Immunoglobulin E—mediated asthma and hypersensitivity pneumonitis with precipitating anti-hapten antibodies due to diphenylmethane diisocyanate (MDI) exposure

lmmunoglobulin E-mediated asthma and hypersensitivity pneumonitis with precipitating anti-hapten antibodies due to diphenylmethane diisocyanate (MDI) exposure C. Raymond M.D., Doris Chicago,

Zeiss, Levitz,

M.D., Theodore M. Kanellakes, M.D., Jack D. Bellone, B.S., J. J. Pruzansky, Ph.D., and Roy Patterson, M.D.

111.

Two workers are presented who were exposed to diphenylmethane diisocyanate (MDI) while coating pipes with a polyurethane foam. After a latent period of exposure, worker A developed immediate-onset asthma and worker B developed a clinical picture of hypersensitivity pneumonitis for which he was hospitalized. The antibody response of these workers to a conjugate of MD1 with human serum albumin (MDI-HSA) was measured by gel precipitation, total antibody binding of lz51 MDI-HSA, and specific IgG and IgE antibody by polystyrene-tube radioimmunoassay (PTRIA). Worker B had precipitating antibody to MDI-HSA by double immunodiffusion in gel. Both workers had high levels of IgG antibody specific for MDI-HSA which had some cross-reactivity with a conjugate of toluene diisocyanate and HSA. Total serum antibody binding of “Y MDI-HSA was 15 pglml in worker A and 900 pglml in worker B. Both workers had serum IgE antibody specific for MDI-HSA as measured by two PTRIA techniques. These results indicate that a marked immunologic response to MDI is possible in exposed workers and that hypersensitivity pneumonitis can occur subsequent to the inhalation of a low-molecular-weight chemical in the industrial setting.

Adverse respiratory reactions to the inhalation of toluene diisocyanate (TDI) were reported in 195 1. l Other isocyanates such as diphenylmethane diisocyanate (MDI) and hexamethylene diisocyanate (HDI) have also been reported to cause respiratory reactions in exposed workers.2 These substances, like TDI, act as polymerizing agents and are used in the manufacture of polyurethanes. Polyurethanes are used as cushion and packing materials, for insulation, and in the manufacture of varnishes, paints, and adhesives. Isocyanates, and particularly TDI, have been known for years to cause pulmonary reactions characterized

From the Department of Medicine, Northwestern University Medical School. Supportedby U.S. Public Health Service Grants HL 15389 and AI 11403. Received for publication June 29, 1979. Accepted for publication Sept. 28, 1979. Reprint requests to: C. Raymond Zeiss, M.D., Department of Medicine, Northwestern University Medical School, 3-03 East Chicago Ave., Chicago, IL 60611. Vol.

65, No.

5, pp. 346-352

by dyspnea, wheezing, and cough. Workers have been identified who have immediate, dual, and late asthmatic reactions to the inhalation of TDI vapor. Inhalation challenge studies have demonstrated that these asthmatic reactions arc provoked by concentrations of TDI which do not affect other exposed workem3 MD1 and HDI have also been shown to cause asthmatic reactions when inhaled under controlled conditions.’ An immunologic basis for these isocyanate-induced asthmatic reactions has been considered because of the latent period of exposure before sensitization, the fact that a minority of exposed workers are affected, and that after sensitization, exposures well below that of the threshold limit value will result in asthmatic reactions .4 Confirmation of the suspected immunologic pathogenesis of these types of asthma seen with TDI or MD1 exposure has been difficult and controversy exists regarding the significance of immunologic mechanisms in isocyanate asthma.5 This report concerns the clinical and immunologic investigation of two workers in the same plant who

0091-6749/80/050346+07$00.7010

@ 1980

The

C. V. Mosby

Co.

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wer.2 exposed to polyurethane foam as pipes were sprayed to make them corrosion resistant. During this exposure, they were sensitized to MD1 and both developed significant clinical and immunologic response to MD1 after a latent period of exposure.

pneumonitis

DI PHENYLMETHANE

to diphenylmethane

DIISOCYANATE

diisocyanate

(MDI)

+

347

PROTEIN

CASE REPORTS Worker A, a 33-yr-old white male, had no chest symptom*, until May, 1977, when he developed tightness in the che.\t and wheezing dyspnea with physical exertion. After sevcml months these symptoms disappeared spontaneously and did not recur until February, 1978, when he developed syniatoms of an upper respiratory tract infection with chest tightness, wheezing dyspnea, and dry nonproductive cough. He ( id not have fever, chills, chest pain, or hemoptysis. He was seen by his family physician, who documented wheezing. At this time a chest roentgenogram was normal and he was placed on tetracycline and aminophylline suppositories. He had continued difficulty through March, 1978, but clerued during the last weeks of March and the first week of April, having been off work for 4 wk. On returning to work, he again had to take bronchodilating medication. In June, 19i’i;, he developed symptoms consisting of chills, fever, cher;t tightness. wheezing, cough, and dyspnea. He was see: by a local physician who again documented wheezing, and he was started on broad-spectrum antibiotics and continti*:d on bronchodilating medication. It was at this time that the patient attributed wheezing dyspnea to the plant envi-onment. He noted that he was able to taper bronchodilator medication over long weekends and was decidedly better on short weekends. In July of 1978, after a I-n i. vacation, he became asymptomatic and required no me&cation. On returning to work, he again became symptomatic with wheezing dyspnea and attributed this to expc-sure to methylene chloride, which was used to flush the instpJments that delivered the polyurethane foam. t!e was seen by one of us (T. K.) in June of 1978. He had worked at the company for 9 yr as a production supervisor, sper;ding 75% of his time in an office environment and 25% in the manufacturing area. He was not directly involved in the *praying of pipes. He made the observation that when the plant was poorly ventilated in the winter months, his condition was worse. He had a 15pack-per-year history of cigarette consumption and had discontinued cigarettes in February. 1978. He had a life-long history of allergic rhinitis Ik:ginning in August, peaking in September, and ending with the first frost. With the onset of wheezing dyspnea, he first noted that strong odors such as perfume, insecticides, detergents. and hair sprays would cause exacerbation of the>: symptoms. On skin testing, he was prick-positive to many allergens, including ragweed. A prick test to MDIHSA (3 mg/ml) was slightly reactive but an intradermal test (O.&J3 mgiml) was negative. Worker B, a 45-yr-old white male, was first seen by one of 111;(J. B.) in March of 1978. He had a complaint of persistent cough of I-mo duration. He stated that in November, 1977, he developed periodic episodes of shortness of breath which he attributed to cigarette smoking. He

FIG. 1. The conjugation of MDI with a carrier protein. A covalent bond is formed with amino groups of the protein. stopped smoking but symptoms persisted. He consulted a physician in January, 1978, and was told that he had asthma. He was given bronchodilating medication and corticosteroids. The symptoms returned after 3 wk and he was again treated with bronchodilators and corticosteroids. The symptoms of dyspnea subsided somewhat but the cough persisted. He noticed that the cough was related to work exposure. At work he would have a gradual increa-se in cough and shortness of breath at times accompanied by fever, myalgia, and arthralgia which would resolve after work hours. He began employment at this particular plant as a welder in August, 1977, and was exposed to the polyurethene foam fumes continuously. On his initiat office visit in March of 1978, he was in no acute distress. Examination of the lungs revealed bilateral basilar rales without wheezing. Chest roentgenogram at this time was normal. He was advised to stay off work for 2 wk and was given bronchodilator therapy. He improved somewhat over this period of time. Upon returning to work on March 13. 1978, he became acutely shart of breath after several hours of exposure and was admitted to an emergency mom. He complained of dyspnea and shortness of breath. His temperature was 101.2” orally. Examination of lungs revealed bilateral basilar rales without wheezing. The white blood cell count was 11,000 (with 85% segmenred forms, 7% lymphocytes, 5% monocytes, and 3% eosinophils). The sedimentation rate was 22. After&l blood gas analysis revealed a pH of 7.35, a Pc.o, of 24, a P,), of SO. A chest roentgenogram revealed infiItrates in the left upper and right lower lung fields. A lung scan demonstrated slightly irregular distribution of activity in both lungs and was not consistent with pulmonary embolus. A ventilation perfusion scan revealed ventilation abnormslities at both bases. On the third day of hospitalization, @manary function studies were done. Forced expiratory volume in 1 set (FEV,) was 2.65 L with a vital capacity of 3.4 L: these were, respectively, 83% and 86% of the predicted value. Normal or negative laboratory studies included hemoglobin and hematocrit, sputum culture, routine blood chemistries, rheumatoid factor, alpha-l -antitripsin level, and sputum for eosinophils. The IgG level was etevated to 1,600 mg%. The serum IgE was 440 rig/ml. After starting on antibiotics and

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10

i

,

> I(: lo:

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: 0

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i 0

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IMMUNOL. MAY 1980

30 min, the conjugate was dialyzed against four changes of 0.1 M NaHC03 and filtered through a 0.20-p filter. As an additional control, HSA was taken through each of the above steps but without the addition of MDI. This was designated as sham-conjugated HSA. To document that MD1 had conjugated with HSA, spectrographic analysis of the conjugate was undertaken. Absorption curves were made of MDI-HSA and HSA from 230 and 280 nm (Fig. 2). There was a marked increase in absorption from 230 to 260 nm, which indicated that the MD1 had become covalently linked to the protein carrier. The ratio of optical density at 240 nm to that at 280 nm was 3.6 for MDI-HSA and 1.6 for HSA. TDI-HSA was conjugated and standardized in an identical manner. Control sera Sera from two asymptomatic workers who had been exposed to MD1 for 3 yr were used as controls as well as sera from nonexposed laboratory personnel. Radioiodination

lo2io

Pi0

2io WAVELENGTH

2;o

2&o

2;o

2bo

(nm)

FIG. 2. The spectra of HSA and HSA conjugated (MDI-HSA) are compared. The increase in optical between 240 and 260 nm indicates the presence conjugated to the HSA carrier.

to MDI density of MDI

aminophylline, he improved. After discharge, he continued to have dyspnea on exertion and persistent basilar rales. He was started on prednisone 40 mg per day with marked improvement. Corticosteroids were tapered over an S-mo period and the patient remains asymptomatic. He was advised to avoid all MD1 exposure and has found other employment. At the time of his acute hospitalization, the etiology of his problem was unclear and one of the authors (T. K.) suggested the possibility of hypersensitivity pneumonitis secondary to chemical inhalation. A visit to the plant site revealed that the isocyanate, MDI, was used in the generation of the polyurethene foam and the possibility of this being the etiologic agent was considered. This worker was found to have marked immunologic reactivity to MD1 which will be outlined in the studies below. Skin tests to MDIHSA were negative. MATERlALS AND METHODS MDI conjugation to human serum (MDI-HSA)

albumin

MD1 may be covalently linked to HSA to form an antigen containing isocyanate determinants (Fig. 1). The method of conjugation has been described previously.6 Briefly, 100 mg MD1 (ICN Pharmaceutical, Inc., Plainview, N. Y .) was dissolved in 1.O ml 1,4-dioxane and added dropwise to 20 ml of 7% NaHC03 containing 100 mg of HSA (Miles Research Laboratories, Inc., Kankakee , Ill .) , After stirring for

IgG, IgE, and MDI-HSA (100 pg) were labeled with lz51 using a modification of the method of Greenwood et a1.i Solutions of chloramine-T, sodium metabisulfite, and potassium iodide (KI) were made in distilled water. To a 2-mCi vial of rz51was added 100 pg of MDI-HSA, followed by 100 pg of chloramine-T. After 1 min, the reaction was stopped with 240 pg of sodium metabisulfite. Thirty seconds thereafter, 0.1 ml of 1% KI was added. The entire contents was pipetted onto a small prepacked Sephadex G25 column (Pharmacia). The vial was washed with 1 ml of KI and this wash was added to the column. To elute the labeled protein from the column, l-ml aliquots of 1% bovine serum albumin in phosphate-buffered saline (BSA-PBS) were added to the column and twelve l-ml samples were collected and counted. An elution profile was thus obtained, the first peak containing the labeled protein and the second peak containing the free iodine. The labeled MDI-HSA was 99% precipitable in 10% trichloroacetic acid, and 97% of the labeled material was bound in antibody excess. The specific activity of the 125I MDI-HSA was 24,000 cpm/ng. Precipitin

reactions

Ouchterlony-type gel double-diffusion analysis was used to detect precipitating antibodies against isocyanate-conjugated proteins. Quantitation of IgG and IgE antibody against MDI-HSA Polystyrene tube radioimmunoassay (PTRIA) was used antibodies of the IgG and IgE class against MDIHSA and TDI-HSA.* The following reagents were added to polystyrene tubes with incubation periods and washing between additions: MD1 or TDI-HSA, 1% HSA in PBS, appropriate dilution of worker’s serum, heavy chain-specific anti-IgG or anti-IgE, and lZ51IgG or rz51IgE. The resutts are expressed as counts per minute of lzsI IgG or i*sI IgE bound

to detect

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by the last layer. The counts per minute bound by a control se’lurn from a normal individual not exposed to isocyanates were

subtracted.

addition IgE antibody to the isocyanates was measured by .1separate radioimmunoassay.g In this method, IgE antibody from a worker’s serum is bound to a polystyrene solid phase through an anti-IgE immunoabsorbent. The MDIHS 4 conjugate which is radiolabeled is then added to the pol;jstyrene tube and the counts per minute bound by the wr,.ker’s IgE is determined. In this assay, high levels of IgG amtbody cannot compete for le51MDI-HSA because only In

Igl~. is bound

to the solid

Total antibody conjugates

phase.

activity

to isocyanate-HSA

was analyzed by > modification technique of Lidd and Farr.“’ Using this assay, the micrograms of MIX-HSA bound per milliliter of serum can be determined. Inc #easing concentrations of radiolabeled MDI-HSA are ad&d to a fixed quantity of serum and the nanograms of labeled isocyanate protein conjugate bound are determined at a point where 20% of the added labeled MDI-HSA is Total antibody

bou,ld.

This

point

in the serum to MDI-HSA of the ammonium sulfate

is in antigen

excess.

All

sera can then be

ranked by their ability to bind the MDI-HSA conjugate at thi:: point of antigen excess. The results are expressed in micrograms of MDI-HSA bound per milliliter of serum. RESULTS Demonstration MDI-HSA

of precipitating

antibody

to

!\.s demonstrated by double-diffusion analysis in agar gel, precipitating antibody to MDI-HSA was found in the serum of worker B (Fig. 3). Precipitating acti city could be found only against MDI-HSA and no{ against the TDI-HSA conjugate. No precipitating activity was seen against untreated HSA, or HSA taken through the conjugation step except for the additrrm of MDI. The serum of worker A was also analyzed and no precipitating antibody activity to either MDI-HSA or TDI-HSA was detected. ToEEal antibody

activity

to ‘*‘I MDI-HSA

1ncreasing quantities of 125I MDI-HSA were added to ‘iliquots of appropriately diluted serum from workers A and B. With increasing amounts of MDIHSA added, there is a decrease in the percent of the added antigen which is bound by antibody; however, there is an increase in the amount of antigen bound with increasing amounts of antigen added. At the 20% binding point, moderate antigen excess is achieved and the amount of antigen bound by antibody is measured at this point (Fig. 4). At the 20% binding point, worker A bound 15 pg of MDI-HSA per milliliter of serum and worker B bound 900 pg of MDI-HSA per millrliter of serum. Sera of asymptomatic workers

Fffi. 3. Precipitating antibody in the serum of worker 6 (center well) to MDI-HSA as detected by gel-diffusion analysis. There is no precipitating antibody detected to TDI-HSA, native HSA, or sham-conjugated HSA /bottom well).

bound less than 3% of the radiolabeled antigen at all levels of antigen added. IgG antibody

to MD1 or TM-HSA

This immunoglobulin class of antibody in workers’ sera can be analyzed by the polystyrene-tube, solidphase radioimmunoassay. In this method, the MDIHSA or TDI-HSA conjugate is bound to the wall of the polystyrene tube, appropriate dilutions of the workers’ sera are added and after appropriate incubation and washing, anti-IgG is then added in excess. The last layer is ‘291-labeled IgG, which will be bound by the anti-IgG in relation to the amount of specific IgG antibody bound to the hapten-protein conjugate on the solid phase. For worker 3, the serum was diluted to 1: 100,000 and was still able to bind 50% of the lz51IgG added. Worker A had lower levels of IgG antibody to MDI-HSA with 50% binding occurring at a serum dilution of 1: 1,000 (Fig. 5). Sera from exposed asymptomatic workers bound less than 5% of the InsI IgG added at a serum dilution of I : 100. In addition, when sham-conjugated HSA was used as an antigen, less than 1% of the added jznl IgG was bound. This assay is useful in determining cross-reactivity between different isocyanates bound to HSA as illustrated in Fig. 6. In this figure, IgCi antibody activity to MDI-HSA and TDI-HSA in the serum of worker B is IgG antianalyzed. This illustrates that the worker’s body is able to bind TDI-HSA but the antibody activity to MDI-HSA is considerably higher. IgE antibody

to MDI-HSA

IgE antibody to MDI-HSA conjugates can be measured by two immunologic methods. In the first, IgE

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loo-

60-

01 100

I 200

ng

FIG. 4. Total antibody point antigen excess

I 1000

5bo

, 2000

MDI-HSA

50’00

binding of radiolabeled MDI-HSA by a worker’s serum. is achieved and maximum binding can be calculated.

01

I 1:104

I:1 03

SERUM

At the 20%

binding

,:1105

DILUTiON

FIG. 5. In this method, MDI-HSA is the first layer on the polystyrene serum, anti-IgG, and 9 IgG. This assay can give a quantitative antibody activity to hapten-protein conjugate as illustrated here

antibody from a worker’s serum is bound to the polystyrene solid phase by an anti-IgE immunoabsorbent. The MDI-HSA conjugate which is radiolabeled is then added to the polystyrene tube and the counts per minute bound by the worker’s IgE is determined. In this immunoassay, high levels of IgG antibody cannot compete for lz51 MDI-HSA because only IgE is bound to the solid phase. Using this assay, IgE antibody activity to MDI-HSA was seen in both workers and the results are given in Table I. The second method also involved a polystyrene-tube solid phase where MDI-HSA is absorbed to the wall of the polystyrene tube, the worker’s serum is added, and after appropriate incubation and washing, anti-IgE is added. The final step is the addition of radiolabeled IgE myeloma protein and the results are expressed as counts per minute bound. The results of this type of assay in both

10’000

ADDED

surface followed by worker’s measure of the amount of IgG with MDI-HSA.

workers are shown in Table I. It is clear that both methods were able to detect IgE antibody activity to MDI-HSA. The sera of two asymptomatic workers exposed to MD1 demonstrated binding levels below that of the nonexposed normal laboratory controls listed in Table I. WSCUSSKbN These two workers represent examples of MDIinduced respiratory reactions where the clinical state can be correlated with immunologic activity to the conjugate of MD1 with human serum albumin, MDI-HSA. Both of these workers had a latent period of exposure to MD1 before developing clinical sensitivity. From that point on they developed quite divergent clinical syndromes. Worker A developed a clinical complex which was

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\ 20-

B

/

T DI-HSA

01

WORKER

/ 1:105

1:104

SERUM

DILUTION

FK;. 6. The cross-reactivity of IgG antibody in the serum of worker B (exposed to MD11 toTDI-HSA is illustrated. TDI-HSA and MDI-HSA form the first layer on two separate polystyrene tubes and the worker’s IgG antibody activity to these two hapten-protein conjugates is determined.

consistent with asthma. He was an atopic individual who had 9 yr of intermittent exposure to MD1 before developing clinical sensitivity. After the development of sensitivity, he gave a classic history for industrial asthma in that he noted that he was better on weekends and became asymptomatic on vacations. On return to work exposure, he again became symptomuric. In contrast to worker A, worker B had continuous exposure to MD1 fumes. In a few months he developed cough, dyspnea, shortness of breath, and occasional wheezing dyspnea. These symptoms were relentless after their first presentation and only partially relieved by bronchodilating drugs and corticosteroids. The tough, dyspnea, and shortness of breath were the predominant symptoms and were occasionally accompanied by fever, myalgia, and arthralgia. He was removed from the work environment for several weeks but immediately upon return to work developed shortness of breath, cough, dyspnea, and fever. This exacerbation required hospitalization. The clinical ,course was consistent with a diagnosis of hypersensitivity pneumonitis. ” Immunologic reactivity to a conjugate of MD1 with human serum albumin also showed differences in these two workers. Worker B had precipitating antibody to MDI-HSA, which was consistent with his clinical picture of hypersensitivity pneumonitis. This striking finding in a worker with industrial lung disease ied us to characterize the quality and quantity of this antibody activity. Using an ammonium sulfateradiolabeled antigen-binding technique, we were able to quantitate the serum antigen binding activity of both workers. Worker B bound 900 pug of MDI-HSA

TABLE worker A

B Control

I. IgE

antibody

-1

assays

Wmt 1,059 1.120 2,533 2,391 260

(PTWA) ‘* l$E @ad 3,200 3,689 1,092 1,212 419

per milliliter while worker A bound 15 pg of MDIHSA per milliliter. This quantitative difference was parallel to the results found on gel-diffusion analysis. Using a polystyrene-tube radioimmurtoassay, it was found that worker A and worker B bad IgG antibody specific for MDI-WSA. However, worker B had over 100 times more IgG antibody activity than worker A, as illustrated in Fig. 5. The IgG antibody activity in the serumof worker B to MDI-HSA was shown to cross-reactwith a conjugate of TDI-HSA (Fig. 6). The IgG activity to MDIHSA was approximately 10 times higher than antibody binding to TDI-HSA. Analysis of IgE antibody activity to MDI-HSA was done (Table I). Using two methodsof analysis, both showed IgE antibody activity to MDI-HSA. In the method where only IgE is allowed to react with radiolabeled MDI-HSA, worker B showed somewhat higher IgE antibody activity than worker A. In the secondassaywhere all immunoglobulin classescompete for solid-phase-bound MDI-HSA, worker B showed less activity than worker A, presumably the result of high levels of precipitating antibody in his serum to MDI-HSA. In addition, the higher levels of

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IgG antibody in the serum of worker B may have acted to block the clinical expression of the IgE antibody that was present. Although the IgE antibody activity in both workers was low and not accompanied by immediate-type skin reactivity, its presence may suggest a pathogenic role. Over the years, several investigators have been able to demonstrate immunologic reactivity to TDIconjugated proteins. 12* l3 Other investigators have been unable to confirm immunologic reactivity to TDI-HSA conjugates. l4 The studies of Butcher et al. have been most extensive in this regard.5 Recently, Karol et al. have demonstrated low levels of IgE antibody activity to p-tolyl (mono) isocyanate human serum albumin in serum of three workers with TDIinduced industrial asthma.r5 Immunologic reactivity to MD1 has not received as much attention. In 1966, Konzen et al. demonstrated antibody activity in seven workers exposed to MD1 but these immunologic studies were not extensive. l6 Although asthmatic reactions to the isocyanates have been described frequently, respiratory reactions which clinically simulate hypersensitivity pneumonia have been reported. I79 l8 Our studies demonstrate that a marked immunologic response can be induced by the inhalation of MDI. The development of hypersensitivity pneumonitis with precipitating antibody to MDI-HSA expands the spectrum of industrial lung disease that can be induced by the inhalation of low-molecular-weight chemicals. REFERENCES 1. Fuchs S, Valade P: Etude clinique et experimentale sur quelques cas d’intoxication par Ie’Desmodur T (diisocyanate de toluene l-2-4- et l-2-6). Arch Ma1 Profess 12:191, 1951. 2. O’Brien IM, Harries MS, Burge PS, Pepys J: Toluene diisocyanate induced asthma. I. Reactions to TDI, MDI, HDI, and histamine. Clin Allergy 9: 1, 1979. 3. O’Brien IM, Newman-Taylor AJ, Burge FS, Harties MG, Fawcett SW, Pepys J: Toluene di-isocyanate-induced asthma. II. Inhalation challenge tests and bronchial reactivity studies. Clin Allergy 9:7, 1979.

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4. Pepys J, Pickering CAC, Breslin ABX, Terry DJ: Asthma due to inhaled chemical agents-Tolylene diisocyanate. Clin Allergy 2~225, 1972. 5. Butcher BT, Salvaggio JE, Weill H, &kind MM: Toluene diisocyanate (TDI) pulmonary disease: Immunologic and inhalation challenge studies. J ALLERGY CLIN IMMUNOL 58:89, 1976. 6. Zeiss CR, Patterson R, Pruzansky JJ, Miller MM, Rosenberg M, Levitz D: Trimellitic anhydride-induced airway syndromes: Clinical and immunologic studies. J ALLERGY CLIN IMMUNOL

&96,

1977.

7. Greenwood FC, Hunter WM, Glover JS: The preparation of ‘3LI-labeled human growth hormone of high specific activity. Biochem J 89:14, 1963. 8. Patterson R, Roberts M: IgE and IgG antibodies against Aspergillusfimigatus in sera of patients with bronchopulmonary allergic aspergillosis. Int Arch Allergy Appl Immunol46: 150, 1974. 9. Zeiss CR, Pruzansky JJ, Patterson R, Roberts M: A solid phase radioimmunoassay for the quantitation of human reaginic antibody against ragweed antigen E. J Immunol llOt414, 1973. 10. Zeiss CR, Metzger WJ, Levitz D: Quantitative relationships between IgE antibody and blocking antibodies specific for antigen E in patients given immunotherapy with ragweed antigen E. Clin Exp Immunol28:250, 1977. 11. Fink JN: Hypersensitivity pneumonitis, in Middleton E, Reed CE, Ellis EF, editors: Allergy principles and practice. Saint Louis, 1978, The C. V. Mosby Co., p. 855. 12. Taylor G: Immune response to tolylene diisocyanate (TDI) exposure in man. Proc R Sot Med 63:379, 1970. 13. Stokinger HE, Scheel LD: Hypersusceptibility and genetic problems in occupational medicine-A consensus report. J Occup Med 15:5&l, 1973. 14. Avery SB, Stetson DM, Pan PM, Mathews KP: Immunological investigation of individuals with toluene diisocyanate asthma. Clin Exp Immunol 4:585, 1969. 15. Karol MH, Ioset HH, Yves CA: Tolyl-specific IgE antibodies in workers with hypersensitivity to toluene diisocyanate. Am Ind Hyg Assoc J 39:454, 1978. 16. Konzen RB, Croft BF, Scheel LD, Gorski CH: Human response to low concentrations of p, p-diphenylmethane diisocyanate (MDI). Am Ind Hyg Assoc J 27:121, 1966. 17. Fink JN, Schlueter DP: Bathtub refinisher’s lung: An unusual response to toluene diisocyanate. Am Rev Respir Dis 118:955, 1978. 18. Charles J, Bernstein A, Jones B, Jones DJ, Edwards JH, Seal RME, Seaton A: Hypersensitivity pneumonitis after exposure to isocyanates. Thorax 31:127, 1976.