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Adaptive immunity of the tympanic membrane
Adaptive Immunity of the Tympanic Membrane JACQUES F. POLIQUrN, M.D.,* ANTONIO CATANZARO, M.D.,t lAMES ROB~, M.D.,* AND MAURICE SCHIFF, M.D.w
In a previous experiment an antiserum was developed in the rabbit from the lamina propria of the tympanic membrane of the guinea pig. In the present study 18 Hartley guinea pigs were used in an in vivo experiment in which the antiserum (RAGYM IgG) served passively to immunize the animals subjected to various 'forms of trauma to the right tympanic membrane. Two groups (18 animals) immunized with normal rabbit IgG or normal saline served as controls. The left tympanic membrane remained untouched in all groups and served as an internal control. Various forms of trauma (infection, cauterization, section), various times (one to 21 days), and diverse techniques of staining (immunofluorescence,.complement, and immunoperoxidase) were studied. The results indicate that the combination of trauma and sensitization in the first group evokes a particular response in the lamina propria of the tympanic membrane in immunized animals (RAGTM IgG). The form of'trauma does not influence the results, but time seems to be an important factor. This work addresses several questions concerning the possible role of the combination of trauma and sensitization in conditions that involve the tympanic membrane and middle ear clinically.
The particular response of the t y m p a n u m to antigenic stimulation has become a perplexing issue. ~.2 Multiple theories have been put forward in order to characterize the specific low grade i m m u n e response of the middle ear as evidenced in the field of homograft surgery and chronic otitis media, a' 4 To cite only a few, the role of preservatives, the small area of contact between, the t y m p a n i c constituents, and the concepts of privileged site and tolerance have been advocated to no avail. More recently the i m m u n o l o g i c role of the eard r u m has deserved particular attention. 5 The structure and biochemical composition of the t y m p a n i c m e m b r a n e are now well characterized,
but the pathophysiologic basis of lesions of the membrane is still poorly understood. Some data are highly suggestive of an immunopathologic event in lesions of the tympanic membrane in such diseases as serous otitis media and tympanosclerosis. 6 In our previous study the antigenic properties of the tympanic membrane were examined. In order to characterize the immune response of the tympanic membrane in the guinea pig, an antiserum that reacts with the guinea pig tympanic membrane had been developed in the rabbit (RAGTM IgG). The present report is concerned with the in vivo experiment in which the antiserum consisting mainly of IgG was studied with
Accepted for publication January 28, 1981. This work was supported by the American Otological Society, the Duaei Research Foundation, and Centre de Recherche M6dicale de l'Universit6 de Sherbrooke. *Chairman and Professor, Department of Otolaryngolagy, Centre Hospitalier Universitaire, Universit6de Sherbrooke, Sherbrooke, Quebec, Canada. +Assistant Professor, Department of Medicine, University of California, San Diego, School of Medicine, San Diego, California. *Associate Professor, Department of Pathology, Scripps Clinic and Research Foundation and Green Hospital, La ]ella, California. {}Clinical Professor, Department of Otolaryngology, University of California, San Diego, School of Medicine, San Diego,
California.
94
American Journal of Otoiaryngology--Volume 2, Number 2, May 1981
TABLE 1.
Material and Methods
GRouP I (18 ANhMALSI*
GROUP[! (18 ANIMALS)
RAGPTMIgG Myringotomy (right tympanic membrane) Cauterization (right tympanic membrane} Infection (right tympanic membrane)
A NH fgG
B Saline
6
3
3
6
3
3
8
3
3
*Left tympanic membrane kept intact in all groups as an internal control.
s t a n d ar d t e c h n i q u e s of i m m u n o f l u o r e s c e n c e staining, complement reactivity, and immunoperoxidase.
MATERIAL AND METHODS
Thirty-six Hartley guinea pigs, weighing 300 to 400 grams, were divided into two groups of 18 animals. All animals were anesthetized according to the m e t h o d of Evans. T In the 18 animals of group I, rabbit antiguinea pig tympanic membrane IgG (RAGTM IgG; 0.5 ml.) was injected by intracardiac puncture. In group II nine animals (group IIa) received, by the same route, passive immunization with normal rabbit IgG (NR IgG), and in nine others normal saline was injected (group IIb). One hour after immunization the f o l l o w i n g m a n i p u l a t i o n s were performed on only the right eardrum (the left earq drum remained untreated as an internal control) in all groups: In group I, six animals u n d e r w e n t a myringotomy on the right t y m p a n i c membrane, in six animals a 10 per cent solution of tricholoacetic acid was applied to the right tympanic membrane, and in six other animals a solution containing 106 viable S t r e p t o c o c c u s v i r i d a n s micro-
TABLE 2.
Group I
organisms in 0.1 ml. of m e d i u m was injected into t h e m i d d l e ear v i a a 22 g a u g e n e e d l e through the tympanic m e m b r a n e (Table 1). Animals of groups IIa and IIb were divided in the same manner (six myringotomies, six cauterizations, six infections). The timing of the anesthesia, immunization, and trauma was similar in groups I, IIa, and IIb. After trauma all groups were again separated according to time and t e c h n i q u e of staining. S p e c i m e n s were thus o b t a i n e d at 24 h o u r s , seven days, and 21 days after trauma (Table 2). With the Cryostat, frozen sections, 5 microns thick, were obtained from the left and right tympanic membranes and used for immunofiuorescence staining with an indirect m e t h o d by the addition of fluoresceinated goat antirahbit IgG. Frozen sections were also e x a m i n e d with rhodomino labeled rabbit antiguinea pig C3. Permanent sections were prepared and studied with peroxidase labeled, goat antirabbit IgG. The reaction was graded for all the stains used from 0 (negative) to v r + (strongly positive) on coded specimens. The tissue sections w ere examined with a Leitz Diavert fluorescent microscope using direct and transmitted light w i t h FITC filters. Photographs were taken with a Leitz Orthomat camera and Ektachrome (200 ASA).
RESULTS
Tables 3 through 6 summarize the results obtained in the different groups. Figure 1 shows a moderate immunof~uorescence staining reaction ( + + ) at the site of a myringotomy in a seven day speci m en of the right t y m p a n i c membrane from group I. Figure 2 shews a moderate (++) reaction in the lamina propria of the tympanic m e m b r a n e using immunoperoxidase staining in a seven day speci-
Material and Methods: Schedule
NUMBEROFANIMALS Group Ha Group lib
r,ME
of Experiment TRAUMA
STAINING
2 2 2
1 1 1
1 1 1
24 hours
[ Myringotomy l Infection Cauterization
Complement C:~ Immunoperoxidase Immunofluorescence
2 2 2
1 1 1
1 1 1
7 days
[ Myringotomy l Infection Cauterization
Complement Ca Immunoperoxidase Immunefluorescence
2 2 2
1 1 1
1 1 1
21 days
[ Myringotomy l Infection Cauterization
Complement C3 Immuneperoxidase Immunofluorescence
JACQUES F. POLIQUIN ET AL.
Volume 2 Number 2 May 1981
95
American
TABLE 3.
Group I, Right Tympanic Membrane, lmmunofluorescence Study*
TABLE 5. Group I, Right Tympanic Membrane, Immunoperoxidase Study*
TIME
MYRINGOTOMY
TIME
Journal of
Otolaryngology
24 h o u r s 7 days 2:1_ d a y s
++1+ ++ ++ +/+ +
CAUTERIZATION ++1+++ ++ +/+ +
INFECTION ++/+++ ++ +/+ +
m e n (infected t y m p a n i c membrane, group I). Figure 3 shows a typical positive C3 reaction (1-1-) at the seventh d a y in the traumatized tympanic membrane from an animal of group I.
DISCUSSION Our antiserum was made from the lamina propria of the guinea pig tympanic membrane. The m i d d l e layer of the eardrmn is composed of fine fibrils and h o m o g e n e o u s interfibrillar ground substanceY These fibrils are neither true collagen nor true elastin, for they differ in both size and chemical contentY The t y m p a n i c finer fibrils contain amino acids that are present in both collagen and elastin. It becomes evident from our observations that the connective tissue elements of the guinea pig tympanic m e m b r a n e are quite antigenic. The antigenic element of the tympanic membrane most probably resides in the fibrous portion or in the ground substance or in both. A microfibrillar protein that is w i d e l y distributed in connective tissue and is associated with both elastin and collagen fibers could be responsible for the antigenicity we observed in the lamina propria. 1~ The immune response in our animals to the combination of t r a u m a and passive immunization to their own t y m p a n i c membrane illustrates the need for comparing the effects of heterologous and autologous antisera. The lamina propria could be the source of hidden antigens, and trauma could precipitate an antigen-antibody reaction in some individuals. Such an immunepathologic event, as evidenced in Hashimoto's thyroiditis, could be the explanation for the hya-
TIME 24 h o u r s 7 days 21 d a y s
4.
G r o u p I, Right T y m p a n i c Complement Study* MYRINGOTOMY ++ ++/+++ 0
+ + / + +, 0
+ +/+ + 0
line degeneration seen in tynnpanosclerosis after multiple myringotomies, tl The t y m p a n i c membrane could be the site of an a u t o i m m u n e process, but more data are necessary in order to apply Witebsy's criteria, t2 Our experiment s h o w e d that t r a u m a alone does not induce a spontaneous i m m u n e reaction. In group I [RAGTM IgG) the c o m b i n a t i o n of immunization and trauma seems to elicit a homing of antibody at the site of trauma. The inflammatory process that occurs w i t h d a m a g e to the lamina propria could increase vascular permeability, leading to leakage of p l a s m a proteins. This could be the mechanism for e n t r y of circulating antibodies into the t y m p a n u m during inflammation or infection. The demonstration of bound antibody in the l a m i n a propria, capable of binding complement and of persisting for weeks, indicates that formation of immune complexes could be involved in t y m p a n i c membrane diseases, especially those related to insult. If the form of trauma does not i n f l u e n c e our results, time, on the other hand, seems to be an important factor. It would be interesting to observe the results of a more prolonged experiment. In conclusion, our experiment has s h o w n that the lamina propria of the guinea pig t y m p a n i c membrane can be antigenic in the rabbit. In sensitized guinea pigs tympanic t r a u m a evokes a particular immune response in the l a m i n a propria during the time of the acute inflammation. These data suggest the possibility of a specific immunopathologic event in the t y m p a n i c membrane t h a t could e n l i g h t e n us r e g a r d i n g the pathophysiology of some ear diseases.
Membrane,
CAUTERIZATION ++ ++t+++ 0
+ +/++ 0
INFECTION
*Left t y m p a n i c m e m b r a n e : n o reaction.
*Left t y m p a n i c m e m b r a n e : no reaction.
TABLE
24 h o u r s 7 days 21 days
MYR/NGOTOMY CAUTERIZATION
INFECTION ++ ++/+++ O
TABLE 6. Groups IIa and lib (Left and Right Tympanic Membranes) TIME
~MMHNOFLUORESCENCE
IMMUNOPERO XIDASE
C:~
24 h o u r s 7 days 21 days
0 0 0
0 0 0
0 0 0
*Left t y m p a n i c r n e m h r a n ~ : ne reaction.
96
ADAPTIVE IMMUNITY OF THE T Y M P A N I C M E M B R A N E
Volume 2 Number 2 May 1981
Figure I. Right tympanic membrane, traumatized, group I, seven days.
Figure 2, RLght t y m p a n i c membrane, traumatized, group I, seven days. (• lO.)
JACQUES F. POLIQUIN ~T AL.
97
American Journal of
Otola ryngology
Figure 3. Moderately positive reaction to C~, right traumatized tympanic membrane, group I, seven days.
References 1. Veldman, J, E., Kuijpers, W., and Overbosch, H. C.: Middle ear implantation: its place in the immunohistophysiology of l y m p h o i d tissue. Clin. Otolaryngol,, 3:93-102, 1978. 2. Poliquin, J,, at el.: [mmunocompetence of the guinea pig's middle ear. J, Otol., 8:385-389, 1979. 3, Gagnon, N. B,, eta].: Homograffs of the middle ear. Arch. Otolaryngo[., 105:35-38, 1977. 4. Marquet, J, F. E.: I-[istorical notes on homografts, Oto[aryngoI, Clin, N. Am,, 10:479-485, 1977. 5. Poliquin, J,, et el.: Antigenicity of the guinea pig's tympattie membrane. Otolaryngol, Head Neck Surg., 87:852-858, 1979. ft. Veltri, R. W., and Sprinkle, P. M,: Secretory otitis media. An immune complex disease. Ann. Otol. Rhinol, Laryngol., 85(Suppl,):135--139, 1976. 7, Evans, E. F.: Neuroleptanesthesia for the guinea pig. Arch. Otolaryngol., 105:185-186, 1979.
98
8. Lim, D. J.: Tympanic membrane: electron microscopic observation. I. Pars tensa. Acta OtolaryngoI, 66:181198, 1968. 9. Johnson, F. R., McMinn, R. tI. M., and At.field, G. N,: Ultrastructural and biochemical observations on the tympanic membrane. ]. Anat,, I03:297-310, 1968. 10. Bystrm, J. C.: Clinical significance of basal cell layer antibodies. Arch. Dermsto[., 113:1380-1382, 1977. 11. Birck, H. G., and Mravec. J, J.: Myringostomy for middle ear effusions. Results of a two-year study. Am~. Otol. Rhinol. Laryngol., 85(Suppl.):263-267, 1976. 12. Barrett, ]. T.: Textbook of Immunology, St. Louis, The C. V, Mosby Company, 1974, Ch. 12.
Department of Otolaryngology Celltro Hospitalier Universitaire 3001 12th A v e n u e North Sherbrooke, Quebec J3H 5N4 Canada (Dr, Poliquin)
ADAPTIVE IMlvI[JNITY OF THE TYMPANIC MEMBRANE
In a previous experiment an antiserum was developed in the rabbit from the lamina propria of the tympanic membrane of the guinea pig. In the present study 18 Hartley guinea pigs were used in an in vivo experiment in which the antiserum (RAGYM IgG) served passively to immunize the animals subjected to various 'forms of trauma to the right tympanic membrane. Two groups (18 animals) immunized with normal rabbit IgG or normal saline served as controls. The left tympanic membrane remained untouched in all groups and served as an internal control. Various forms of trauma (infection, cauterization, section), various times (one to 21 days), and diverse techniques of staining (immunofluorescence,.complement, and immunoperoxidase) were studied. The results indicate that the combination of trauma and sensitization in the first group evokes a particular response in the lamina propria of the tympanic membrane in immunized animals (RAGTM IgG). The form of'trauma does not influence the results, but time seems to be an important factor. This work addresses several questions concerning the possible role of the combination of trauma and sensitization in conditions that involve the tympanic membrane and middle ear clinically.
The particular response of the t y m p a n u m to antigenic stimulation has become a perplexing issue. ~.2 Multiple theories have been put forward in order to characterize the specific low grade i m m u n e response of the middle ear as evidenced in the field of homograft surgery and chronic otitis media, a' 4 To cite only a few, the role of preservatives, the small area of contact between, the t y m p a n i c constituents, and the concepts of privileged site and tolerance have been advocated to no avail. More recently the i m m u n o l o g i c role of the eard r u m has deserved particular attention. 5 The structure and biochemical composition of the t y m p a n i c m e m b r a n e are now well characterized,
but the pathophysiologic basis of lesions of the membrane is still poorly understood. Some data are highly suggestive of an immunopathologic event in lesions of the tympanic membrane in such diseases as serous otitis media and tympanosclerosis. 6 In our previous study the antigenic properties of the tympanic membrane were examined. In order to characterize the immune response of the tympanic membrane in the guinea pig, an antiserum that reacts with the guinea pig tympanic membrane had been developed in the rabbit (RAGTM IgG). The present report is concerned with the in vivo experiment in which the antiserum consisting mainly of IgG was studied with
Accepted for publication January 28, 1981. This work was supported by the American Otological Society, the Duaei Research Foundation, and Centre de Recherche M6dicale de l'Universit6 de Sherbrooke. *Chairman and Professor, Department of Otolaryngolagy, Centre Hospitalier Universitaire, Universit6de Sherbrooke, Sherbrooke, Quebec, Canada. +Assistant Professor, Department of Medicine, University of California, San Diego, School of Medicine, San Diego, California. *Associate Professor, Department of Pathology, Scripps Clinic and Research Foundation and Green Hospital, La ]ella, California. {}Clinical Professor, Department of Otolaryngology, University of California, San Diego, School of Medicine, San Diego,
California.
94
American Journal of Otoiaryngology--Volume 2, Number 2, May 1981
TABLE 1.
Material and Methods
GRouP I (18 ANhMALSI*
GROUP[! (18 ANIMALS)
RAGPTMIgG Myringotomy (right tympanic membrane) Cauterization (right tympanic membrane} Infection (right tympanic membrane)
A NH fgG
B Saline
6
3
3
6
3
3
8
3
3
*Left tympanic membrane kept intact in all groups as an internal control.
s t a n d ar d t e c h n i q u e s of i m m u n o f l u o r e s c e n c e staining, complement reactivity, and immunoperoxidase.
MATERIAL AND METHODS
Thirty-six Hartley guinea pigs, weighing 300 to 400 grams, were divided into two groups of 18 animals. All animals were anesthetized according to the m e t h o d of Evans. T In the 18 animals of group I, rabbit antiguinea pig tympanic membrane IgG (RAGTM IgG; 0.5 ml.) was injected by intracardiac puncture. In group II nine animals (group IIa) received, by the same route, passive immunization with normal rabbit IgG (NR IgG), and in nine others normal saline was injected (group IIb). One hour after immunization the f o l l o w i n g m a n i p u l a t i o n s were performed on only the right eardrum (the left earq drum remained untreated as an internal control) in all groups: In group I, six animals u n d e r w e n t a myringotomy on the right t y m p a n i c membrane, in six animals a 10 per cent solution of tricholoacetic acid was applied to the right tympanic membrane, and in six other animals a solution containing 106 viable S t r e p t o c o c c u s v i r i d a n s micro-
TABLE 2.
Group I
organisms in 0.1 ml. of m e d i u m was injected into t h e m i d d l e ear v i a a 22 g a u g e n e e d l e through the tympanic m e m b r a n e (Table 1). Animals of groups IIa and IIb were divided in the same manner (six myringotomies, six cauterizations, six infections). The timing of the anesthesia, immunization, and trauma was similar in groups I, IIa, and IIb. After trauma all groups were again separated according to time and t e c h n i q u e of staining. S p e c i m e n s were thus o b t a i n e d at 24 h o u r s , seven days, and 21 days after trauma (Table 2). With the Cryostat, frozen sections, 5 microns thick, were obtained from the left and right tympanic membranes and used for immunofiuorescence staining with an indirect m e t h o d by the addition of fluoresceinated goat antirahbit IgG. Frozen sections were also e x a m i n e d with rhodomino labeled rabbit antiguinea pig C3. Permanent sections were prepared and studied with peroxidase labeled, goat antirabbit IgG. The reaction was graded for all the stains used from 0 (negative) to v r + (strongly positive) on coded specimens. The tissue sections w ere examined with a Leitz Diavert fluorescent microscope using direct and transmitted light w i t h FITC filters. Photographs were taken with a Leitz Orthomat camera and Ektachrome (200 ASA).
RESULTS
Tables 3 through 6 summarize the results obtained in the different groups. Figure 1 shows a moderate immunof~uorescence staining reaction ( + + ) at the site of a myringotomy in a seven day speci m en of the right t y m p a n i c membrane from group I. Figure 2 shews a moderate (++) reaction in the lamina propria of the tympanic m e m b r a n e using immunoperoxidase staining in a seven day speci-
Material and Methods: Schedule
NUMBEROFANIMALS Group Ha Group lib
r,ME
of Experiment TRAUMA
STAINING
2 2 2
1 1 1
1 1 1
24 hours
[ Myringotomy l Infection Cauterization
Complement C:~ Immunoperoxidase Immunofluorescence
2 2 2
1 1 1
1 1 1
7 days
[ Myringotomy l Infection Cauterization
Complement Ca Immunoperoxidase Immunefluorescence
2 2 2
1 1 1
1 1 1
21 days
[ Myringotomy l Infection Cauterization
Complement C3 Immuneperoxidase Immunofluorescence
JACQUES F. POLIQUIN ET AL.
Volume 2 Number 2 May 1981
95
American
TABLE 3.
Group I, Right Tympanic Membrane, lmmunofluorescence Study*
TABLE 5. Group I, Right Tympanic Membrane, Immunoperoxidase Study*
TIME
MYRINGOTOMY
TIME
Journal of
Otolaryngology
24 h o u r s 7 days 2:1_ d a y s
++1+ ++ ++ +/+ +
CAUTERIZATION ++1+++ ++ +/+ +
INFECTION ++/+++ ++ +/+ +
m e n (infected t y m p a n i c membrane, group I). Figure 3 shows a typical positive C3 reaction (1-1-) at the seventh d a y in the traumatized tympanic membrane from an animal of group I.
DISCUSSION Our antiserum was made from the lamina propria of the guinea pig tympanic membrane. The m i d d l e layer of the eardrmn is composed of fine fibrils and h o m o g e n e o u s interfibrillar ground substanceY These fibrils are neither true collagen nor true elastin, for they differ in both size and chemical contentY The t y m p a n i c finer fibrils contain amino acids that are present in both collagen and elastin. It becomes evident from our observations that the connective tissue elements of the guinea pig tympanic m e m b r a n e are quite antigenic. The antigenic element of the tympanic membrane most probably resides in the fibrous portion or in the ground substance or in both. A microfibrillar protein that is w i d e l y distributed in connective tissue and is associated with both elastin and collagen fibers could be responsible for the antigenicity we observed in the lamina propria. 1~ The immune response in our animals to the combination of t r a u m a and passive immunization to their own t y m p a n i c membrane illustrates the need for comparing the effects of heterologous and autologous antisera. The lamina propria could be the source of hidden antigens, and trauma could precipitate an antigen-antibody reaction in some individuals. Such an immunepathologic event, as evidenced in Hashimoto's thyroiditis, could be the explanation for the hya-
TIME 24 h o u r s 7 days 21 d a y s
4.
G r o u p I, Right T y m p a n i c Complement Study* MYRINGOTOMY ++ ++/+++ 0
+ + / + +, 0
+ +/+ + 0
line degeneration seen in tynnpanosclerosis after multiple myringotomies, tl The t y m p a n i c membrane could be the site of an a u t o i m m u n e process, but more data are necessary in order to apply Witebsy's criteria, t2 Our experiment s h o w e d that t r a u m a alone does not induce a spontaneous i m m u n e reaction. In group I [RAGTM IgG) the c o m b i n a t i o n of immunization and trauma seems to elicit a homing of antibody at the site of trauma. The inflammatory process that occurs w i t h d a m a g e to the lamina propria could increase vascular permeability, leading to leakage of p l a s m a proteins. This could be the mechanism for e n t r y of circulating antibodies into the t y m p a n u m during inflammation or infection. The demonstration of bound antibody in the l a m i n a propria, capable of binding complement and of persisting for weeks, indicates that formation of immune complexes could be involved in t y m p a n i c membrane diseases, especially those related to insult. If the form of trauma does not i n f l u e n c e our results, time, on the other hand, seems to be an important factor. It would be interesting to observe the results of a more prolonged experiment. In conclusion, our experiment has s h o w n that the lamina propria of the guinea pig t y m p a n i c membrane can be antigenic in the rabbit. In sensitized guinea pigs tympanic t r a u m a evokes a particular immune response in the l a m i n a propria during the time of the acute inflammation. These data suggest the possibility of a specific immunopathologic event in the t y m p a n i c membrane t h a t could e n l i g h t e n us r e g a r d i n g the pathophysiology of some ear diseases.
Membrane,
CAUTERIZATION ++ ++t+++ 0
+ +/++ 0
INFECTION
*Left t y m p a n i c m e m b r a n e : n o reaction.
*Left t y m p a n i c m e m b r a n e : no reaction.
TABLE
24 h o u r s 7 days 21 days
MYR/NGOTOMY CAUTERIZATION
INFECTION ++ ++/+++ O
TABLE 6. Groups IIa and lib (Left and Right Tympanic Membranes) TIME
~MMHNOFLUORESCENCE
IMMUNOPERO XIDASE
C:~
24 h o u r s 7 days 21 days
0 0 0
0 0 0
0 0 0
*Left t y m p a n i c r n e m h r a n ~ : ne reaction.
96
ADAPTIVE IMMUNITY OF THE T Y M P A N I C M E M B R A N E
Volume 2 Number 2 May 1981
Figure I. Right tympanic membrane, traumatized, group I, seven days.
Figure 2, RLght t y m p a n i c membrane, traumatized, group I, seven days. (• lO.)
JACQUES F. POLIQUIN ~T AL.
97
American Journal of
Otola ryngology
Figure 3. Moderately positive reaction to C~, right traumatized tympanic membrane, group I, seven days.
References 1. Veldman, J, E., Kuijpers, W., and Overbosch, H. C.: Middle ear implantation: its place in the immunohistophysiology of l y m p h o i d tissue. Clin. Otolaryngol,, 3:93-102, 1978. 2. Poliquin, J,, at el.: [mmunocompetence of the guinea pig's middle ear. J, Otol., 8:385-389, 1979. 3, Gagnon, N. B,, eta].: Homograffs of the middle ear. Arch. Otolaryngo[., 105:35-38, 1977. 4. Marquet, J, F. E.: I-[istorical notes on homografts, Oto[aryngoI, Clin, N. Am,, 10:479-485, 1977. 5. Poliquin, J,, et el.: Antigenicity of the guinea pig's tympattie membrane. Otolaryngol, Head Neck Surg., 87:852-858, 1979. ft. Veltri, R. W., and Sprinkle, P. M,: Secretory otitis media. An immune complex disease. Ann. Otol. Rhinol, Laryngol., 85(Suppl,):135--139, 1976. 7, Evans, E. F.: Neuroleptanesthesia for the guinea pig. Arch. Otolaryngol., 105:185-186, 1979.
98
8. Lim, D. J.: Tympanic membrane: electron microscopic observation. I. Pars tensa. Acta OtolaryngoI, 66:181198, 1968. 9. Johnson, F. R., McMinn, R. tI. M., and At.field, G. N,: Ultrastructural and biochemical observations on the tympanic membrane. ]. Anat,, I03:297-310, 1968. 10. Bystrm, J. C.: Clinical significance of basal cell layer antibodies. Arch. Dermsto[., 113:1380-1382, 1977. 11. Birck, H. G., and Mravec. J, J.: Myringostomy for middle ear effusions. Results of a two-year study. Am~. Otol. Rhinol. Laryngol., 85(Suppl.):263-267, 1976. 12. Barrett, ]. T.: Textbook of Immunology, St. Louis, The C. V, Mosby Company, 1974, Ch. 12.
Department of Otolaryngology Celltro Hospitalier Universitaire 3001 12th A v e n u e North Sherbrooke, Quebec J3H 5N4 Canada (Dr, Poliquin)
ADAPTIVE IMlvI[JNITY OF THE TYMPANIC MEMBRANE