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Tissue, Tear, and Serum IgE Concentrations in Vernal Conjunctivitis
TISSUE, TEAR, AND SERUM IgE CONCENTRATIONS IN VERNAL CONJUNCTIVITIS M A T H E A R. ALLANSMITH,
M.D.
Boston, Massachusetts GARY S. H A H N ,
B.S.
Stanford, California AND M E R E D I T H A. S I M O N ,
M.S.
Boston, Massachusetts
We postulated that perhaps vernal con junctivitis had as part of its mechanism a localized hyperplasia of IgE plasma cells in the cobblestone excrescences; and also that perhaps locally produced IgE raised the tear IgE level. To evaluate these pos tulations, tarsal conjunctiva from patients with vernal conjunctivitis was stained for all immunoglobulins, and IgE was meas ured in tears and serum of patients and controls. M A T E R I A L AND M E T H O D S
Subjects—Eleven patients with vernal conjunctivitis were selected for the tearserum portion of the study on the basis of availability. All had severe itching, tear ing, disease for more than two seasons, typical cobblestone excrescences over the entire upper tarsal conjunctiva (Fig. 1), and many eosinophils in scrapings of the conjunctiva. Ten patients (seven males and three females) were aged 6 to 33 years From the Department of Cornea Research, Eye Research Institute of Retina Foundation (Dr. Allansmith and Ms. Simon), and the Department of Oph thalmology, Harvard Medical School (Dr. Allansmith), Boston, Massachusetts arid the Division of Ophthalmology, Stanford University School of Medicine, Stanford, California (Mr. Hahn). This study was supported in part by training grant EY000208 and Research grant EY-00043 from the Na tional Eye Institute, by National Institutes of Health grant EY-00445 and Research Career Developmen tal Award EY-11682 (Dr. Allansmith), and by the Massachusetts Lions Eye Research Fund, Inc. Reprint requests to M. R. Allansmith, M.D., 20 Staniford St., Boston, MA 02114.
with an average age of 14 years. The last patient was a 75-year-old man with clini cally typical disease including many eo sinophils in the conjunctival scraping. Four patients had tissue removed from the upper tarsal conjunctiva. One of these, a 23-year-old man, was also in the tear-serum group. Two other males and one female, aged 8 to 33, comprised the group. All had at least moderately severe disease. Ten normal subjects, six females and four males, served as controls for the tear-serum group and were aged 15 to 53 years. None had ocular disease. Three had mild hay fever with no ocular symptoms. Upper tarsal tissue from two cadavers served as controls for the tissue group. Both subjects had died of cancers not involving the eyes or orbits at 31 and 53 years of age.
Fig. 1 (Allansmith, Hahn, and Simon). Clinical picture of vernal conjunctivitis. The upper palpebral conjunctiva has many giant papillary excrescences resembling cobblestones (arrow).
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Tissue — Cobblestone excrescences were shaved from the upper tarsal plates under topical anesthesia. Tissues were fixed for at least four hours in a freshly prepared mixture of 19 parts absolute ethanol and 1 part glacial acetic acid. Fixed specimens were embedded in de gassed paraffin, cut at 4 μ, and stained by the immunofluorescence method as previ ously described for IgG, IgA, IgM, IgD, and IgE with appropriate controls. 1 Tears and serum—Paired samples were obtained. Blood was taken from an arm vein, allowed to clot, and serum was removed. Tears were collected by posi tioning a chip of Week cell sponge about 1 x 1 x 3 mm between the upper and lower lacrimal puncta. The sponge ex panded with accumulated tears. The tears were extracted by recompressing the satu rated sponges into the bottom of a small self-capping microcentrifuge tube with a metal probe. Approximately six soaked sponges yielded several hundred microliters of tears that were stored at —5°C until analyzed. Serum was collected from venous blood. Serum and tear IgE concentrations were determined using the radioimmunosorbent technique (Phadebas IgE test). The mean error of the technique, express ed as the standard deviation of 44 dupli cated assays, was 15%. The value used for the amount of IgE corresponding to one unit was taken to be 2.39 ng/ml. This value was derived from the numerical average of the value 2.35 ng/unit, ob tained from 20 analyses of World Health Organization IgE reference standard 68-341 containing 10,000 units/ml as measured by Yunginger and Gleich 2 and the value 2.42 ng/unit measured by Bazaral and Hamburger. 3 All mean values, correlation coefficients, and statistical comparisons were calculated with the amounts transformed to their common logarithm. This type of transformation, which has precedence in biological sys
507
tems, 4 resulted in a more normal distribu tion and a relatively linear relationship between serum and tear IgE concentra tions. RESULTS
Plasma cells were seen in all tissues stained with hematoxylin and eosin. Plas ma cells of three types were seen by immunofluorescence stain in tissues from two of the four patients. There was no predominance of IgE staining cells. Rath er, cells staining for IgA, IgD, and IgE were found (Figs. 2 and 3) with plasma IgA cells predominating over others in a ratio of approximately 4:1:2 for IgA: IgD:IgE. No staining plasma cells were seen in the upper tarsal conjuhctival tissues from the two normal subjects. Control staining was negative. 1 In the control-stained slides, the many eosinophils were easily visible (Fig. 4). Abundant extracellular immunoglobulin was present in both groups (Fig. 5). This was thought to be extravascular serum protein as is present in the normal conjunctiva. 1 More IgE seemed to be in the extracellular stroma of the tissues
Fig. 2 (Allansmith, Hahn, and Simon). Upper tarsal cobblestone tissue stained for IgE. Arrow indiates plasma cell containing IgE (x250).
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Fig. 3 (Allansmith, Hahn, and Simon). Tissue stained for IgA. Arrow in dicates plasma cells con taining IgA (x250).
from the patients than of the normal sub jects. The mean serum IgE concentration of the patient group was 1,031 ng/ml with a 95% confidence interval of 18 to 59,346
Fig. 4 (Allansmith, Hahn, and Simon). Tissue from patient with control stain shows no specific fluorescence. Many autofluorescent eosinophils are seen (arrow) (x250).
ng/ml (Table 1). The control group had a mean serum IgE concentration of 201 ng/ml with a 95% confidence interval of 52 to 781 ng/ml (Table 2). Student's i-test revealed a significant difference between the means of the two groups (P<.05). The mean tear IgE concentration in the patient group was 130 ng/ml with a 95% confidence interval of 5 to 3,538 ng/ml. The control group had a mean tear IgE concentration of 61 ng/ml. No significant difference between the means was found with the Student's f-test (P>.05<.2). The correlation coefficient of the pa tient group was r = +.79 (Fig. 6) (P<.01). The correlation coeficient calculated from the paired serum and tear IgE concentra tions in the normal group was r = +.61 (Fig. 7) (P<.05). The two patients (Cases 1 and 2, Table 1) with high IgE values had the most severe systemic and local disease. One patient (Case 1) was a 22-year-old Mexican-American man with severe atopic dermatitis, asthma, and hay fever. His dense atopic cataracts had been re-
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VERNAL CONJUNCTIVITIS TABLE 1
S E R U M AND T E A R I G E C O N C E N T R A T I O N S ΓΝ P A T I E N T S W I T H VERNAL CONJUNCTIVITIS
Fig. 5 (Allansmith, Hahn, and Simon). Tissue stained for IgG. Note bright stromal fluorescence (arrow), but no IgG-containing plasma cells (x250).
moved when he was in his teens, and one eye developed keratoconus for which he received a transplant at age 2 1 . No vernal ulcers had occurred. The other patient (Case 2) was a 19-year-old black man with atopic dermatitis, asthma, and severe long-standing bilateral vernal corneal ul cers that had thinned his stroma to one half the normal thickness and extended over the visual axes. One other subject, a 17-year-old boy originally selected as a control, was re moved from the group because of mark edly raised serum levels of 5,247 ng/ml
Case No.
Serum IgE, ng/ml
Tear IgE, ng/ml
1 2 3 4 5 6 7 8 9 10 11
67,020 18,960 2,552 1,908 1,049 570 301 281 246 162 112
16,240 148 124 67 165 53 62 55 74 55 74
Mean ±2S.D.
1,031 18-59,346
130 5-3,538
and tear levels of 387 ng/ml. Our exami nation included detailed questioning for symptoms of atopy or other disease, ques tioning for out-of-country travel (worm infestation), a physical examination, total blood eosinophil counts, repeated exami nation of the stool for ova and parasites, a red blood cell sedimentation rate and determination of parents' serum IgE lev els. All were within normal limits. The subject was perfectly healthy. Repeated measurements of tear and serum IgE con centrations showed consistently high levTABLE 2 S E R U M AND T E A R I G E C O N C E N T R A T I O N S IN N O R M A L SUBJECTS
Subject No. Serum IgE, ng/ml Tear IgE, ng/ml 1 2 3 4 5 6 7 8 9 10
978 298 267 243 181 157 148 146 105 88
100 83 72 67 90 24 76 50 52 40
Mean ±2S.D.
201 52-781
61 26-144
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SERUM (ng IgE/ml) Fig. 6 (Allansmith, Hahn, and Simon). Correlation of tear and serum IgE concentra tions in patients with vernal conjunctivitis.
els. Other apparently normal nonatopic patients with high serum IgE levels have been described. 5 However, since this sub ject's IgE levels were outside of the 99.0% confidence interval for normal IgE levels r= + .61 p< .05
DISCUSSION
<
10J
10'
in serum as determined by others, 2 he was excluded from the statistical tests. If this subject's tear and serum levels had been included in the statistical tests, neither serum nor tear IgE levels would have been significantly different from those of the patient group.
SERUM Cng IgE/ml) Fig. 7 (Allansmith, Hahn, and Simon). Correlation of tear and serum IgE levels in normal subjects.
The patients with vernal conjunctivitis had higher mean serum and tear levels of IgE than the control subjects, although only the serum IgE levels were signifi cantly greater (P<.05). The tear and serum levels of each group showed a significant correlation, suggesting that tear IgE levels are a function of the serum IgE levels (Figs. 6 and 7). Local synthesis of IgE was found in cobblestone excrescences from the upper tarsal plates of some of the patients with vernal conjunctivitis. However, synthesis
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of IgA and IgD was also found. Neither of these two immunoglobulins is thought to be involved in atopic processes. 6 The molecules of IgE being synthe sized in the patients' tissues were either too few to alter total tear IgE concentra tions, or unable to escape into the tear film in sufficient numbers to raise the tear IgE level significantly. Strong evidence suggests that vernal conjunctivitis is an atopic disease, 7,8 and our results of elevated serum IgE levels support this. However, elevated tear IgE levels are not consistently found. The cobblestone excrescences, although they contain IgE-making cells, do not repre sent primarily a local hyperplasia of IgEforming plasma cells. Although local IgE-mediated damage may occur in vernal conjunctivitis, a local hyperplasia of only the IgE system is not present. SUMMARY
Abnormal upper tarsal tissue from four patients with vernal conjunctivitis stained immunofluorescently contained abundant IgA-, IgD-, and IgE-forming plasma cells in two of four patients. These were absent in normal upper tarsal conjunctiva. The IgE levels of paired tear-serum samples of 11 patients with vernal conjunctivitis and ten control sub jects were 130 ng/ml in patients that did not differ significantly from the normal
511
tear IgE value of 61 ng/ml. The serum IgE means of 1,031 ng/ml for patients and 201 ng/ml for normal subjects differed significantly (P<.05). Tear and serum IgE values correlated significantly for both groups (P<.01 for patients, P<.05 for normal subjects). These findings are consistent with a hyperplasia of IgA, IgD, and IgE antibody-forming cells in tarsal conjunc tiva of some patients with vernal conjunc tivitis, and tear IgE levels being a func tion of serum IgE levels. REFERENCES 1. Allansmith, M. R., Whitney, C. R., McClellan, B. H., and Newman, L. P.: Immunoglobulins in the human eye: location, type and amount. Arch. Ophthalmol. 89:36, 1973. 2. Yunginger, J. W., and Gleich, G. J.: Seasonal changes in serum and nasal IgE concentrations. J. Allergy Clin. Immunol. 51:175, 1973. 3. Bazaral, M., and Hamburger, R. N.: Standardi zation and stability of immunoglobulin E (IgE). J. Allergy Clin. Immunol. 49:189, 1972. 4. Sokal, R., and Rohlf, F. J.: Biometry. San Francisco, W. H. Freeman and Company, 1969, p. 476. 5. Fitch, K. D., Turner, K. J., and Morton, A. R.: The relationship between serum IgE levels and exercise-induced asthma. Ann. Allergy 30:497,1972. 6. Ishizaka, K., Ishizaka, T., and Hornbrook, M.: Allergen binding activity of IgE, IgG and IgA antibodies in sera from atopic patients: in vitro measurements of reagenic antibody. J. Immunol. 98:490, 1967. 7. Allansmith, M., and Frick, O. L.: Antibodies to grass in vernal conjunctivitis. J. Allergy 34:535, 1963. 8. Beigelman, M. N.: Vernal Conjunctivitis. Los Angeles, University of Southern California Press, 1950, p. 278.
M.D.
Boston, Massachusetts GARY S. H A H N ,
B.S.
Stanford, California AND M E R E D I T H A. S I M O N ,
M.S.
Boston, Massachusetts
We postulated that perhaps vernal con junctivitis had as part of its mechanism a localized hyperplasia of IgE plasma cells in the cobblestone excrescences; and also that perhaps locally produced IgE raised the tear IgE level. To evaluate these pos tulations, tarsal conjunctiva from patients with vernal conjunctivitis was stained for all immunoglobulins, and IgE was meas ured in tears and serum of patients and controls. M A T E R I A L AND M E T H O D S
Subjects—Eleven patients with vernal conjunctivitis were selected for the tearserum portion of the study on the basis of availability. All had severe itching, tear ing, disease for more than two seasons, typical cobblestone excrescences over the entire upper tarsal conjunctiva (Fig. 1), and many eosinophils in scrapings of the conjunctiva. Ten patients (seven males and three females) were aged 6 to 33 years From the Department of Cornea Research, Eye Research Institute of Retina Foundation (Dr. Allansmith and Ms. Simon), and the Department of Oph thalmology, Harvard Medical School (Dr. Allansmith), Boston, Massachusetts arid the Division of Ophthalmology, Stanford University School of Medicine, Stanford, California (Mr. Hahn). This study was supported in part by training grant EY000208 and Research grant EY-00043 from the Na tional Eye Institute, by National Institutes of Health grant EY-00445 and Research Career Developmen tal Award EY-11682 (Dr. Allansmith), and by the Massachusetts Lions Eye Research Fund, Inc. Reprint requests to M. R. Allansmith, M.D., 20 Staniford St., Boston, MA 02114.
with an average age of 14 years. The last patient was a 75-year-old man with clini cally typical disease including many eo sinophils in the conjunctival scraping. Four patients had tissue removed from the upper tarsal conjunctiva. One of these, a 23-year-old man, was also in the tear-serum group. Two other males and one female, aged 8 to 33, comprised the group. All had at least moderately severe disease. Ten normal subjects, six females and four males, served as controls for the tear-serum group and were aged 15 to 53 years. None had ocular disease. Three had mild hay fever with no ocular symptoms. Upper tarsal tissue from two cadavers served as controls for the tissue group. Both subjects had died of cancers not involving the eyes or orbits at 31 and 53 years of age.
Fig. 1 (Allansmith, Hahn, and Simon). Clinical picture of vernal conjunctivitis. The upper palpebral conjunctiva has many giant papillary excrescences resembling cobblestones (arrow).
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Tissue — Cobblestone excrescences were shaved from the upper tarsal plates under topical anesthesia. Tissues were fixed for at least four hours in a freshly prepared mixture of 19 parts absolute ethanol and 1 part glacial acetic acid. Fixed specimens were embedded in de gassed paraffin, cut at 4 μ, and stained by the immunofluorescence method as previ ously described for IgG, IgA, IgM, IgD, and IgE with appropriate controls. 1 Tears and serum—Paired samples were obtained. Blood was taken from an arm vein, allowed to clot, and serum was removed. Tears were collected by posi tioning a chip of Week cell sponge about 1 x 1 x 3 mm between the upper and lower lacrimal puncta. The sponge ex panded with accumulated tears. The tears were extracted by recompressing the satu rated sponges into the bottom of a small self-capping microcentrifuge tube with a metal probe. Approximately six soaked sponges yielded several hundred microliters of tears that were stored at —5°C until analyzed. Serum was collected from venous blood. Serum and tear IgE concentrations were determined using the radioimmunosorbent technique (Phadebas IgE test). The mean error of the technique, express ed as the standard deviation of 44 dupli cated assays, was 15%. The value used for the amount of IgE corresponding to one unit was taken to be 2.39 ng/ml. This value was derived from the numerical average of the value 2.35 ng/unit, ob tained from 20 analyses of World Health Organization IgE reference standard 68-341 containing 10,000 units/ml as measured by Yunginger and Gleich 2 and the value 2.42 ng/unit measured by Bazaral and Hamburger. 3 All mean values, correlation coefficients, and statistical comparisons were calculated with the amounts transformed to their common logarithm. This type of transformation, which has precedence in biological sys
507
tems, 4 resulted in a more normal distribu tion and a relatively linear relationship between serum and tear IgE concentra tions. RESULTS
Plasma cells were seen in all tissues stained with hematoxylin and eosin. Plas ma cells of three types were seen by immunofluorescence stain in tissues from two of the four patients. There was no predominance of IgE staining cells. Rath er, cells staining for IgA, IgD, and IgE were found (Figs. 2 and 3) with plasma IgA cells predominating over others in a ratio of approximately 4:1:2 for IgA: IgD:IgE. No staining plasma cells were seen in the upper tarsal conjuhctival tissues from the two normal subjects. Control staining was negative. 1 In the control-stained slides, the many eosinophils were easily visible (Fig. 4). Abundant extracellular immunoglobulin was present in both groups (Fig. 5). This was thought to be extravascular serum protein as is present in the normal conjunctiva. 1 More IgE seemed to be in the extracellular stroma of the tissues
Fig. 2 (Allansmith, Hahn, and Simon). Upper tarsal cobblestone tissue stained for IgE. Arrow indiates plasma cell containing IgE (x250).
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Fig. 3 (Allansmith, Hahn, and Simon). Tissue stained for IgA. Arrow in dicates plasma cells con taining IgA (x250).
from the patients than of the normal sub jects. The mean serum IgE concentration of the patient group was 1,031 ng/ml with a 95% confidence interval of 18 to 59,346
Fig. 4 (Allansmith, Hahn, and Simon). Tissue from patient with control stain shows no specific fluorescence. Many autofluorescent eosinophils are seen (arrow) (x250).
ng/ml (Table 1). The control group had a mean serum IgE concentration of 201 ng/ml with a 95% confidence interval of 52 to 781 ng/ml (Table 2). Student's i-test revealed a significant difference between the means of the two groups (P<.05). The mean tear IgE concentration in the patient group was 130 ng/ml with a 95% confidence interval of 5 to 3,538 ng/ml. The control group had a mean tear IgE concentration of 61 ng/ml. No significant difference between the means was found with the Student's f-test (P>.05<.2). The correlation coefficient of the pa tient group was r = +.79 (Fig. 6) (P<.01). The correlation coeficient calculated from the paired serum and tear IgE concentra tions in the normal group was r = +.61 (Fig. 7) (P<.05). The two patients (Cases 1 and 2, Table 1) with high IgE values had the most severe systemic and local disease. One patient (Case 1) was a 22-year-old Mexican-American man with severe atopic dermatitis, asthma, and hay fever. His dense atopic cataracts had been re-
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VERNAL CONJUNCTIVITIS TABLE 1
S E R U M AND T E A R I G E C O N C E N T R A T I O N S ΓΝ P A T I E N T S W I T H VERNAL CONJUNCTIVITIS
Fig. 5 (Allansmith, Hahn, and Simon). Tissue stained for IgG. Note bright stromal fluorescence (arrow), but no IgG-containing plasma cells (x250).
moved when he was in his teens, and one eye developed keratoconus for which he received a transplant at age 2 1 . No vernal ulcers had occurred. The other patient (Case 2) was a 19-year-old black man with atopic dermatitis, asthma, and severe long-standing bilateral vernal corneal ul cers that had thinned his stroma to one half the normal thickness and extended over the visual axes. One other subject, a 17-year-old boy originally selected as a control, was re moved from the group because of mark edly raised serum levels of 5,247 ng/ml
Case No.
Serum IgE, ng/ml
Tear IgE, ng/ml
1 2 3 4 5 6 7 8 9 10 11
67,020 18,960 2,552 1,908 1,049 570 301 281 246 162 112
16,240 148 124 67 165 53 62 55 74 55 74
Mean ±2S.D.
1,031 18-59,346
130 5-3,538
and tear levels of 387 ng/ml. Our exami nation included detailed questioning for symptoms of atopy or other disease, ques tioning for out-of-country travel (worm infestation), a physical examination, total blood eosinophil counts, repeated exami nation of the stool for ova and parasites, a red blood cell sedimentation rate and determination of parents' serum IgE lev els. All were within normal limits. The subject was perfectly healthy. Repeated measurements of tear and serum IgE con centrations showed consistently high levTABLE 2 S E R U M AND T E A R I G E C O N C E N T R A T I O N S IN N O R M A L SUBJECTS
Subject No. Serum IgE, ng/ml Tear IgE, ng/ml 1 2 3 4 5 6 7 8 9 10
978 298 267 243 181 157 148 146 105 88
100 83 72 67 90 24 76 50 52 40
Mean ±2S.D.
201 52-781
61 26-144
510
AMERICAN JOURNAL OF OPHTHALMOLOGY
APRIL, 1976
SERUM (ng IgE/ml) Fig. 6 (Allansmith, Hahn, and Simon). Correlation of tear and serum IgE concentra tions in patients with vernal conjunctivitis.
els. Other apparently normal nonatopic patients with high serum IgE levels have been described. 5 However, since this sub ject's IgE levels were outside of the 99.0% confidence interval for normal IgE levels r= + .61 p< .05
DISCUSSION
<
10J
10'
in serum as determined by others, 2 he was excluded from the statistical tests. If this subject's tear and serum levels had been included in the statistical tests, neither serum nor tear IgE levels would have been significantly different from those of the patient group.
SERUM Cng IgE/ml) Fig. 7 (Allansmith, Hahn, and Simon). Correlation of tear and serum IgE levels in normal subjects.
The patients with vernal conjunctivitis had higher mean serum and tear levels of IgE than the control subjects, although only the serum IgE levels were signifi cantly greater (P<.05). The tear and serum levels of each group showed a significant correlation, suggesting that tear IgE levels are a function of the serum IgE levels (Figs. 6 and 7). Local synthesis of IgE was found in cobblestone excrescences from the upper tarsal plates of some of the patients with vernal conjunctivitis. However, synthesis
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of IgA and IgD was also found. Neither of these two immunoglobulins is thought to be involved in atopic processes. 6 The molecules of IgE being synthe sized in the patients' tissues were either too few to alter total tear IgE concentra tions, or unable to escape into the tear film in sufficient numbers to raise the tear IgE level significantly. Strong evidence suggests that vernal conjunctivitis is an atopic disease, 7,8 and our results of elevated serum IgE levels support this. However, elevated tear IgE levels are not consistently found. The cobblestone excrescences, although they contain IgE-making cells, do not repre sent primarily a local hyperplasia of IgEforming plasma cells. Although local IgE-mediated damage may occur in vernal conjunctivitis, a local hyperplasia of only the IgE system is not present. SUMMARY
Abnormal upper tarsal tissue from four patients with vernal conjunctivitis stained immunofluorescently contained abundant IgA-, IgD-, and IgE-forming plasma cells in two of four patients. These were absent in normal upper tarsal conjunctiva. The IgE levels of paired tear-serum samples of 11 patients with vernal conjunctivitis and ten control sub jects were 130 ng/ml in patients that did not differ significantly from the normal
511
tear IgE value of 61 ng/ml. The serum IgE means of 1,031 ng/ml for patients and 201 ng/ml for normal subjects differed significantly (P<.05). Tear and serum IgE values correlated significantly for both groups (P<.01 for patients, P<.05 for normal subjects). These findings are consistent with a hyperplasia of IgA, IgD, and IgE antibody-forming cells in tarsal conjunc tiva of some patients with vernal conjunc tivitis, and tear IgE levels being a func tion of serum IgE levels. REFERENCES 1. Allansmith, M. R., Whitney, C. R., McClellan, B. H., and Newman, L. P.: Immunoglobulins in the human eye: location, type and amount. Arch. Ophthalmol. 89:36, 1973. 2. Yunginger, J. W., and Gleich, G. J.: Seasonal changes in serum and nasal IgE concentrations. J. Allergy Clin. Immunol. 51:175, 1973. 3. Bazaral, M., and Hamburger, R. N.: Standardi zation and stability of immunoglobulin E (IgE). J. Allergy Clin. Immunol. 49:189, 1972. 4. Sokal, R., and Rohlf, F. J.: Biometry. San Francisco, W. H. Freeman and Company, 1969, p. 476. 5. Fitch, K. D., Turner, K. J., and Morton, A. R.: The relationship between serum IgE levels and exercise-induced asthma. Ann. Allergy 30:497,1972. 6. Ishizaka, K., Ishizaka, T., and Hornbrook, M.: Allergen binding activity of IgE, IgG and IgA antibodies in sera from atopic patients: in vitro measurements of reagenic antibody. J. Immunol. 98:490, 1967. 7. Allansmith, M., and Frick, O. L.: Antibodies to grass in vernal conjunctivitis. J. Allergy 34:535, 1963. 8. Beigelman, M. N.: Vernal Conjunctivitis. Los Angeles, University of Southern California Press, 1950, p. 278.