center are supported in part by National Center for Advancing Translational Sciences, NIH Grant UL1 TR000439. The research was funded with support from a University Hospitals Center for Clinical Research and Technology grant, internal support from the UHCMC Department of Emergency Medicine, and funds from Henry Myer III Faculty Fellowship Award.
Funding statement: The research was funded with support from a University Hospitals Center for Clinical Research and Technology grant and internal support from the University Hospitals Cleveland Medical Center Department of Emergency Medicine.
Abbreviations
- HBSS
Hanks balanced salt solution
- Ig
immunoglobulin
- IL
interleukin
- LC-MS/MS
liquid chromatography with tandem mass spectrometry
Footnotes
Disclosures: None.
Data availability statement: The authors do not have permission to submit additional data.
Conflict of interest: The authors have stated explicitly that there are no conflicts of interest in connection with this article.
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