allergy to insect stings

Allergy to insect stings I. Diagnosis of IgE-mediated Hymenoptera sensitivity by venom-induced histamine release*

A. K. Sobotka, M. D. Valentine, A. W. Benton, and 1. M. Lichtenstein**

Ii’crltivlore, Md., a,nd [Jxiversity Park, Pa.

The failure to distinguish sensitive from normal individuals by skin. testing ZL’itf) whole body extracts of Hymenoptera led us to study whether “allergic” reactions to these insects were based on an immunologic mechanism rind to attempt the deveL opment of a useful diagnostic test for this Condition. Pure venoms of honeybee, yellow jncket, yrllow and white-faced horn<Q and bumbl&ecs were used as antigens for leukocyte histamine release. The leukocytes from 13’/16 patients judged clinica&I to haue h,ad systemic reactions to a stinging insect released > 50 per cent of th,eir histamine with 0.001 to 1.0 ,ag per milliliter of venom. Whether the few patients wh,o had a negati?;e response were truly allergic or sensitive to venoms not used fwj testing remains to be seen. None of the leukocytes of the 12 control patients, howez;es, relensed histamine at l,OOO-fold greater concentrations of venom. We confirmed previous studies which showed that skin testing with Hymenoptera whole body

extracts failed to separate these two groups. Passive sensitization of norm&l leu.%o- cytes for venom-in&iced histamine release was accomplished with sera from several of the Hymenoptera-sensitive patients. Heating these sera to .5tY C. or passing them through an IgE immunoabsorbent column marlcedly reduced their sensitizing ability. We conclude that venom-induced histamine release from the leukocytes of Bymenop- tern-sensitive patients is mediated by antibodies of the IgE class and suggest that the great majority of reactions to Hymenoptera stings are truly allergic. The clear-cut diagnostic utbity of pztre venoms in vitro warrants further exploration of their diagnostic utility by in viva slci7t testing.

Each year a significant number of people in the United States die as a result of being stung by insects of the cdlass 1Tymcnoptera.l As many as 4 people per thousand2* 3 may have serious systemic reactions and thereafter live in real fear of the sequelae of a subsequent sting. The clinical presentation of these reactions resembles anaphylaxis, but there has been little solid evidence that the pathogenesis involves an immunologic event. The greatest problem in assum- ing a reaginia antibody basis for these reactions is the failure, in repeated studies, to clearly differentiate the putatively (‘bee” allergic patient from the nonallergic patient by skin testing.4. 5

Skin testing studies began with the work of Benson and Semenov,” who

From Johns Hopkins University School of Medicine and Pennsylvania State University. Supported by Grants AI 07290 and AI 08270 from the National lnstitute of AllerFFy and

Infectious Diseases, National Institutes of Health.

Received for publication July 23, 1973. Reprint requests to: Dr. A. K. Sobotka, The Good Samaritan Hospital, 5601 Loch Raven Rlvd.,

Baltimore, Md. 21239. “Publication No. 97 of the O’Neill Research Laboratories. **Recipient, of a Research Career Development Award from the National Institute of Allergy

and Infectious Diseases, National Institutes of IIealth.

vol. cc?, hTo. 9, pp. 170-184

VOLUME 53 NUMBER 3

Allergy to insect stings 171

found a beekeeper sensitive not only to venom, but also to bee body extract. Others have assumed that whole body extracts might be useful in the diagnosis of all Hymenoptera-sensitive paGents. Controlled studies, however, have shown that 38 to 98 per cent of normals have positive skin reactions when tested with whole body extracts in the same concentration range required to elicit a response in patients with a clear, recent history of a systemic reaction following a sting by a defined insect. This failure may well be due to the use of whole body extracts rather than venom in testing patients whose only Hymenoptera exposure has consisted of stings, as opposed to the more general exposure of the beekeeper. An equally likely explanation for such disparate results can be found in examining the toxic nature of the extracts themselves.7

A number of investigators have approached this question using immunochemical analyses of the whole body extracts and venoms, relying heavily on the immunogenicity of these materials in animals. Leaders in this work have been Foubert and Stier,s Arbesman, Langlois, and Shulman,s Langlois, Shulman, and Arbesmanl”s I1 and Shulman and associates .I* The interpretation of these investigators’ results has been complicated by the ability of normal serum constituents (largely lipoproteins) to bind some venom components.*3 Moreover, immunochemical analysis did not yield useful diagnostic reagents for human skin testing.

Relying heavily on anecdotal experience, conventional medical wisdom (with which we agree) has called for immunotherapy in patients who have suffered a systemic reaction following Hymenoptera stingsI The results of this therapy have been chronicled by a registry of Hymenoptera-sensitive patients initiated in 1963 under the auspices of the American Academy of Allergy.ls This study has of necessity been largely retrospective and uncontrolled. While the results suggest the utility of such therapy, they cannot be accepted as rigorous proof. Also there is no scientific basis for any particular course of immunotherapy, defined end point for therapy, or an objective test which indicates that protection has been achieved.

This study was designed to answer two unresolved questions: (1) Does the systemic reaction to insect stings have an immunologic pathogenesis, i.e., is it mediated by IgE (reaginic) antibody? and (2) Can a useful diagnostic test be devised which clearly separates the allergic from the nonallergic individual? Using purified Hymenoptera venoms and the leukocyte-histamine release model of “in vitro anaphylaxis,” our results suggest that both questions can be answered in the affirmative.

MATERIALS AND METHODS Patients

Using clinical criteria only, 16 patients who had experienced symptoms suggestive of systemic anaphylaxis following an insect sting were selected. The reactions were described as rapid in onset, occurring within the 6rst hour, and were usually severe, several patients

requiring hospitalization. All 16 patients exhibited one or more of the following: (1) generalized urticaria or angioneurotic edema; (2) respiratory difficulty due either to laryngeal

edema or bronchospasm; (3) vascular collapse, with or without loss of consciousness; and (4) generalized pruritus and erythema. The reaction to the insect sting had occurred at least two weeks before this study began and in most cases, within the preceding 2 to 6 month

period; the patients selected had not previously undergone immunotherapy with Hymenoptera

172 Sobotko et al. J. ALLERGY CLIN. IMMUNOL. MARCH 1974

rb-2 lb-l 1 YELLOW JACKET(pg/ml venom)

Non Dialyzed

o Allergic

A Control

Dialyzed

l Allergic

A Control

yg/ml. HONEY BEE VENOM

FIG. 1. A, Histamine release by dialyzed and nondialyzed yellow jacket venom from the

leukocytes of a donor sensitive to yellow jackets. B, Histamine release by dialyzed and nondialyzed honeybee venom from the leukocytes of a honeybee-allergic (0, 0) and a

normal donor (A, A). Note the toxic release of histamine at 1 Y per milliliter of nondialyzed

venom.

extracts. Twelve hospital employees with no history of Hymenoptera sensitivity volunteered as controls. Informed consent was given by patients and controls prior to the study. Desensitization was recommended for the patients following the guidelines established by the

American Academy of Allergy,‘4 and treatment was begun without delay after initial studies were completed. Patients were instructed in the avoidance of stings and were advised to obtain Medic Alert emblems (Medic Alert Foundation, Turlock, California) and Center

insect sting first aid kits (Center Laboratories Inc., Port Washington, New York).

Antigens

Antigens used for skin testing included individual whole body extracts of honeybee, yellow jacket, wasp, and hornet obtained from several commercial sources. All whole body extracts were standardized in protein nitrogen units (PNU). The antigens used for histamine release

studies consisted of these whole body extracts and pure venoms from honeybees, yellow jackets,

VOLUME 53 NUMBER 3


80

1 Allergic cells 0 A l

,w Control cells o A 0 \,-1,. 1_-a._.

lb Iki2 lb I I

pg VENOM /ml.

FIG. 2. Histamine release from the leukocytes of a donor sensitive to several insect venoms and from the ceils of a normal donor.

yellow hornets, white-faced hornets, and bumblebees. The honeybee venom was collected by

electrical stimulation,16 the others by capillary withdrawal from the venom sacs. The venoms were lyophilized after collection, subsequently weighed in milligram quantities, and diluted

in tris-buffered saline (TACM). After preliminary studies, all of the venoms used were dialyzed prior to use to remove histamine and other low molecular weight substances that

interfered with the fluorometric assay for histamine or that were detrimental (toxic) to the leukocytes. Ten-milliliter aliquots of the venoms were dialyzed using No. 18 Vieking tubing

(Union Carbide) for 48 hours against 4 changes of 500 ml. of TACM. As is seen in Fig. 1, dialysis did not significantly decrease the allergenicity of the venoms while removing inter-

fering substances. The venoms were then aliquoted and stored at -20” C. until used.

Histamine release studies

Determinations of hypersensitivity to venoms were done simultaneously on the leukocytes

of allergic patients and normal controls. Blood was collected, dextran sedimented and washed as described previously.17 The leukocytes, resuspended in TACM, were mixed with appropriate

concentrations of venom usually covering a range of 0.001 to 10 fig per milliliter venom,

allowed to react for 60 minutes at 37“ C., and the histamine release determined fluorometrically by May’s modification of the method of Lichtenstein and Osler.lT9 1s In passive sensitization

experiments, recipient cells, which had previously been shown to be nonreactive to venoms and ragweed antigen E, were used following the techniques described by Levy and Osler.19

Skin testing

Patients and controls were initially scratch tested with the antigens. If scratch tests

(range 0.01 to 1.0 PNU) were negative, intradermal tests were begun, starting with a eoneen- tration of 0.01 PNU and proceeding in most instances in tenfold increases to 1,000 PNU or

until a positive test was obtained. Diluent controls were run in all persons tested. A positive skin test was defined as any reaction consisting of a wheal 5 mm. greater than the diluent

control and erythema of 15 mm. diameter.

RESULTS

The leukocytes of 16 patients who had suffered systemic reactions associated with Hymenoptera stings were challenged in vitro with the several venoms

174 Sobotka et al. J. ALLERGY CLIN. IMMUNOL. MARCH 1974

60

/ White Hornet

w;dfy

Yellow Hornet

0 g f-

BumMe Bee _-__ _--- ---__ -__- ----.

HaRyb

0 B I I 10-3

I 10-z

I 1 -1

pg/ml”VENOtvl I IO

FIG. 3. A, Histamine release from the leukocytes of a donor sensitive to yellow jacket/

yellow hornet (vespid) venoms. Note the lack of a response to honeybee and bumblebee

venoms. B, Histamine release from the leukocytes of another donor sensitive to white and yellow hornet venoms. Note the lack of a response to bumblebee or honeybee venoms.

C, Histamine release from the leukocytes of a donor sensitive to honeybee venom; note the lack of a response to bumblebee, yellow jacket, or yellow hornet venoms.

listed above. In each of the original experiments the cells of an allegedly allergic donor were studied together with a normal control. A typical experiment is shown in Fig. 2. This patient’s leukocytes were most responsive to yellow jacket venom, showing 50 per cent histamine release at < 0.1 y per milliliter, but also gave a positive response to the venoms obtained from honeybees and bumblebees. Control results are included in Fig. 2 ; they are negative in this instance, as they

VOLUME 53 NUMBER 3


Fig. 3, C. For legend see opposite page.

CELL SENSITIVITY TO VENOMS

loo-

BO-

f _ Q !Y s! 60- z” r - : ff 40-

!5

ti -

! 20-

-

l

.

.

.

3

0.001 yg/ml

-

aa 0.001 yg/ml 0.1 pg/ml

VENOM CONCENTRATION 0 = Allergic 0 =Cmtml

FIG. 4. Summary of all histamine release data indicating the degree of sensitivity to

Hymenoptera venoms in 13 allergic and 12 control patients.

were in every experiment, and are not further plotted. Leukocytes of the 12 normal controls used in this study were nonreactive to pure venoms at concentrations up to 10 pg per milliliter. On the other hand, 13 of the 16 patients defined as allergic by clinical criteria alone showed a significant ( > 50 per cent) histamine release response. Typical patterns of this response are shown in Fig. 3. Most patients, concordant with their clinical history, had leukocytes which were

176 Sobotko et al. J. ALLERGY CFIN. IMMUNOL. MARCH 1974

PASSIVE SENSITIZATION

100

?

Hwey Bes Al Brgrc Serum Honey Bee Venom

Normal Serum Honey Bee Venom

10.5 10-4 10-z 10-2

pq ANTIGEN /ml

I00 1

60

1 Celk EG Serum W S.

1 Unheated Serum

/’ 10.' I IO

@ pg/ml YELLOW JACKET VENOM

FIG. 5. A, Histamine release following the passive sensitization of normal cells with: (1)

serum from a honeybee-sensitive donor followed by challenge with honeybee venom; (2) serum from a ragweed-allergic donor followed by challenge with ragweed antigen E;

and (3) serum from a normal donor followed by challenge with honeybee venom. B, Effect of heating at 56” C. for 2 hours on the subsequent ability of a serum from a donor sensitive to yellow jackets to passively sensitize normal leukocytes for histamine release by

yellow jacket venom. C, Effect of absorbing a serum from a donor sensitive to yellow jackets with anti-IgE on the ability of the serum to passively sensitize normal leukocytes

for histamine release by yellow jacket venom.

reactive to the yellow jacket/yellow hornet/white-faced hornet (vespid) venoms and not to the honeybee and bumblebee venoms (Fig. 3, A and B). Of those studied, 10 showed sensitivity to the cross-reacting vespid venoms with concentrations required for 50 per cent histamine release ranging from 10”’ to 10-l y per milliliter. Only 3 of these 10 patients also responded to the bumblebee and/or

VOLUME 53 Allergy to insect stings 177 NUMBER 3

50

1 i+ 40 9 i: LL i

y 30-

5

iz I 20-

2 8 cc IO - k!

O-

Serum W S (IgE 132ng/ml 1

0 t

I I I C lO-2 10-l I IO

YELLOW JACKET VENOM (Ng/ml)

FIG. 5, C. For legend see opposite page.

honeybee venoms and then in more concentrated solutions. The 3 other patients responded to only honeybee venom (Fig. 3, C). The results from the venom- sensitive patients and the normal controls are summarized in Fig. 4.

Passive sensitization

In order to ascertain whether these reactions were mediated by IgE antibody, attempts were made with a number of sera to passively sensitize the leukocytes of normal donors. In 4 instances this was possible. In the experiment shown in Fig. 5, A, the cells of a normal donor were sensitized with serum from the honeybee-sensitive patient, a ragweed-allergic patient, and a normal control patient. When sensitized with normal serum, the donor was unreactive to honeybee and ragweed antigens (not shown) ; when sensitized with the serum of the venom-sensitive patient, the cells responded to venom challenge, and when sensitized with the serum of the ragweed-allergic donor, the cells released histamine when exposed to antigen E. It is of interest that the amount of unfrac- tionated venom required to elicit a response was comparable to that amount of the highly purified antigen E which initiated a corresponding response.

Further passive sensitization studies were carried out with the serum from another Hymenoptera-sensitive patient. The serum had been heated at 56” C. for 2 hours (Fig. 5, B) or run over a column made of an immunosorbent linked to anti-IgE* (Fig. 5, C). Both treatments markedly reduced the ability of the sera to passively sensitize normal leukocytes. In the latter instance, the total

*We are indebted to Dr. Kimishige Ishizaka for absorption of the eera using the method described in the Jomal of Inwnzmology, volume 104, page 335, 1970, and for measurement of the total IgE antibody levels using a radiolabeled double-antibody inhibition technique described in the Journal of Zmmzmology, volume 105, page 1459, 1970.


Yellow Jacket Venom

YellowJacket WBE YelbwJacket WBE +----TJ--~+---* +----TJ--~+---*

yg/ml VENOM------- -PNU/ml WHOLE BODY EXTRACl yg/ml VENOM------- -PNU/ml WHOLE BODY EXTRACl

~ 10-3 10-Z lo-’ I IO 100 1003

pg/ml VENOM----- PNU/ml. WHOLE BODY EXTRACT

FIG. 6. A, Histamine release by yellow jacket venom and yellow jacket whole body extract from the leukocytes of a donor sensitive to yellow jacket venom. B, Histamine re-

lease by yellow jacket venom and yellow jacket whole body extract from the leukocytes of another donor sensitive to yellow jacket venom.

IgE concentration of the serum was decreased from 132 to less than 10 ng. per milliliter with a marked drop in the level of sensitizing antibody.

In vitro studies with whole body extracts

In order to compare our in vitro histamine release studies with in vivo skin testing, it was necessary to study the histamine release patterns obtained with whole body extracts. In most instances (4/6) the patients who released histamine to the purified venoms failed to respond to the whole body extract in concentrations of up to 100 PNI; per milliliter (Fig. 6, A). Higher concentrations were often toxic. In some instances, however, (e.g., Fig. 6, 3) there was a significant response at the 100 PNC level. In other instances we found that patients responded in vitro to whole body extracts from one commercial source and not to others purportedly made from the same type of insect (Fig. ‘7). It is im- possible to accurately compare the concentrations of the venoms, quantitated as micrograms solid per milliliter, with the whole body extracts standardized in terms of PNU per milliliter. It is clear, however, that the whole body extract had a much lower specific activity. When active they required 100 PNU or about,

VOLUME 53 NUMBER 3

o-

I I I I I

0 A 10-S 10‘2 10-I I IO

&ml. VENOM PNU/ml. W HOLE BODY EXTRACT

Yellow Jacket Cells: J.G.

I /

0 B 10-2

I 10-l

I I -TF--xo M/ml. VENOM ---------- PNU/ml. WHOLE BODY EXTRACT

FIG. 7. A, Sensitivity of leukocytes from a vespid-sensitive donor to yellow hornet venom

and to whole body extracts of yellow hornet from three commercial sources. 6, Sensitivity of the leukocytes from a vespid-sensitive donor to yellow jacket venom and to whole body extracts from three commercial sources.

7 pg of protein per milliliter, whereas the venoms were usually active at < 0.018 pg solids per milliliter. It is not unexpected that the whole body extracts would have some activity since the venom sac is included in processing the insects for extraction. What is surprising is how often they were negative.

To further assess the importance of the body antigens in the whole body extract, a special preparation was made by first removing the abdominal portion of honeybees containing the venom sac and then extracting only the head and thorax. Four honeybee-sensitive patients were tested with this material and the

180 Sobotka et al. J. ALLERGY CLIN. tMMltNO1. MARCH 1974

pg ANTIGEN /ml

FIG. 8. Sensitivity of the leukocytes of 2 honeybee-sensitive donors to honeybee venom

and to a preparation made from honeybee head and thorax.

venom simultaneously (Fig. 8). Three did not respond to the head and thorax extract at concentrations lOO- to l,OOO-fold greater than the concentration of venom required for 50 per cent histamine release (e.g., Patient N. G.), Patient E. B. had a single positive point that is questionable; even if this is accepted, the head and thorax extract is seen in this patient to contain less han 0.1 per cent as much antigen as the venom.

Skin testing

The 13 patients who were reactive to venoms in vitro and appropriate controls were skin tested with the usual clinical mixtures containing equal parts of whole body extract of honeybee, yellow jacket, wasp, and hornet (Fig. 9, A). Subsequent skin testing was carried out on several allergic patients and controls with the individual whole body extracts (Fig. 9, B). Scratch tests were uni- formly negative. The thresholds for skin reactions were in the range of 1 to 1,000 PNlY per milliliter in both the allergic and control patients. While there is a tendency for the allergic patients to respond at lower concentrations, the overlap between the groups was extensive. The absence of a clear separation (such as is seen between ragweed-allergic patients, who react to 1O-5 to l@” pg per milliliter ragweed extract and nonallergic controls who show no response to 10 to 100 pg per milliliter of ragweed) makes the materials of little diagnostic utility. The skin tests were repeated using the extracts of whole body prepared by three different commercial companies with essentially the same results.

DlSCUSSfON

This report provides conclusive evidence, for the first time, that the large majority (N 80 per cent in this study) of patients with anaphylactic reactions following Hymenoptera stings have a reaginic response to venom antigens ; it is, therefore, reasonable to assume that IgE antibodies are the cause of this response. The reaginie nature of reactions to insect stings had rightly been brought into question by the failure of skin testing with crude whole body

VOLUME 53 NUMBER 3


MIXED WHOLE BODY EXTRACT

PNU/ml. FOR I+ SKIN REACTION

0 Allergic

0 0 Control

INDIVIDUAL WHOLE BODY EXTRACT

YELLOW JACKET I 1

> 1000 1000 ’ Source 100 ’ IO

PNU/ml. FOR I+ SKIN REACTION

FIG. 9. A, Skin sensitivity of 9 allergic patients and 12 controls to mixed Hymenoptera

extracts from three commercial sources. B, Skin sensitivity of 8 allergic patients and 6

controls to individual whole body extracts from three commercial sources. No difference was noted between skin sensitivity to the insects to which the patient was allergic, as

judged by histamine release, and those to which, by the same criterion, the patient was

not allergic.

extracts to distinguish allergic from normal patients. This failure has been reported by numerous investigators including Schwartz,4 Bernton and Brown,5 and Mueller,*O and is probably due to the nature of the extracts. At low concentrations the whole body extracts may contain too little venom to induce a response or, perhaps more likely, the venom proteins may be unstable on storage. At higher concentrations, on t,he other hand, toxic venom proteins, such as the histamine-releasing mellitin or the vasoactive amines of the venoms, such as histamine and serotonin, or the vasoactive peptide bradykinin, can cause a positive response in normal skin.

Whatever the cause, the literature clearly demonstrates that there is sufficient overlap in the concentration of whole body extracts which will induce positive skin tests in atopic and normal man to make this procedure diagnostically use- less. Our results confirm this previous work. There was no significant difference between the concentration of whole body extract required to induce a positive reaction in the 13 selected Hymenoptera-allergic patients versus the 12 controls. This was true with mixed whole body extracts obtained from 3 different manu-


facturers as well as with the specific whole body extracts, likewise obtained from several commercial sources.

Using venom preparations and in vitro histamine release, however, there was w overlap between the normal group and the allergic group. All patients responded to 1 7 per milliliter of venom or less, and most responded well to 0.1 to 0.001 y per milliliter of this material. On the other hand, no normal patient had leukocytes which released histamine on challenge with up to IO y per milliliter of the various venoms. We do not feel that this diagnostic advantage is due to the use of the in vitro technique, but rather reflects the superiority of venoms to whole body extracts. Our opinion is based on extensive experience comparing histamine release and skin test studies using grass and ragweed extracts and fractions in allergic and normal patients : the correlation between the two procedures is extremely high. 21 It follows from this that skin testing with venoms should be evaluated. This is not currently possible because these materials, with the excep- tion of honeybee venom, have not been approved by the Food and Drug Ad- ministration and honeybee venom was approved only recently.

We have not yet begun to study the allergenic components of the venoms Simple dialysis does not diminish their potency as antigens, so that the major allergen must have a molecular weight of at least 5 to 10,000. Since dialysis re- moves the vasoactive amines and some of the toxic peptides, it seems likely that dialyzed venoms will be most appropriate for skin testing.

We have no ready explanation for the 3 patients judged clinically to have suffered systemic reactions following an insect sting who failed to evidence a positive in vitro test. Two of these were unable to positively identify the stinging insect and, as we are currently testing with but a limited panel of venoms, it is possible that they would have reacted specifically to other venoms (i.e., Polistes) . We do not, however, feel that all responses to stings are IgE-mediated and are presently content to conclude that the great majority of reactions involve this mechanism.

The patterns of the patients’ reactions to the various venoms are also of interest. It would appear that there is only limited cross-reactivity between vespid and honeybee venoms in our study. The widely accepted notion that clinical hypersensitivity may result from exposure to an antigen shared by all

of the Hymenoptera is primarily based on studies involving antisera raised in laboratory animals.8, 9, I1 These studies are of interest but, unfortunately, not strictly relevant to human hypersensitivity. Passive transfer and cross-neutrali- zation studies in man by Loveless and Fackler,Zza using crushed venom-sac solutions, support our view that there is little cross-reactivity between honeybee and vespid venoms. Studies by Langlois, Shulman, and ArbesmanlO in human skin with whole body extracts are less conclusive but suggest a similar interpretation. In our study, 7 patients reacted only to the yellow jacket/hornet family of venoms and 3 patients reacted only to honeybee venom. Although 3 patients reacted to both venoms, they were evidently far more sensitive to the former. This puts into question the common practice of skin testing and treating with a mixture of Hymenoptera extracts prepared from the several families,

The in vitro histamine release reaction used in these experiments has been

VOLUME 53 NUMBER 3


studied extensively ; it has not been possible to demonstrate that antibodies of any class other than IgE can mediate leukocyte histamine release. This strongly

implies that IgE is involved in the reactions herein reported. This conclusion is strengthened by the passive sensitization experiments, in which the responsible antibody was removed by heating or by passage through an anti-IgE immunosorbent. This argument is not weakened by the failure of several sera to cause passive sensitization. The ability of sera to passively sensitize normal cells for histamine release is based on the level of sensitivity of the patient. Many adult ragweed- and grass-sensitive patients similarly fail to passively sensitize leukocytes,22 and these patients are often more sensitive (as judged by the concentration of antigen required to elicit histamine release) than most of our Hymenop- tera-allergic patients.

This study has not addressed itself to evaluating the treatment of Hymenop- tera-sensitive patients. This is in progress. The most critical goal is to ascertain whether immunotherapy leads to blocking antibodies, and to determine what constitutes a protective level of blocking antibody. The in vitro system has been used for this purpose in pollen allergies,23 and preliminary studies indicate that blocking antibodies to Hymenoptera venoms are easily detectable with this procedure.

As a result of this study, we are able to conclude that most systemic reactions to Hymenoptera stings are mediated by IgE antibodies and that sensitive patients can be easily and clearly distinguished from normals with the use of pure venoms and leukocyte histamine release. We do not doubt that the venoms

will be of equal diagnostic utility in skin testing and urge that these materials be made generally available so that this hypothesis can be properly tested.

REFERENCES

1

2

3

4 5

6

7 8

9

10

11

12

13

Parish, H. M.: Analysis of 460 fatalities from venomous animals in the United States, Am. J. Med. Soi. 245: 129, 1965. Chafee, F. H.: Prevalence of bee sting allergy in an allergic population, Acta Allergol. 25: 292, 1970. Settipane, G. A.: Prevalence of bee sting allergy in 4,992 Boy Scouts, Acta Allergol. 25: 286, 1970. Schwartz, H. 5.: Skin sensitivity in insect, J. A. M. A. 194: 703, 1965. Bernton, H. S., and Brown, H.: Studies on the Hymenoptera. I. Skin reactions of normal persons to honey bee (Apis mellifera) extract, J. ALLERQY 36: 315, 1965. Benson, R. L., and Semenov, H.: Allergy in its relation to bee sting, J. ALLERGY 1: 105, 1930. Habermann, E.: Bee and wasp venoms, Science 177: 314, 1972. Foubert, E. L., Jr., and Stier, R. A.: Antigenic relationships between honeybees, wasps, yellow hornets, black hornets, and yellow jackets, J. ALLERGY 29: 13, 1958. Arbesman, C. E., Langlois, C., and Shulman, S.: The allergic response to stinging insects. IV. Cross-reactions between bee, wasp, and yellow jacket, J. ALLERGY 36: 147, 1965. Langlois, C., Shulman, S., and Arbesman, C. E.: The allergic response to stinging insects. II. Immunologic studies of human sera from allergic individuals, J. ALLERGY 36: 12, 1965. Langlois, C., Shulman, S., and Arbesman, C. E.: The allergic response to stinging insects. III. The specificity of venom sac antigens, J. ALLERGY 36: 109, 1965. Shulman, S., Bigelson, F., Lang, R., and Arbesman, C. E.: The allergic response to stinging insects, biochemical and immunologic studies on bee venom and other bee body preparations, J. Immunol. 96: 29, 1966. Dirks, T. F., Jr., and Sterburg, J. G.: Non-immunochemical complexing between Hymenop- teran venoms and rabbit serum, Toxicon 10: 381, 1972.


14 Levine, M. I.: Insect stings, J. A. M. A. 217: 964, 1971. 15 Insect Allergy Committee of the American Academy of Allergy. Insect sting allergy

questionnaire study of 2,606 cases, J. A. M. A. 193: 109, 1965. 16 Benton, A. W., Morris, R. A., and Stuart, J. D.: Venom collection from honey bees,

Science 142: 228, 1963. 17 Lichtenstein, L. M., and Osler, A. G.: Studies on the mechanisms of hypersensitivity

phenomena. IX. Histamine release from human leukocytes by ragweed pollen antigen, J. Exp. Med. 120: 507, 1964.

18 May, C. D., Lyman, M., Alberto, R., and Cheng, J.: Procedures for immunochemical study of histamine release from leukocytes with small volume of blood, J. ALLERGY 46: 12, 1970.

19 Levy, D. A., and Osler, A. G.: Studies on the mechanisms of hypersensitivity phenomena. XIV. Passive sensitization in vitro of human leukocytes to ragweed pollen antigen, J. Immunol. 97: 203, 1966.

20 Mueller, H. L.: Insect allergy, in Samter, M., editor : Jmmunologieal disease, ad. 2, Boston, 1971, Little, Brown & Company.

21 Lichtenstein, L. M., Norman, P. S., and Connell, J. T.: Comparison between skin-sensitizing antibody titers and leukocyte sensitivity measurements as an index of the severity of ragweed hay fever, J. ALLERGY 40: 160, 1967.

22 Levy, D. A., and Osler, A. G.: Studies of the mechanism of hypersensitivity phenomena: In vitro assays of reaginic activity in human sera: Effect of therapeutic immunization on seasonal titer changes, J. Immunol. 99: 1068, 1967.

22a Loveless, M., and Fackler, W. R.: Wasp venom allergy and immunity, Ann. Allergy 14: 347, 1956.

23 Lichtenstein, L. M., and Osler, A. 0.: Studies on the mechanisms of hypersensitivity phenomena. XII. An in vitro study of the reaction between ragweed pollen antigen, allergic human serum and ragweed sensitive cells, J. Immunol. 96: 169, 1966.

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