Immunological and biochemical studies in beekeepers and their family members

mmunological and biochemical studies beekeepers and their famil . W. Yunginger, M.D., R. T. Jones, B.S., K. . W. Welsh, B.A., and G. J. Gleich, M.D. Ro

. Leiferman,

B.S.,

To determine the immunological and biochemical consequences of chronic honeybee venom administration to humans, we studied beekeepers and their family members in two separate investigations. In the first of these studies serums were analyzed for their content of antibodies to honeybee constituents. IgE antibody levels to honeybee venom (HBV), venom sac (HBVS), and phospholipased (PLA) were significantly elevated in beekeepers and their family members as compared to non-beekeeper controls. However, IgE antibody levels to HBVS, HBV, and PLA did not distinguish sting-sensitive from non-sensitive beekeepers. IgE antibody levels to honeybee whole body (HBWB) were highest in beekeepers with histories suggestive of inhalant sensitivity to bee body dust. IgG antibody levels to PLA were highest in non-sensitive beekeepers stung daily or weekly; there was a significant positive correlation between IgE and IgG antibody levels to PLA in these beekeepers. In the second study the potentially harmful effects of chronic venom administration were sought, especially evidence of chronic renal disease. While a number of beekeepers and family members showed minor abnormalities in their urinalysis or blood chemistry determinations, in most instances the incidence of abnormalities was not greater among persons receiving a larger number of stings. Commercial beekeepers had a sign$cantly greater incidence of urinary free lipids and hyaline casts, but in no case were large amounis of these substances found. The results discourage the belief that chronic honeybee venom administration is associated with any clinical disease in humans.

Although venom immunotherapy has been used by Loveless1 for more than twenty years, only recently has it been tested by others.2* 3 These recent reports have provided data on the immunologic consequences of venom immunotherapy and have reawakened interest in its use. ‘Honeybee venom is the best-characterized of the Hymenoptera venoms,4 and the allergenicity of certain of its components has been reported recently.5 We measured the levels of serum IgE and IgG antibodies to honeybee venom and

venom components and investigated the consequences of chronic honeybee venom ad~nistra~on to humans. In order to discover if any subclinical biochemical, hematological, or renal abnormalities were present in persons who had been stung repeatedly by honeybees for many years, we studied beekeepers and their family members. MATERIALS AND METH Studies of beekeepers a Two separatestudies were conducted in 1974 and 1976. In both years we attended the summer meetings of the Mlnnesota Beekeepers Association, where we interviewed as many beekeepers and their family members as possible. During 1974 we obtained serum samples from approximately 50% of the registrants at the summer meeting. Our intent was to secure serums that could be expected to contain large quantities of either IgG antibody (frequently stung, non-sensitive beekeepers) or IgE antibody (stingsensitive family members). One hundred thirty-two serums were collected; patients were assigned to either a nonsensitive group or a sting-sensitive group based on their clinical history. Patients reporting the development of generalized urticaria, angioedema, laryngospasm, wheezing, or vascular collapse following bee stings were considered

From the Departments of Pediatrics, Internal Medicine, and Immunology, and the Allergic Diseases Research Laboratory, Mayo Medical School, Mayo Clinic, and Mayo Foundation. Supported in part by a grant (AI-l 1483) and contract (AI-42546) from the National Institute of Allergy and Infectious Diseases; a contract from the Food and Drug Administration (223-77-1202); and by Mayo Foundation. Dr. Yunginger is the recipient of an Allergic Diseases Academic Award (AI-00107). Presented in part at the Thirty-first Annual Meeting of the American Academy of Allergy, San Diego, Calif., Feb. 17, 1975. Received for publication June 16, 1977.

Accepted for publication Nov. 1, 1977. Reprint requests to: J. W. Yunginger, Research Laboratory, Mayo Clinic,

QOW-6749/78/0261-0093$00.90/O

M.D., Allergic Rochester, Minn.

0

1978

The

Diseases 55901.

C. V. Mosby

Co.

Vai.

61, No.

2, pp. 93-101

Yunginger

E 1. Clinical

T

et al.

characteristics

J. ALLERGY CLIN. IMMi!NOL. FEBRUARY 1978

of the 1974 study groups Beekeepers

and family

Non-sensitive

Total Male

Female Occupation Active beekeepers Beekeeper family members Bee contact No bee contact Other (entomologist, honey packer, retired, etc.) Coexisting asthma or hay fever *Four persons in this group were presently receiving mixed Hymenoptera such treatment in the past. Twelve persons in this group had reacted patients the sting reaction had occurred more than one year previously.

sting-sensitive.* A group of ten non-atopic laboratory workers were also studied as a non-beekeeper control group. The characteristics of the 1974 study groups are shown in Table I. During the 1976 summer meeting (when beekeepers were regularly being stung), we secured blood and urine specimens from beekeepers and their family members to determine if any hematological, biochemical, or renal abnormalities were associated with chronic bee venom administration. Seventy-two blood specimens and 68 urine samples were obtained; patients were classified into five groups on the basis of their occupation and degree of contact with bees. Blood chemistry determinations were done on fasting specimens and included sodium, potassium, calcium, phosphorus, total protein, glucose, alkaline phosphatase, glutamic-oxaloacetic transaminase (GOT), total bilirubin, uric acid, creatinine, and direct bilirubin tests. A complete biood count and leukocyte differential were also obtained. A freshly voided urine specimen was tested for specific gravity, pH, glucose, and protein; the urine sediment was also examined. Urine tests were performed within one hour after collection. Blood tests were performed in the Mayo Clinic laboratories within 6 to 32 hr after collection; plasma and serum were isolated within one hour after collection and kept refrigerated at 4” C until assayed. Informed consent was obtained from all volunteers in both studies. History forms were completed which noted age, sex, occupation, past medical history, and reactions (if any) to bee stings, along with the number and frequency of bee stings.

*One beekeeper reported experiencing generalized urticaria following the first few stings he received each spring. However, he was able to tolerate multiple stings during the summer and autumn months with no systemic symptoms. For statistical purposes he was considered to be non-sensitive.

members

Sting-sensitive

91 14

27” 3 24

91

1

6 3 5 22

25 0 8

105

whole

~~~~b~ek~~p~~a 10

5 5

1

body

immunotherapy,

0 and 5 other patients

bad received

to a bee sting within the previous year, while in the remaining 15

Reagents 1. Honeybee allergens. Honeybee whole body (HBWB) extract was purchased as a commercial aqueous 1: 10 dilution (Greer Laboratories, Inc., Lenoir, N. 6.). Phospholipase-A (PLA) purified from bee venom (Sigma Chemical Co., St. Louis, MO.) was prepared at 2.5 mg/ml in 0.2 M HaBOa-0.04 M NaOH-0.16 M NaCl (boratesaline, pH 8.0). Honeybee venom (HBV) (Champlain Valley Apiaries, Middlebury, Vt.) was prepared at 25 mg/ml in borate-saline; a few hairlike particles and some insoluble gray residue were removed by centrifugation. Honeybee venom sac material was included as a test preparation which would theoretically contain intermediate quantities of venom and body antigens as compared to venom itself on one hand and whole body extract on the otber. Honeybee venom sacs (HBVS) (Davis Insect Venoms, Gshawa, Ontario, Canada) were homogenized in borate-saline (41 mg/ml) for 6.5 min at 15,000 rpm in a model 60M Virtis homogenizer (The Virtis Company, Inc., Eardiner, N. 79.). Particulate matter was removed by centrifugation, and the protein contents of the HBV and HBVS supernatant fluids were measured by the biuret reaction using human serum albumin as the standard.6 Serum IgE antibodies to HBVS, HBV, and PLA were measured by the radioallergosorbent test (RAST) using allergens coupled to microcrystalline cellulose. The quantities of honeybee allergens reacted per gram of cellulose were 9.2 ml (HBWB); 121 mg protein (HB VS); 169 mg protein (HB ‘17); and 10 mg (PLA) Coupling yields were calculated from spectrophotometric absorbance measurements of coupling supernatants and washings at 280 nm for the latter three allergens, and were 71%, 83%, and 94%, respectively. The RAST was performed as previously reported7; results were expressed as the percent of total radioactive counts bound to the RAST polymer. 2. IgG antibody. Serum IgG antibodies to PLA were measured by radioimmunoprecipitationa~ g PLA (1 mg) was radiolabeled with r311 (950 ,uCi) by the chloramine-T pro-

VOLUME 61 NUMBER 2

Serum ~JI C 0

ICKI

hay fever

2.5 1.25 0.62 I 4

13’I-PLA added, yg

0’

cl

I

I

I

I

I

I

1

2

3

4

5

6

Serum,yl FIG. 1. :Measurement of IgG antibody to PLA.A, Determination of maximum binding values. B, Relationship between maximal binding levels and quantity of serum. The correlation coefficient (r) = +a.99.

cedurel”; unreacted radioiodine was removed by gel filtration through Sephadex G-25 which was equilibrated with 1% bovine serum albumin (BSA) in 0.1 M phosphate buffer (pH 7.5). Void volume fractions were pooled and dialyzed for 3 days at 4” C against 0.15 M NaCl. Phosphotungstic acid precipitated 97.6% total radioactive counts in this preparation, which was then diluted with additional 1% BSA. A standard IgG antibody pool was prepared using equal quantities of serum from five non-allergic, frequently stung beekeepers. This pool was diluted to 1: 20 using 1% BSA; further two-fold dilutions were made to 1: 1,280, using a 1: 20 dilution of a normal non-beekeeper control serum as the diluent. Radiolabeled PLA was added to 100 ~1 patient serum (1: 20) or to 100 ~1 of the standard IgG antibody pool (7 points from 1: 20- 1: 1,280) and diluted to 0.3 ml with 1% BSA in 10 x 75 mm glass tubes. Tubes were incubated for 1 hr at 37” C, then overnight at 4” C, after which 0.25 ml of burro anti-IgG (Fc) was added. After a second incubation for 1 hr at 37” C and overnight at 4” C, the resulting precipitates were mixed throroughly with 0.5 ml 0.1 M phosphate buffer (pH 7.5) to break up the precipitate. An additional 2.5 ml 0.1 M phosphate buffer was then added and the tubes were centrifuged; three such washing steps were used. The radioactivity associated with the washed precipitates was counted in a gamma spectrometer. As shown in Fig. 1, A, addition of increasing amounts of radiolabeled PLA resulted in the formation of plateau or maximum binding regions. As shown in Fig. 1, B, the

FIG. 2. IgE antibody

levels to honeybee venom sac in beekeepers and non-beekeepers. in this and subsequent figures, “non-sensitive beekeepers”’ are primarily persons actively involved with beekeeping on a day-to-day basis. “Sensitive beekeepers” are, with one exception, beekeeper family members ratherthan active beekeepers. See Table I for details. Geometric mean antibody levela for each group are indicated by horizontal bars in Figs. 2 to 6.

quantity of PLA bound was linearly related to the quantity of serum analyzed. In the assay of serum from the patients, 1.5 pg of radiolabeled PLA was added to 5 ~1 patient serum. Our standard IgG antibody pool contained 347 fig antibody to PLA per milliliter, as calibrated against a standard serum in an immunoprecipitation experiment at Johns Hopkins University. * IgG antibody levels to PLA were consequently expressed as pg antibody/ml .

Statistical

analyses

In the 1974 study the relationship between IgE and IgG antibody levels in the patient groups was examined using the Spearman rank correlation test, while the IgE antibody levels in the various patient groups were compared using the Mann-Whitney rank sum test. Geometric mean IgE antibody levels between groups were compared using the t test. In the 1976 study the incidence of selected blood and urine abnormalities in patient subgroups was tested by x2 analysis.ll A programmable Hewlett-Packard 9810A calculator was used for the statistical evaluations. *We thank Dr. L. M. Lichtenstein and Dr. A. I(. Sobotka for their kindness in performing this calibration.

J. ALLERGY Cl-IN. IMtWJNQL. FEBRUARY 1978

40.0

30.0 8Nonatopic 2 20.0 I 0 Asthma / hoy fever 2

Beekeepers

/

Nonsensitive

/

n=l05 FIG. 3. beekeepers

IgE

antibody levels and non-beekeepers.

50.0 / 40.0 *Nonatopic 30.0 oAsthmo/hay

to

honeybee

venom

Non-

in

onsensitize i=i=lO CT*

fever

0

FIG.

5. IgE antibody

beekeepers 2 0 0 0. -7

. : 1 .. ..*. .. .

BgE antibody levels and non-beekeepers.

owes

l. .

Nonleekeepers Nonsensitive n=lO

Beekeepers

FIG. 4. beekeepers

.‘.’

to

phospholipase-A

in

in beekeepers

In 1974 we determined the responsiveness of beekeepers and their family members to a variety of honeybee antigens. IgE antibody levels to HBVS, HBV, and PLA are shown in Figs. 2 to 4, respectively. In all cases the geometric means of the IgE antibody levels in both beekeeper groups were significantly elevated as compared to the non-beekeeper control group (p < 0.001). Although the mean IgE antibody level in the sensitive beekeeper group was higher in each case than that of the non-sensitive beekeeper group, the difference was significant only with respect to the HBV (p < 0.025). Within the

levels

to

honeybee

whole

body

in

and non-beekeepers.

sting-sensitive beekeeper group patients with coexisting asthma or hay fever had significantly higher IgE antibody levels to HBVS, HBV, and PLA than did their non-atopic, sting-sensitive cohorts (p < 0.01). Within the nonsensitive beekeeper group, atopic patients had significantly higher IgE antibody levels only to HBVS (p < 0.001). We were unable to demonstrate a significant correlation between IgE antibody level and the interval since the last sting reaction in the sting-sensitive group. Finally, there was a highly significant correlation among an i~divid~al~s IgE antibody levels to HBVS, HBV, and PLA in both the sting-sensitive and non-sensitive beekeeper groups. The IgE antibody levels to HBWB are shown in Fig. 5. Once again, there was a significant difference in mean IgE antibody levels between both beekeeper groups as compared to the non-beekeeper controls (p < 0.01). Of note was the relatively large number of non-sensitive beekeepers with elevated IgE antibody levels to HBWB. Moreover, it was apparent that the atopic members of the non-sensitive group (having asthma or hay fever) had much higher BOWL IgE antibody levels than their non-atopic cohorts. Two of these atopic beekeepers had volunteered the information that they experienced asthma when cleaning out hives or when extracting honey; both individuals had high IgE antibody to HBWB. Each of the 22 atopic members of the non-sensitive group were subsequently queried by mail questionnaire; 14 stated that their rhinitis or asthma symptoms were worsened

VOLUME 61 NUMBER 2

tudies

l* . . . . l

in beekeepers

500.0

loo.0

. . .

9. .

. .

. . . 0.. %

a.5

.

.

Group with Group with AIHF AIHF f rom bee dusit n-22 n=l4

ma-hay

IgE antibody levels ups of non-sensitive fever.

to

honeybee beekeepers.

whole body in AiHF = asth-

by exposure to bee body dust; 5 patients denied such exacerbations; and 3 persons did not respond to the questionnaire. The IgE antibody levels to HBWB in these subgroups of beekeepers are shown in Fig. 6. A significant elevation in mean HBWB IgE level was seen in the beekeepers reporting a clinical history of inhalant sensitivity to honeybee body dust as compared to the non-atopic beekeeper group (p < 0.001). The PgG antibody levels to PLA are shown in Fig. 7. As the assay was performed, the lower limit of sensitivity was 0.2 p&/ml. IgG antibody levels were markedly elevated in the non-sensitive beekeeper group as compared to the sting-sensitive group. Within the former group, mean IgG antibody levels were significantly elevated in the beekeepers who were stung daily or weekly as compared to the mean IgG antibody levels in the beekeepers who were stung monthIy or annually (t test, p < 0.001). The atopic members of the non-sensitive beekeeper group did not have significantly different IgG antibody levels than their non-atopic cohorts. There was a highly significant positive correlation between IgG and IgE antibody levels to PLA in the non-sensitive beekeeper group (rS = +0.36, p < 0.0005). Measurable IgG antibodies to PLA were found in only 6 of the stingsensitive persons; therefore, no correlations between IgG and IgE antibodies to PLA could be made in this group. 1

$~~~~

In this study we attempted to identify any potentially harmful effects of chronic venom administration. The 72 patients were grouped according to oc-

FIG. 7. IgG antibody levels to ~hosphol~~~s~-A in both beekeeper groups, noting the frequency with which individuals were stung. o = persons stung daily or weekly: . = persons stung monthly; X = persons stung once or twice yearly or less.

cupation and the degree of exposure to bees; the clinical characteristics of the study groups are shown in Table II. The blood chemistry findings are listed in Table III. The blood Na+, K+, and Ca+’ elevations probably represent biological and analytical vtialions which were probably of no clinical consequence. Thirteen of the 20 elevations in blood glucose values were 3 mg/dl or less; the highest value of 113 mgldl was measured in a known diabetic patient. Five of the 7 elevations in GOT were 4 U/L or less; the value of 40 U/L was measured in a woman taking desipramine HC I, a drug which has been reported to cause elevated serum transaminase values. Two of the 5 elevations in uric acid were measured in patients reporting either gout or arthritis. Finally, 16 of the 21 elevations in creatinine values in men, and 8 of the 9 elevations in creatinine values in women, were 0.2 mg/dl or less. The 2.9 mgldl value was measured in a hobby beekeeper with known renal disease. The urinalysis findings are listed in Table IV; 4 beekeepers did not submit urine specimens. The specimen containing glucose and reducing substances was from a commercial beekeeper with a fasting blood glucose of 111 mg/dl. A repeat urinalysis in his home community was entirely normal: but a repeat fasting blood glucose was 120 mg/dl; the patient is probably a chemical diabetic. Nine of the 1L specimens containing protein showed only grade 1 pro-

J. ALLERGY CLIN. IMMUNOL. FEBRUARY ?978

T

Ii. Clinicai

characteristics

of the 1976 study groups Contact

roup

(male/female)

-

with

bees

Mean

Active beekeeper (57 / 1) Commercial (45 / 1) Hobby (1210) Beekeeper family member (2/ 10) Bee contact (O/4) No bee contact (2/6)* Retired beekeeper (2/O)

(yr ) Range

Stings

per day

Mean

23 12

2-57 l-50

10 1

O-X! o-2

11 0 48

3-30 0 41-55

2 0 0

O-8 0 0

*Includes 4 persons with systemic anaphylaxis after honeybee stings load chemistry

results

in 72

TABLE

IV. Urinalysis

results

in 68 beekeepers*

beekeepers Outside 95% interval Units

Sodium Potassium

Calcium Phosphorus Total protein Glucose Alkaline phosphatase Male Female COT Total bilirubie Uric acid Male Female Creatinine Male Female Direct bilirubin

mEq/L mEq/L mgidl mgldl Gmidl mg/dl

95% confidence interval-adults

135-145 3.6-4.8 8.9-10.1 2.5-4.5 6.2-8.0

70- 100

No.

Range

11 146-148 2 4.9-5.0 1 10.2 0 3 8.1-8.3 20 101-113

U/L

mgidl mg/dl

5 1 1 7

256-339 79 210 21-40

0.1-1.1

4

1.2-1.6

4.3-8.0 2.3-6.0

4 3 1

8.1-9.0 3.6-3.9 7.1

0.8-1.2 0.6-0.9

21 9

1.3-2.9 1.0-1.2

Neg.

0

76- 196 8-20

mgidl

the +3 protein specimen was obtained from the hobby beekeeper with known renal disease. Ten of the 18 specimens containing RBCs had only 1 RBC per high-power field. Occasional urinary casts and/or occasional urinary free lipids were noted in a relatively high percentage of beekeepers. Hematologic tests were unremarkable in most patients Four patients had leukocyte counts in excess of 10,0Q0/mm3. Leukocyte differential counts and RBC indices were unremarkable, and all hemoglobin determinations were normal.

teinuria;

Determination

Glucose (enzyme) Sugar (Benedict’s)

Protein

NO.

1

ange

11

Positive +2 4-I - +3

18

I-50

1

Microscopic

Erythrocytes Leukocytes Male Female Bacteria

5 3 2

4-12” 8-750” Present

Casts 90-239

U/L

Ab~ormai~tiee

-

Hyaline Granular Free lipids Oval bodies

29 21 9 1

Occasional Occasional Occasional Occasional

*Less than 4 leukocytes per high-power field in males and less than 8 leukocytes per high-power field in females were considered normal.

To determine if there was an association between multiple bee stings and the development of any specific disease state, blood abnormality, or urine abnormality, the incidence of selected abnormalities in each patient subgroup was tested by x2 analysis; the results are shown in Table V. The incidence of proteinuria was significantly less in commercial beekeepers and significantly increased in hobby beekeepers. However, the occurrence of hyaline casts was seen more often in commercial and retired beekeepers. The occurrence of urinary free lipids was seen exclusively in the commercial and retired beekeeper groups. We examined the question of whether or not proteinuria, hematuria, and elevated serum creatinine values were occurring randomly in the study groups or were associated with one another. There was a significant association between proteinuria and hematuria (x2 = 5.31, p < 0.025); how-

VOLUME 61 NUMBEI? 2

Studies

Clinical,

blood

chemistry,

and urine

abnormalities

among Patient

beekeeper

~~~am@ter§

Total

History of arthritis

IO

fBlood

11

Na+

TBlood uric acid TBlood glucose ?Blood creatinine ~Alkaline phosphatase Pmteinuria Pyuria (males)

THyaline casts TGranular casts Free lipids

5 20 30 6 11 5 29 21 9

~ubgro~~$

group

Beekeepers Test

Family

Commercial

Hobby

Retired

7 9 4 12 15 2 2 2 22 15 7

2 1 0 2 6 2 4 2 4 3 0

0 0 0 0 1 1 1 1 2 2 2

in beekeeper5

members

Bee contact

No bee contact

1 0 1 3 3 0 1 0 0 0 0

value

0 1 0 3 5 1 3 0 i 1 0

NS” NS NS NS NS NS CO.01 NS
*NS: not significant (p > 0.05).

ever, there was no significant association between elevated serum creatinine and either proteinuria or hematuria. Finally, patients were grouped into 5 categories based on average number of stings received per day, rather than occupation or degree of contact with bees; we could not demonstrate any significant relationship between the abnormalities listed (Table VI) and the frequency of stings. SSIQ We have shown that beekeepers and their family members exhibit significantly elevated IgE antibody levels to all four honeybee components tested, as compared to non-beekeeper controls. The mean IgE antibody level to HBV was significantly higher in the sting-sensitive group than in the non-sensitive beekeeper group, although there was much overlap in HBV IgE antibody levels between patients in these groups. Therefore, an elevated serum IgE antibody to HBVS, HBV, or PLA by itself is not diagnostic of sting sensitivity in beekeepers or their family members. Non-sensitive beekeepers with clinical histories suggestive of inhalant sensitivities to bee body dust showed elevated serum IgE antibodies to honeybee whole body components. Inhalant sensitivity to bee body dust was first reported over 40 years ago,12 but has apparently received little notice in recent years. The HBWB RAST and the results of our questionnaire survey suggest that occupational exposure to honeybee body dust results in the inhalant sensitization of many beekeepers. Further studies involving skin testing and inhalation challenge testing are necessary to establish this point, however. In previous studies whole body extracts were not found to be useful in diagnostic skin testing for sting sensitivi-

TABLE VI. Clinical, blood chemistry, abnormalities in patient subgroups to sting frequency*

and urine according

Mean

daily

10 History of arthritis 11 TBlood Na+ TBlood uric acid 5 20 TBlood glucose TBlood creatinine 30 TAlkaline phosphatase 60

2

4

0

3

1

3 1

2 3

2 0

2 0

2 1

5 6

3 I

2 2

5 IO

5 11

Proteinuria Pyuria (males) THyaCnecasts TGranular casts Free lipids

2

0

0

3

6

6 2 5 2 2322

10 3

5 5

II

5 29 21 90

1023 11012 6 6

*There was so significant association in any of the subgroups. tY. 13, I4 Our findings are consistent with these

ous studies, in that we found no difference in I@ antibody levels to whole body materials between the sting-sensitive and non-sensitive beekeeper groups. The sting-sensitive group in the 1974 study was a heterogeneous group. Four sting-sensitive persons were receiving mixed Hymenoptera whole body immunotherapy, and 5 had received such therapy in the past. However, the IgG antibody ievels to PLA in these patients were not significantly different from those of the other 1X sting-sensitive persons; neither were any of the IgE antibody levels different in the whole body-treated patients. Because previous serum specimens were not available from these persons, the effect of whole body hyposensitization on the IgE and

J. ALLERGY CLIN. IMMUNOL. FEBRUARY 1978

IgG antibody levels in these patients must remain conjectural. Serum IgG antibodies to PLA were highest in the non-sensitive beekeepers who had been stung most frequently. Moreover, there was a significant positive correlation between IgG and IgE antibodies to PLA in this group. We had previously noted a correlation between IgG and IgE antibodies to ragweed antigen E in hyposensitized ragweed-sensitive patients.$ The finding in both studies that IgG and IgE antibody levels were positively correlated suggests that IgG antibodies do not serve to “turn off” IgE antibody production. The IgG antibody levels in our nonsensitive beekeeper group were comparable to those reported by Light and co-workers, who used a competitive binding radioimmunoassay to measure total serum antibodies to PLA in 34 beekeepers.15 However, our findings differ from those of Light and co--workers16 in that we found elevated levels of IgE antibody to HBV and PLA in almost all beekeepers, regardless of their clinical reactions to bee stings. Clinical experiments involving venom immunotherapy have been hampered in part by the scarcity of some venoms and by the Food and Drug Administration (FDA) proscription of interstate shipment of venoms for in vivo therapeutic use. Persons who apply for Investigational New Drug notices for the therapeutic use of honeybee venom are requested by the FDA to supply experimental data on long-term safety experiments in animals. The 1976 study was undertaken to secure data from beekeepers who had received numerous bee stings for many years. We felt that it was unnecessary to reproduce in laboratory animals the same “experiment of nature” that has been occurring in humans since man began beekeeping centuries ago. However, we are unaware of any long-term hematological or immunological studies in beekeepers. It has been recognized for some time that serums from beekeepers contain antibodies against PLA17 and hyaluronidase, I* but in the latter instance, the IgG antibodies were specific for bee venom hyaluronidase and did not react with hyaluronidase derived from two human sources (testis and synovium). lg Many persons have received long-term honeybee venom injections for treatment of arthritis; unfortunately most of these patients have not been monitored with tests of hepatic, renal, or hematological functions.20 An article in the French literature reports the administration of bee venom to 14 patients for treatment of arthritis. 21 Patients were monitored before and after therapy with blood counts, urinalyses , serum cortisol levels, serum calcium levels, and venom skin tests. No striking abnormalities were noted in these patients, who received

cumulative venom doses of 3 to 4 mg with maximal single injections of 70 pg. The average honeybee sting contains about 50 pg solids.2 The overall incidence of urine abnorm~ities and elevated serum creatinine values in the 1976 study appears quite high. However, the magnitudes of increase of many of these abnormalities were small. For example, the elevations in serum creatinine were 0.2 mg/dl or less in 24 of 30 patients with values above the normal range. Similarly, 9 of 11 urine specimens containing protein showed only grade 1 proteinuria, and 10 of 18 urine specimens containing erythrocytes had only one erythrocyte per high-power field. Chronic venom administration could conceivably produce an immune-complex type of kidney disease, but if so the degree of proteinuria and hematuria suggests that the effects are not marked. We postulated that if any subclinical untoward effects were produced by long-term honeybee venom administration, these effects would be most marked in commercial beekeepers, who receive many stings daily. Indeed, there was a significant increase in the incidence of free urinary lipids in commercial and retired beekeepers, as well as an increase in urinary hyaline casts of borderline significance. We emphasize, however, that only occasional casts and lipids were found in all urines tested. While there was a significant association between the presence of hematuria and proteinuria in the 1976 study, the association between elevated serum creatinine and either hematuria or proteinuria appeared random. When patients were grouped according to the frequency with which they were stung rather than by occupation, the incidence of blood and urinary abnormalities was similar in persons stung frequently and in persons who did not receive many bee stings. On the basis of this ‘“onetime” study of a large number of beekeepers, it seems unlikely that chronic honeybee venom administration produces clinical disease in humans. We stress, however, that additional studies employing more control groups are necessary to exclude the presence of subclinical renal abnormalities in beekeepers. We are indebted to Ms. Jane Zubler and Ms. Carol White for technical aSsistaxe, and to J. V. Donadio, Jr., M.D.,

Division of Nephrology, Department of Internal Medicine, for reviewing the manuscript. We also ihank the officers and members of the Minnesota Beekeepers Association, whose enthusiastic support made these studies possible. REFERENCES 1. Loveless, M. H., and Fackler, R. W.: Wasp venom allergy and immunity, Ann. Allergy 14~347, 1956. 2. Lichtenstein, L. M., Valentine, M. D., and Sobotka, A. I<.: A case for venom treatment in anaphylactic sensitivity to Hymenoptera sting, N. Engl. J. Med. 29&l 123, 1974.

VOLUME 61 NUMBER 2

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