Combined alveolitis and asthma due to hexamethylene diisocyanate (HDI), with demonstration of crossed respiratory and immunologic reactivities to diphenylmethane diisocyanate (MDI)

Combined alveolitis and asthma due to hexamethylene diisocyanate (HDI), with demonstration of crossed respiratory and immunologic reactivities to diphenylmethane diisocyanate (MDI) Jean-Luc Malo, M.D., Gilles Ouimet, M.D., And& Car-tier, M.D., D. Levitz, B.S., and C. Raymond Zeiss, M.D. Montreal, Canada, and Chicago,

Ill.

A worker exposed intermittently

to hexamethylene diisocyanate (HDI) developed episodes of dyspnea, wheezing, and fever on working days. Complete lung function tests performed when the subject was asymptomatic were normal except for increased airway responsiveness to histamine, which significantly improved after a 3 wk period off work. At that time, specific inhalation challenges with HDI were carried out. After being exposed for 5 min, the subject developed general malaise, cough, fever, and leukocytosis, together with a mixed restrictive and obstructive breathing defect. We demonstrated a subsequent increase in airway hyperexcitability, which lasted for 2 mo. The subject was also challenged with diphenylmethane diisocyanate (MDI) for I5 min. A late obstructive reaction was documented. Increased levels of specific IgG antibodies against HDI-human serum albumin (HSA) and MDI-HSA were demonstrated. (J

ALLERGY CLINIMMUNOL 72:4/3-419, 1983.)

Fuchs and Valade’ first described the respiratory effect of isocyanate exposure. Since then, several reports of bronchiaP6 and possible alveolar reactions7-lo to isocyanateshave been published. We here describea subject who developed respiratory and systemic symptomsafter exposure to HDI at work. Specific inhalation challenges with I-ID1 suggesteda mixed alveolar and bronchial response.We also documenteda late obstructive reaction after challenge with MDI. Specific IgG antibodiesto HDI-HSA and MDI-HSA were found. CASE REPORT The subject was a 56 yr old man working as a foreman in a garage where painting was done. One of the paints used

From the Service de Pneumologie, Hopital du Sack-Coeur, Montreal, Canada, and the Section of Allergy-Immunology, Department of Medicine, Northwestern University Medical School, Chicago, 111. Dr. Zeiss was supported by U.S. Public Health Service Grant HL 15389 and the Veterans Administration. Received for publication Dec. 7. 1982. Accepted for publication May 16, 1983. Reprint requests: Doctor Jean-Luc Malo, Service de Pneumologie, Hopital du Sack-Coeur, 5400 West Gouin Blvd., Montreal, Quebec, Canada H4J 1C5.

consisted of an enamel containing aliphatic ester (14%) and ether ester (29%). This enamel was mixed 3 parts for 1 with a polyisocyanate activator, which was a mixture of aliphatic ester (27%), ether ester (21%), xylene (14%). toluene (6%), and polymeric HDI (7%). When first seen, this patient reported that he had had, in the past 5 to 6 yr, episodes of shortness of breath, wheezing, malaise, and chills. These symptoms occurred on the late afternoons of working days and lasted for several hours. He also reported wheezing at night, which on one occasion lasted several days after cessation of work exposure. The subject had stopped smoking 8 yr before and had no personal nor familial atopic history. He did not live on a farm and had no birds at home. In the past, he had an episode of pulmonary infiltrates with peripheral eosinophilia, which was attributed to ampicillin. At the time the subject was first seen, he was asymptomatic. Chest auscultation showed no added sound. The chest radiograph was interpreted as normal.

METHODS Lung function Lung

volumes

tests were

measured

in a flow-displacement

body plethysmograph as described by Leith TLC and its subdivisions were determined Boyle’s law after the technique of DuBois et electrically integrated to derive FEV, Pleural estimated

with

a 10 cm eosphageal

balloon

and Mead.” according to al.” Va was pressure was

positioned

in the 413

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et al.

Abbreviations used HDI: Hexamethylene diisocyanate MDI: Diphenylmethane diisocyanate HSA: Human serum albumin PBS: Phosphate-buffered saline FE V, : Forced expiratory volume- 1 set FVC: Forced vital capacity VC: Vital capacity FRC: Functional residual capacity TLC: Total lung capacity PCp,: Concentration of histamine causing a 20% fall in FEV, SC,,: Specific lung conductance DL,.(): CO transfer factor of the lung V,: Buccal Bow R,: Lung resistance ELISA: Enzyme-linked immunosorbent assay

lower third of the esophagusI and connected at one side of a Validyne differential pressure transducer (mean pressure 45 ? 100 cm H20) via a 100 cm polyethylene catheter. The other side of the transducer was connected to an oral pressure tap to give transpulmonary pressure. Transpulmonary pressure compensated for lung volume (TPPc) and Va were continuously monitored on an oscilloscope and recorded on paper. The average RL value was calculated from the paper recordings by relating peak-to-peak changes in V, and TPP, from five or more consecutive spontaneous breaths. sGr was derived from R, and FRC. Transfer factor (DLco) was obtained from the steady-state measurement of the end-tidal carbon monoxide concentration. I4 Reference values were obtained from Goldman and Becklakels for lung volumes, from Cotes et al.r6 for FEV,, and from Bates et al. I4 for DL,, Histamine inhalation challenges were performed at the same time of the day with a Wright’s nebulizer according to a standardized procedure.rr

Specific inhalation

challenges

These tests were carried out in a large (15 by 20 ft) painting room in the hospital workshop. Measurement of isocyanate concentration was performed with a MDA Autospot instrument (MDA Instruments, Poole, England). The subject was asked to reproduce the working environment by painting a board with an aerosolized liquid mixture. This aerosol was produced by a sprayer activated by compressed air. The enamel and activator containing polymeric HDI were mixed as suggested by the manufacturer. The following sequence of tests was done on three different days: (1) control day (no exposure); (2) 5 min exposure to the mixture of enamel and activator containing HSI; (3) 5 min exposure to enamel only. The tests were started after a 3 wk period away from work. Inhalation challenge with the enamel only (day 3) was performed 3 wk after the challenge with the enamel and activator (day 2). so that lung volumes, FEV,. FVC, and DL,, were back to baseline values.

CLIN. IMMUNOL. OCTOBER 1983

3.8 34. z ” 2

30-

261 221

9 ~~~~~~

-10-T---W’““” 0 40 MINUTES

60

13

24

HOURS

FIG. 1. Changes of body temperature, FVC, and FEVJFVC after exposure to enamel and activator containing HDI (closed circles) as compared with exposure to enamel alone (open circles).

In addition, challenges with polymeric MDI, to which the subject had not been exposed previously, were carried out 14 mo after the initial challenge with HDI. The subject was then exposed for 5 and 15 min on two consecutive days. On the control and challenge days, FEV, and FVC were measured before the test, every 10 min for 1 hr. and every hour for at least 7 hr afterwards. These tests were performed on a Vitalograph apparatus (Vitalograph Ltd., Buckingham, England). Body temperature was recorded hourly. Complete lung function tests were performed on each day. On days 2 and 3 of HDI and enamel exposure and on both days of MD1 exposure, those were done 5 to 6 hr after the end of challenge.

Immunologic

tests

Blood was drawn at a time of the reaction to HDI (day 2), 5 mo later during which time the patient had been continuously away from work, and before the challenge with MD1 (14 mo after the challenge with HDI). Serum was stored at -20” C. Two methods were used to determine antibody activity to MD1 and HDI: total antibody binding of HDIHSA conjugate, using an ammonium sulfate precipitation techniqueg; and an ELISA providing measurements of IgG and IgE antibodies to MDI-HSA and HDI-HSA.rR Conjugation of MDI-HSA and HDI-HSA. The MD1 was covalently linked to HSA (Cappel Laboratories, Cochranville, Pa.) as previously described.g The HDI-HSA conjugate was kindly supplied by I. Leonard Bernstein, M.D..

VOLUME NUMBER

TABLE

72 4

Alveolitis

I. Lung function

and asthma due to hexamethylene

diisocyanate

415

results Day Predicted

TLC (L) vc CL) FRc CL) RV/TLC (%) FEV, CL) FEV,/FVC (%) DLc() (ml/min/mm Hg) SC,, (set-’ cm H,O-i)

Day

5.99 3.90 3.48 35 3.05 72 15.1 -

-26

Day

5.58 3.95 2.42 29 2.90 77 13.3 ND

1

-1

Day 6

BBDT

ABDT

Day 3

ND ND ND ND 2.83 81 13.4 ND

5.15 2.15 3.10 47 1.83 72 12.2 .07

5.33 3.45 2.48 35 2.31 72 ND .I4

5.58 3.40 2.48 39 2.58 80 ND .I7

5.50 3.80 2.30 31 2.58 75 ND ND

Day

17

5.80 3.80 2.70 34 2.80 78 12.9 ND

BBDT = before bronchodilator (2.5 mg of salbutamol in aerosol); ABDT = after bronchodilator; ND = not done; RV = residual volume. Day 0 is the challenge day with enamel plus HDI. Twenty-six days earlier (day -26), the subject had lung function tests at the end of a day spent at work; the subject worked until day -21 and was off work afterwards. Day 17 is the challenge day with enamel only. Values of day 0 and day 17 were obtained 5-6 hr after exposure to enamel plus HDI and to enamel alone, respectively.

University of Cincinnati Medical Center, Cincinnati, Ohio, and was prepared as previously described.lg Rudioiodinarion of HDZ-HSA. HDI-HSA (100 pg) was labeled with iz51 by a modification of the chloramine T method.g The radiolabeled HDI-HSA had a specific activity of 25,000 counts per minute per nanogram, and 97% of the labeled protein was bound in antibody excess. ELISA reagents. Alkaline phosphatase-conjugated antihuman IgG and p- nitrophenyl phosphate substrate were obtained from Sigma Chemical Co., St. Louis, MO. Total antibody activity HDI-HSA Total serum antibody binding of HDI-HSA was measured in the following manner. Increasing concentrations of radiolabeled HDI-HSA (2000 to 20,000 ng) were added to 0.1 ml of 1: 5 dilution of serum and the micrograms of labeled HDI-HSA bound were determined at a point of antigen excess. The results were expressed in micrograms of HDI-HSA bound per milliliter of serum after subtraction of control binding. ELISA activity.

method

for

detection

of IgG

and

IgE

antibody

A modification of the ELISA method of Engvall and PerlmanniR using rigid microtiter plates (Dynatech Laboratories, Inc., Alexandria, Va.) was used to measure IgG antibody to MDI-HSA and HDI-HSA. The wells were coated with MDI-HSA and HDI-HSA (200 @g/ml) in O.lM NaHCO, for 24 hr. Serial dilutions of the subject’s and control sera (from unexposed healthy laboratory staff and from one individual with asthma on exposure to HDI without evidence of alveolitis) from 1: 100 to 1: 10,000 with 5% bovine serum albumin in PBS was added to the wells and incubated at room temperature for 2 hr. Alkaline phosphatase-conjugated anti-human IgG and IgE diluted 1: 100 in PBS was added and incubated for 2 hr at room temperature. The plates were washed three times with PBS between each addition. The substrate (p-nitrophenyl phosphate) was added to the wells for 30 min at 37” C. The color developed was measured in a Dynatech Microelisa Minireader at 405 nm. The uptake of enzyme-labeled antiserum, and hence optical density, increased with

the amount of IgG and IgE antibodies in the patient’s serum that reacted with the solid-phase absorbed MDI-HSA and HDI-HSA.

RESULTS Initial investigation

The subject was tirst given a mini-Wright peak flowmeter (Clement Clarke International, Ltd., London, England). Daily measurement (two to four times per day) of peak flow rates for 1 mo, including 1 wk off work and 3 wk at work when he was exposed to HDI only intermittently (a few minutes two to three times a week), failed to demonstrate significant variations. At the end of this period, after a day spent at work, complete lung function tests were done at a time the subject was asymptomatic. Except for a reduced value for FRC, these tests could be interpreted as normal (day -26, Table I). Five days later (day -21), after a day spent at work, the histamine inhalation test showed that the provocative PCzO was 3 mg/ml, thus showing mild airway hyperexcitability. Leukocyte count was 5400/mm3, with 3% eosinophils . Sedimentation rate was 18 mm/hr. Specific inhalation challenges to HDI

The subject had been off work for 3 wk when the specific inhalation challenges were started. On the control day when the patient was not exposed to any of the products (day - 1, Table I), no significant changes of body temperature, FEV,, or FVC were seen. When the subject was challenged with enamel and activator containing I-RX (maximum air concentration of isocyanate = 0.02 ppm) for 5 min (day O), no symptoms were recorded in the first hour. There was no nausea, headache, rhinitis, conjunctivitis, or

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END OF EXPOSURE

CLIN. IMMUNOL. OCTOBER 1983

AT WORK

INHALATION

CHALLENGE

22 67 I/:‘!

2 58\2-80 I

10

2 72 I

30

1

I

50

{ 1

BASELINE FEV,(Il

DAYS

FIG. 2. Changes in the provocative PC,, after histamine inhalation, (day -21), after a period off work (day - I), and after inhalation Accepted range of reproducibility of the test, corresponding to difference around the value of day -1; asterisk, 6% change nebulization of 16 mglml, so that the precise value of PC,,, was mglml.

cough. Sixty minutes after the end of exposure, the subject reported a burning sensation in his chest, which was accompanied by cough. In the two following hours, the FVC dropped but the FEV,/FVC ratio did not change significantly (Fig. 1). The subject then had a productive cough. From the third hour onward, the subject experienced chills, headache, and malaise, and 6 hr after the exposure he was prostrated. Physical examination revealed bibasal inspiratory crackles. No wheezes were heard. There was a marked increase in body temperature together with a significant fall in FVC. Although the maximum variation in the FEV,/ FVC ratio was 6% on the control day, the greatest change after HDI exposure was 11%. Leukocyte count was 16,500 (89% segmented forms, 0% eosinophils). Sedimentation rate was 35 mm/hr. The chest radiograph was normal. Six hours after exposure, complete lung function tests were performed (day 0, Table I). TLC and VC were decreased and FRC was higher. sGL was low and Pao, was 53 torr. Thereafter, the clinical condition improved and physical signs regressed. On the day after the challenge (day 1, Table I), lung volumes, FPV, , and sGr, improved. FPV, and sGL gained 12% and 20%. respectively, after bronchodilator. Leukocyte count was down to 12,200 (81% segmented forms, 2% eosinophils) and sedimentation rate was 39 mm/hr. Pao? had improved to 81 torr. When FEV, and FVC were back to baseline values (day 17, Table I), the patient was challenged with the enamel alone. No significant reaction occurred Fig. 1).

at the end of exposure at work challenge to HDI. Shaded area, a single twofold concentration in FEV, was documented after not obtained but was above 16

Fig. 2 shows the results of PCzOfrom the time the patient stopped working until 53 days after the challenge. A reduced PCzO (3 mg/ml) was documented when the subject stopped work, thus showing increased bronchial hyperexcitability. Three weeks later, PCzOimproved to 16 mg/ml. However, after the challenge, PCzOagain fell, even when baseline FEV, was within 10% of the prechallenge value. There was a progressive recovery afterwards. Specific

inhalation

challenges

to MDI

Because the initial immunological investigation suggested high levels of antibody to MDI-HSA, we decided to perform specific inhalation challenges with MDI. These tests were carried out 14 mo after the initial tests with HDI, at a time he was asymptomatic and had not been unexposed to HDI since day 0. The subject first had a control day when he was not exposed to any product. He was then exposed to MD1 for 5 and 15 min on two consecutive days. On each occasion, the maximum level of isocyanate measured in the air of the challenge room was 0.02 ppm. Whereas he reported no symptoms after being exposed for 5 min to MDI, he had dyspnea and cough 5 to 6 hr after the end of the challenge for 15 min. There was a fall in FEV,, FVC, and the FEV,/FVC ratio several hours after the challenge (Fig. 3). There was no significant change in DLco . PC,, dropped from 14 mg/ml before challenge to 3.7 mg/ml the day after the challenge, at a time when baseline FEV,‘s were 2.82 and 2.60 L, respectively. PC,,, was back to 13 mg/ml 2 wk later, with a baseline FEV, of 2.86 L.

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Alveolitis

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HDI-I-ISA

diisocyanate

417

MDI-HSA

.22.8 0

* I

0 1

500

2500 RECIPROCAL

.#+I/ 0

20

40

60 90

I 2

4HO”&

a

,

t 24

MINUTES

FIG. 3. Changes of FEV,, FVC, and FEV,/FVC ratio after exposure to MDI (closed circles) as compared with a control day (closed circles) when there was no exposure to any product.

Immunologic

results

The immunological investigation included the assessment of total antibody binding of HDI-HSA by using an ammonium sulfate precipitation technique. The levels were 100 pug of HDI-HSA bound per milliliter at the time of challenge with HDI, which dropped to 28 and 24 vg 5 and 14 mo later, respectively Specific IgG antibodies to HDI-HSA and MDIHSA were detected by the ELISA method. These results are illustrated in Fig. 4 for two different serum dilutions, 1:500 and 1:2500. At the 1:500 dilution, the levels of antibodies to HDI-HSA and MDI-HSA were very high. These levels dropped at the two subsequent visits when the subject was no longer exposed to HDI. The levels at the 1: 2500 serum dilution were lower against MDI-HSA than HDI-HSA. At these serum dilutions the control with MD1 asthma had the same low optical density readings as unexposed laboratory workers. Specific IgE antibodies to HDI-HSA and MDI-HSA were not detected. DISCUSSION Asthmatic reactions to isocyanates have been well described.‘. 5, 6 The hypersensitivity pneumonitis type of response has also been more recently docu-

OF SERUM

DILUTION

FIG. 4. Assessment of specific IgG antibodies by the ELISA method at two different serum dilutions, and expressed as optical density. Closed circles, triangles, and squares, Values obtained at the time of challenge with HDI and 5 and 14 mo later, respectively; open circles, control values.

mented.7-‘0 We have evidence that our subject presented both an alveolar and bronchial reaction to HDI. Alveolitis was suggested by the symptomatology, fever (maximum 38.8” C), inspiratory crackles, and high leukocyte count ( 16,500/mm3) after specific inhalation challenge. We also found a restrictive breathing defect together with a marked reduction in Paoz. The chest radiograph remained normal and transfer factor by the steady-state method did not decrease significantly. Single-breath CO measurements might have been more sensitive to detect the reaction. However, Hendrick et al. 2o showed that a temperature above 37.2” C, increase in leukocyte count of 2500 or more, and a fall in FVC of 15% or more were sensitive indices of a positive alveolar response after inhalation challenge and that chest radiograph and transfer factor were less sensitive. We also found evidence of a bronchial reaction to HDI. The FEV,/FVC ratio dropped slightly after exposure but more than on the control day. There was an increase in FRC together with a fall in sG,, which suggests hyperinflation and airway obstruction. The presence of a satisfactory response to bronchodilator on the day after the challenge also suggests airway involvement. Monitoring of peak expiratory flow rates at work and off work did not reveal significant fluctuations, probably because the subject was exposed only intermittently to HDI at work. However, we were able to document an increase in bronchial hyperexcitability at the time the patient left

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work. After recovery, we induced a similar increase in hyperexcitability after inhalation challenge. These changes were followed by progressive recovery. In occupational asthma, similar increase in nonallergic bronchial hyperexcitability after a challenge has been documented by others ” in the case of nonimmediate reactions. Our subject was not exposed to “pure” HDI either at work or in the laboratory. Indeed. the enamel activator also contained aliphatic and ether esters, xylene, and toluene. Since aliphatic and ether esters were also included in the enamel paint to which the subject was exposed on the control day (day 17), we can reasonably assume that these agents were not responsible for the reaction. It would also be unlikely that xylene and/or toluene were the offending agents, since they have never been described as causing any specific asthmatic or alveolar reactions. Specific IgE antibodies to isocyanates are demonstrated occasionallyz2 or not*” in occupational asthma due to this group of agents. Specific IgE antibodies were not found in our subject. The presence of specific IgE antibodies would have been unlikely due to the nature of the reaction, which was nonimmediate alveolitis and asthma. Specific IgG antibodies have been found in cases of alveolitis due to isocyanates.“, lo We were able to document the presence of these antibodies in our subject at levels comparable to those in cases previously described. Furthermore, the IgG antibody levels dropped significantly 5 mo after cessation of exposure. Our worker experienced a late obstructive and restrictive breathing defect after exposure to HDI and a late obstructive reaction after challenge to MDI. Crossed asthmatic reactions to different types of isocyanates have been described.” These crossed bronchial reactions may be linked with the crossed immunoreactivity , which was found in the present study and recently by others al~o.*~ Since the molecular structure of HDI, which is aliphatic, and MDI, which is aromatic, are not related, the most likely explanation for this crossed immunologic reactivity could be that the isocyanate-protein linkage is the major antigenic determinant to which antibody is directed . We

thank

Miss

Lyette

Rochon

for secretarial

assistance.

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CLIN. IMMUNOL. OCTOBER 1983

3. Adams WGF: Lung function of men engaged in the manufacture of tolylene diisocyanate (TDI). Proc R Sot Med 63:378. 1970. 4. Diem JE. Glindmeyer H, Dharmarajan V. Buthcer B. Jones RN, Weill H: Longitudinal evaluation of the respiratory health effects of diisocyanate exposure in a new manufacturing plant. Am Rev Respir Dis 191(Suppl.):231. 1980 (abst.). 5. O’Brien IM, Harries MG, Burge P.S. Pepys J: Toluene diisocyanate-induced asthma. I. Reactions to TDI. MDI. HDI and histamine. Clin Allergy 9: 1. 1979. 6. Butcher BT, Salvaggio JE. Weill H. Ziskind MM: Toluene diisocyanate (TDI) pulmonary disease: immunologic and inhalation challenge studies. J ALLERGY CLIN IMMUNOL 58:89. 1976. 7. Charles J. Bernstein A. Jones B, et al: Hypersensitivity pneumonitis after exposure to isocyanates. Thorax 31: 127. 1976. 8. Fink JN, Schlueter DP: Bathtub refinisher’s lung: an unusual response to toluene diisocyanate. Am Rev Respir Dis 118:955, 1978. 9. Zeiss CR. Kanellakes TM, Bellone JD. Levitz D, Pmzansky JJ. Patterson R: Immunoglobulin E-mediated asthma and hypersensitivity pneumonitis with precipiting anti-hapten antibodies due to diphenylmethane diisocyanate (MDI) exposure. J ALLERGY CLIN IMMUNOL 65:346. 1980. 10. Malo JL. Zeiss CR: Occupational hypersensitivity pneumonitis after exposure to diphenylmethane diisocyanate. Am Rev Respir Dis 125:113, 1982. 11. Leith DE, Mead J: Principles of body plethysmograph. In Procedures for standardized measurements of lung mechanics. Distributed by the Division of Lung Disease, National Heart, Lung and Blood Institute, Bethesda, Md., 1974. 12. DuBois AB, Botelho SY, Bedell GN. Marshall R, Comroe JH: A rapid plethysmographic method for measuring thoracic gas volume: a comparison with a nitrogen washout method for measuring functional residual capacity in normal subjects. J Clin Invest 35:322. 1956. 13. Milic-Emili J. Mead J. Turner JM, Glauser EM: Improved techniques for estimating pleural pressure from eosphageal balloons. J AppI Physiol l&207. 1964. 14. Bates DV. Macklem PT. Christie RV: Respiratory function in disease, ed. 2. Philadelphia. 1971, W. B. Saunders Co.. pp. 80-81, 93. 15. Goldman HI. Becklake MR: Respiratory function tests: normal values at median altitudes and the prediction of normal results. Am Rev Tuberc 79:457. 1959. 16. Cotes JE, Rossiter CE. Higgins ITT, Gilson JC: Average normal values for the forced expiratory volume in white Caucasian males. Br Med J 1:1016, 1966. 17. Cockcroft DW, Killian DN. Mellon JJA , Hargreave FE: Bronchial reactivity to inhaled histamine: a method and clinical survey. Clin Allergy 2235, 1977. 18. Engvall E. Perlmann P: Enzyme linked immunosorbent assay, Elisa. III. Quantitation of specific antibodies by enzymelabeled anti-immunoglobulin in antigen-coated tubes. J Immunol 109:129, 1972. 19. Chen SE, Bernstein IL: The guinea pig model of diisocyanate sensitization. I. Immunologic studies. J ALLERGY CLIN IMMUNOL 70:383, 1982. 20. Hendrick DJ , Marshal R. Faux JA , Krall JM: Positive “alveolar” responses to antigen inhalation provocation tests: their validity and recognition. Thorax 35415, 1980. 21. Cockcroft DW, Cotton DJ, Mink JT: Non-specific bronchial

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hyperreactivity after exposure to western red cedar: a case report. Am Rev Respir Dis 119:505, 1979. 22. Butcher BT, O’Neil CE, Reed MA, Salvaggio JE: Radioallergosorbent testing of toluene diisocyanate-reactive individuals using p-tolyl isocyanate antigen. J ALLERGY CLIN IMMUNOL 66:213. 1980. 23. Danks JM. Cromwell 0. Buckingham JA. Newman-Taylor

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AJ. Davies RJ: Toluene-diisocyanate induced asthma: evahration of antibodies in the serum of affected workers against tolyl-monoisocyanate protein conjugate. Clin Allergy 11:161, 1981. 24. Baur X: Immunologic cross-reactivity between different albumin-boundisocyanates. JALLERGYCLINIMMUNOL~~:~~~, 1983.