Pathologic pulmonary alterations in long-term human heart-lung transplantation

Pathologic Pulmonary Alterations in Longterm Human Heart-Lung Transplantation SAMUEL A. YOUSEM, MD,* CONOR M. BURKE,MB, MRCEI AND MARGARET E. BILLINGHAM, MB, BS, FCAP, FACC* Twenty-one patients with end-stage pulmonary hypertension underwent combined allograft h e a r t - l u n g transplantation after 1980. Almost 50 per cent of these patients survived beyond the immediate postoperative period, with the longest survival period more than 389 years at the time of this report. Five patients died in the perioperative or immediate postoperative period, and 11 r e t u r n e d to normal lives with essentially normal p u l m o n a r y function. In the remaining five allograft recipients recurrent respiratory infections and progressive obstructive airway disease developed, with superimposed restrictive deficits in three of them. Two open lung biopsies, two autopsies, and one retransplantation were performed in these recipients. Morphologically, these allograft recipients showed extensive bronchiolitis obliterans, interstitial and pleural fibrosis, and accelerated arterial and venous arteriosclerosis. Bronchiolitis obliterans may prove tO be a significant complication of h e a r t - l u n g transplantation. HUM PATIIOL 16:911-923, 1985.

The resuhs of medical treatment for pulmonary hypertension, whether idiopathic or secondary to congenital heart disease, have been uniformly poor. During the last two decades, surgical options, most often nnilateral h, ng trausplantation, have been explored. Only recently has combined b e a r t - l t , ng transplantation been attempted. 1-4 In the earl)" 1970s numerous investigators, 5-1-" particularly Veith, documented the changes of acute rejection in unilateral lung allografts in both animal models and ltuman recipients. Initial attention was focnsed on the exquisite perivascular nature of the mononuclear inflammatory infihrate in acute rejection. The infiltrate was associated with an alveolar exudate containing desquamated pneumocytes attd an admixture of inflammatory cells. Later, a second form--so-called alveolar or atypical rejection--was described; this form was characterized by fibrinous alveolar exudates with a paucity of both mononuclear cells and perivascular cuffs of lymphocytes and plasma cells. More recently, a third fortn, 13 perhaps a variation of the first, consisting primarily of perivascular lymphocytic cuffs without interstitial or alveolar manifestations, has been seen, particularly in cyclosporine-treated patients. Reported late restdts of uncontrolled pulmolaary rejection were interstitial fibrosis and honeycombing. 6-12 Beginning in 1980, investigators at S t a n f o r d Received December 3, 1984, from the Departments of *Pathology and tRespiratory Medicine, Stanfurd University Medical Ccqtcr, Stanford, California. Revision accepted for publication February 1, 1985. Supported in part by research grant HI. 13108 from the National lleart, Lung and Blond Institute, National Institutes of llealth, Bethesda, Maryland. Address corrcsppndence a n d r e p r i n t requests to Dr. Yousem: Department of Puli1)onary and Mcdiastinal Pathology, Armed Forces Institute of l'athology, Washington, DC 20306.

911

University Hospital performed 21 co,nbined heartlung transplantations. When this r e p o r t was prepared, the longest survival period was almost four )'ears. However, five of the 16 patients (31 per cent) who survived the immediate perioperative and postoperative period experienced progressive pulmonary dysfimction. In this report we describe the pulmonary changes in these five long-term h e a r t - h m g transplant survivors. The clinical course of these five patients, who had survived for 3.0, 1.3, 1.25, 1.0, and 0.,t )'ears following transplantation at the time of this study and in whom progressive airflow obstruction and recurrent respiratory tract infections had developed, may impact on the fimu-e success of combined h e a r t - l u n g transplantation. MATERIALSAND METHODS

O f the h e a r t - h m g transplant recipients (N = 2 l) at Stanford University Medical Center, only five of the 16 who survived the immediate postoperative period subseqt, ently underwent open lung biopsy, retransplantation, or autopsy. T h e clinical histories of each o f these patients were reviewed, with special concern for infectious processes, pulmonary timction status, and radiographic changes. All tissue was fixed in 10 pet" cent fornmlin and stained routinely with hematoxylin-eosin. In all cases pertinent blocks were stained with additional stains, including elastic van Gieson, Gomori's methenanfine sih'er, Ziehl-Neelsen, and trichrome stains. Sections o f lung for transmission electron microscopy were initiall)' l)laced in a solution of 2 per cent paralormaldehyde, 2.5 per cent glutaraldehyde, and 0.1 .xt sodium cacodylate huffer. T h e samples were postfixed in 2 per cent osmium tetroxide and then stained en hloc with uranyl acetate. After dehydration and infihration, the tissue was embedded in epoxy resin. Thin sections were cut on an uhramicro'tome, stained with lead citrate, and screened in an electron microscope. Sections of hmg for scanning electron microscol) )9 were fixed in the same manner as for transmission electrola, microscopy. After deh)'dration in a graded alcohol series, the specimens were prepared by the critical point procedure, sputter-coated with gold for 1 naint, te, giving a 20-nm coating, and screened with an International Scientific Instrument (ISI-'t0). Tissue for immunologic studies was processed and stained as described previot, sly. 13 Briefly, firststage incubatinta with one of the monoclonal anti-

HUMAN PATHOLOGY

Volume 16, No. 9 (September 1985)

bodies listed below was tollowed by the application o f b i o t i n - c o n j u g a t e d g o a t a n t i m o u s e F(ab'),_, a n t i b o d y (Tago, lhn'lingante, California) or h o r s e atltimouse a n t i b o d y ( V e c t o r Labs, B u r l i n g a m e , C a l i f o r n i a ) . A third stage o f avidinzconjugated h o r s e r a d i s h peroxidase (Vector) was t h e n applied and subsequently inc u b a t e d in d i a m i n o b e n z i d e n e followed by c o p p e r sulfate a n d c o u n t e r s t a i n e d with m e t h y l e n e blue. Monocl/3nal a n t i b o d i e s to B a n t i g e n s ( T 0 1 5 ; D. Mason, Oxfi>rd, England); T-cell antigens ( L e u - l , - 2 , - 3 , - , t ; Becto~.Dickinson, Stmnyvale, California) a n d macr o p h a g e antigens (Leu-M3; Becton Dickinson) were used. I n t m t m o f h t o r e s c e n c e s t u d i e s with g o a t antih u n t a n atttibodies directed against l g G , IgM, IgA, c o m p l e n l e n t (C'3), a n d fibrin were also p e r f o r m e d by s t a n d a r d m e t h o d s . B r i e f l y , f r o z e n s e c t i o n s o f tissue e n t b e d d e d in O C T e m l ) e d d i n g m e d i u m were fixed in acetone a n d sttbsequently incubated with fluoresceinated antisera at roont t e m p e r a t t t r e . T h i s was followed by n m h i p l e washes in I+hosl)hate-bttffered saline p r i o r to m o u n t i t i g and exantixtation t m d e r a f l u o r e s c e n c e nticroscope.

REPORT OF FIVE CASES Case 1. The Hoh-Oram syndrome (ventricular septal defect, atrial septal det~'ect, and skeletal abnormalities) in a 30-year-old man resuhed in end-stage pulmonary hypertension. The clinical course following h e a r t - l u n g transplantation from a 21-year-old donor was complicated by bleeding fiom posterior mediastinal vessels, requiring reexploration and ligation. Repeat cardiac biopsies demonstrated no evidence of rejection, ,utd the patient was discharged on the 42nd postoperative day. At four ntonths, l)ulmonary function studies showed a restrictive ventilatory defect witlt no airflow obstrttction. I)uring tire following 21/., years the Imtient was well, with stable puhnonary flutction, l)uring this time he had three lower respiratory tract infections tlmt responded to empiric antibiotic treatment, and ire presented with persistent rhinitis with mucopurulent Itypopllaryngeal secretions. One episode precipitated bronchoscnpy, which, 29 montlls after surgery, showed mucus phtgging of the anterior segntent of the right upper lobe and acute cxudative bronchitis with sqnanlous metaplasia at biopsy. Approximately three years after transplantation, he complained of dyspnea on exertion for the first time since receiving the transplant. Radiographic findings included new peripheral patchy areas of consolidation, most prominent in the upper lobes, and peribronchial thickening. Pulmonary function stndies perfiu'med at that time showetl a marked nbstrvctive aml restrictive ventilatory defect. Cardiac biopsy sltowed no evidence of rejection. Open ltlng biopsy performed three )ears after transplantation showed marked pleural and interstitial tibrosis with mucus plugging. Following a lack of response to tre:ttment with steroids, aminophylline, antibiotics, and bronclmdilators, he underwent a second heart-lung transplantation. He returned Imtne attd was without pulmoliary complaints tltree months after receiving the second trans-" 1)lant. Case _9. A ventrict,lar septal defect with Eisenmenger's syndronte in a O2-year-old man rest,hed in nearly terminal pt, lmonary llypertension. Following heart-lung transplan-

912

tation from air 18-year-ohl donor, postoperative lmeumonia was treated successfully with antibiotics. On postoperative days 15 to 23 cardiac biopsies showed acute rejection with necrosis. He was treated with a bolus of steroids, and the rejection resoh'ed on day 35. Puhnonary fitnction stttdies showed a restrictive ventilatory defect with no airflow obstruction, and tire patient was discharged on tire 39th postoperative day. Following this episode he rentained well for seven months, although he experienced severe rhinitis associated with postnasal aspiration. Chest radiographs showed a new diffuse reticulonodular infiltrate, most marked in the upper lobes. I'nlmonary function tests showed progression of the restrictive defect. Following a nondiagnostic transbronchial biopsy, open Itmg biopsy was performed on postoperative day 248. A cardiac biopsy showed no evidence of rejection at titis time. High-dose steroid therapy was instituted for two mouths following tire biopsy, but tlo clinical or physiologic response was seen. Repeat transbroncltial biopsy showed nonspecific chaqges, and the dosage of prednisone was gradually reduced. Tire patient subsequently complained of dyspnea on exertion and had recurrent respirator)" infectious. Serial puhnonary flntction tests showed a stable restrictive defect with progressive, nonresoh'ing airflow obstruction, despite treatment with I>ronchor and pnlmonary toilet therapy. Repeated chest radiograplrs showed an tmchanging bilateral reticular infiltrate, and repeat cardiac biopsies were unremarkable. Case 3. Truncus arteriosus (type I) with features of Eisenmenger's syndrome in a 41-year,old man resulter in end-stage pulmonary hypertension. Following h e a r t - h m g transplantation front a 19-year-old donor, fever and a right lower lobe infiltrate that responded to broad-spectrum antibiotics developed on the sixth l>OStoperative day. Twentyone days after transplantation a new fever was accompanied by bilateral fluffy infiltrates that progressed rapidly to complete opacification on chest tilms; severe Iwpoxemia was present. Resuhs of open hmg biopsy perforated at that time suggested p t d m o n a r y rejection. T h e patient was treated with empiric broad-spectrum antibiotics, biglt-dose steroids, anti antithymocyte globulin, and an intmediate clinical response was seen. Cytomegalovirus (CMV) titers at this time, when contpared with previous titers, suggested recent infection. Repeat cardiac biopsy on the 59th postoperative day showed rejection, which resolved following steroid therapy. He was well when disclmrged on the 63rd postoperative day. Four months after transplantation a productive cnuglt and dyspnea deveh)ped, anti pulnmnary function tests showed a new obstructive ventilatory defect. Despite bronchodilators and antibiotic therapy and a pulmooary toilet program, repeat l)uhnolmry flmction tests two mouths later showed progression of the airflow obstruction. Repeat heart biopsies failed to show abnormalities. Case 4. A 26-year-old man with a ventricular septal defect attd Eisenmenger's syndrome received tire heart and hmgs of a 22-year-old donor. Bilateral exud,ttive plet, ral effusions and a respiratory tract infection were treated with arrtibioti~,s on the ninth postoperative r Endomyocardial biopsy on Imstoperative day 23 showed cardiac rejection, which was treated with steroids and resolved on postoperative day 160. The patient was discharged on postoperative day 29; ptdmonary fnnction studies showed mild restriction. During the following three months he was clinically kvell but COml~lained of persistent rhinitis and postnasal aspiration. By the fourtll nmnth Ire complained of dyspnea on exertion. One month later he presented with cough and purulent Slmtum; chest radiograplls showed new peribron-

HEART-LUNG TRANSPLANTATION(Yousem et al.)

chial irtfihration in both lower zones. Bronchoscopy and biopsy showed an organizing pneumonia. Cuhures were negative, but the patient responded well to broad-spectrum antibiotic treatment. Cardiac biopsy showed no evidence of rejection. During tile following eight months, he camplained of persistent prodnctive cough and increasing dyspnea. Pulmonary flmction tests showed a progressive obstrttctive and restrictive ventilatory defect, despite bronchodilator and antibiotic treatment. Fourteen months after trart~plantation, steroids were added to the regimen for presumptive bronclfiolitis obliterans, with some clinical response. ChesLradiographs obtained after two weeks of stereid tlterapy~showed a new consolidation in the left upper zone, and two days later the patient died suddenly of a suspected myocardial infarct. Case 5. A 33-year-old man who had an end-stage ventricular septal defect atad Eisenntenger's syndrome received the heart and hmgs o f t 32-year-old donor. A postoperative Pseudomonas pneumonia responded to antibiotic therapy. No evidence of cardiac rejection was found on serial endontyocardial biopsies, and tile patient was discharged on tile ,t7th postoperative day. Ptdmonary function tests performed two months after transplantation showed a mild restrictive defect. His subsequent clinical course was remarkable for persistent rhinitis, a chronic cough productive of copious ntucopurulent sputum, and intermittent respiratory tract infections. Despite a puhnonary toilet prograxn and treatment with bronchodilators, lmhnonary flmction tests showed progressive obstructive and restrictive defects, and chest radiographs showed progressive, coarse, z~odular densities in both lower zones, with bronchial dilation and peribronchial and pleural thickening. Clinically, tile patient corttinued to experience chronic expectoration of mucopurulent spntunl and dyspnea on moderate exertion. Ltmg biopsy was not performed, but the clinical and physiologic observations suggested obliterative bronchiolitis. Eleven months after surgery, the patient presented with new bilateral pt, lmonary infiltrates, whiclt progressed rapidly despite antibiotic therapy. He died approximately 1.25 )'ears after transplantation, and autopsy was performed at an outside institution.

FIGURE 1. Case 1. Cylindrical bronch!ectasis. Specimen bronchogram of the left lower lobe shows fusiform dilation of the bronchial tree, with peripheral tapering and pruning. These features were also observed in cases I and 4.

aspects, had shaggy white fibrous adhesions. Tile bronchial tree showed cylindrical dilation, confirnted by specimen b r o n c h o g r a p h y (fig. 1), with' extensive mucus plugging. T o w a r d tile peripllery tile broncltioles narrowed abruptly and appeared occluded or mucus-filled on cross section. T h e p u l m o n a r y arteries and veins were tmremarkable, although tile elastic arteries showed mild intimal thickening. T h e pulntonary p a r e n c h y m a was fluff)' and sponge-like, with a single consolidated region, 1.5 cm in diameter, in the posterior aspect o f the right u p p e r lobe. Tlais finding suggested broncltopneumonia. T h e left lung weiglted 270 g and had at smooth, glistening pleural surface, except for dense scar tissue bridging the u p p e r and lower lobes along the major fissure. T h e bronchial tree resembled that of the right lung, witlt finsiform dilation and ntncosal pitting. Mttcus plugging was striking. T h e lingtda had been excised 1)reviously. T h e vascular tree was unremarkable, as were the hilar lylnplt nodes. Case 4. T h e severe proliferative coronary graft arteriosclerosis with a large, recent left ventricular myocardial infarct seen at autopsy was the presumed cause of death. Dense adhesions and diffuse pleural fibrosis, particularly in the posterior thorax, bound botll'lungs to the chest wall. Tile tracheal anastomosis was intact and well healed. T h e bronchial tree in both lungs showed mncus pltlgging and cylindrical bronchiectasis, with abrupt tapering of the bronchi peripherally. T_,he p u h n o n a r y parenchyma was diffilsely firnt, and n u m e r o u s sntall subpleural scars were centered on tile interlobular septa. A hemorrhagic abscess cavity (Aspergillus abscess microscopically) was present in the left u p p e r lobe. T h e p u h n o n a r y arteries and veins were smooth and glistening, without large vessel atherosclerosis or throntboemboli. Case 5. A t autopsy, the lungs were densely adherent to the chest wall, particularly in the posterior

RESULTS

Gross P a t h o l o g i c Findings

Case 1. T h e specinten obtained by open h m g biopsy front the left u p p e r lobe on the 1,085th postoperative day was firm and nteasured 1.5 • 1.0 • 0.3 cnt. Mucus plugs were expressed easily. At retransplantation, tile heart a n d lungs were rentoved individually. T h e heart weighed 350 g and showed mild pericardial fibrosis. T h e ventricles were ntildly dilated. T h e left ventricle contained several sntall organizing infarcts, ranging in diameter fl'om 0.4 to. 1.5 cm and located particnhtrly along the posterior and lateral walls. T h e left anterior descending and left circuntflex coronary arteries showed severe arteriosclerosis, with 90 per cent luminal compromise and no thromboemboli. T h e r i g h t l u n g weiglted 290 g. T h e visceral pleura was coated with a tltin, glistening, o p a q u e brown m e m b r a n e that encased tile lung and bridged both m a j o r an'd n t i n o r "f'issures laterally. T h e remaining pleura~ particularly the medial and posterior 913

HUMAN PATHOLOGY

Volume 16, No. 9 {September 1985]

TABLE 1. Pathologic Findings in Five Long-term Survivors of Heart-Lung Transplantation

Case

Time after Transplantation ()'r)

i

3.0

3+

3+

1+

II

2+

1+

0

2 3

!.0 0.4 1.3 1.25

1+ 2+ 3+ 2+

2+ 1+ 3+ 2+

3+ 3+ 2+ 2+

II I It It

2+ I+ 2+ 2+

2+ 1+ 2+ NA

2+ 3+ 0 NA

5

Bronchiolitis Obliterans

Mucus Pluggillg

Obstructive Change

Arterial lntimal }I)perplasia

Venous Intimal H)'perplasia

Interstitial Fibrosis

Perivascttlar Cuffing

Gr,'-gling: 0 - 3 + (absent to severe); obstructive change: for description, see text; arterial intimal hyperplasia: grading according to IIeath and EdwardsI4; NA, not applicable (see Results section).

tltorax. The anterior aspects of the right ttpper and middle lobes attd the left ttpper lobe were also filsed to the epicardiunt and residual pericardiuna. The tracheal anastotnosis was intact, as were all vasctdar anastomotic sites. Tire bronchial tree showed diffuse ectasia witll ntucus plugging and puruletlt exudates. T h e puhnonary parenchynm was consolidated in all lobes and had a granular, cheesy qttality. No abscesses or cavities were found, ahhough the gross appearance was that of the gra)' hepatization stage of extensive bilateral brotachotmettmonia. Dense pericardial and chest wall adhesions bound the anterior aspects of the right and left ventricles to the chest wall. The pericardiuna had a g~anular qualit)', especially where it abutted the adhereht lungs. Four-chamber dilation o f the heart was observed, as was severe circuntferential atherosclerosis of all major coronary arteries. Despite these findings, no lnyocardial infarcts were seen.

The gross appearance of the open lung biopsy specitnens in cases 2 and 3 was tmremarkable.

Histologic Findings The major pathologic findings in these tire longterm sttrvivors o f I t e a r t - l u n g transplantation are summarized in table 1. All patients showed brotlchiolitis obliterans to varying degrees, frequently in association with nltlCnS inspissation and distal obstructive change. In all patients the bronchiolitis was patchy, ahhouglt extensive (pan-lobar) in distribution. Early changes consisted o f ulcerated bronchiolar epitheliutn with necrotic epithelial and inflammatory cells enineshed in lunfinal mttcus (fig. 2). O t h e r bronchioles contained aggregates of fi),ttny lipid-laden histioc)'tes in tire lutnina and distal ;tit- spaces, sttggesting ntore proximal obstrttction, which often appeared on deeper sectioning of paraffin blocks. Later, as organization occurred, fresh acid mucopolysacclmriderich granulation tissue formed onion skin-like phtgs within the bronchiolar lumina (Masson bodies) (tig. 3). In these areas the respiratory epithelitm~ was attenttated and displayed sqtmmous metaplasia. Elastic tissue stains showed interrttption of the basal elagtic layer at the base of these polypoid plugs. Chronic" injury was reflected in dense submucosal scars lined b)' cuboidal epitllelintn with an interrupted ttnderlying elastic latnina and, frequently, scar tissue re914

placing portions of the smooth muscle wall (fig. ,t). A mononuclear subnmcosal and peribronchial infiltrate of l)'mphocytes and plasma cells diminished with time after injury. Frequentl)' (especially in cases 1, 4, and 5) entire bronchioles had been replaced b)" scar tissue and were identified only b)' their location and elastic tissue stains (fig. 5). One patient (case 2) had only focal bronchiolitis obliterans but marked obstructive change, consisting of air spaces distended b)' foatny, lipid-laden macrophages. This aheration suggested nlore extensive airwa)' disease. I~ulnlonar) ' ftmction studies supported tltis interpretation. Another patient (case 1) showed severe bronchiolitis obliterans but lacked obstructive phenomena. All five patients showed arteriosclerotic cltanges in the muscular pulmonar)' arterioles and elastic arteries; these changes were graded according to the scheme proposed b)" Heath and Edwards. H These aherations freqnentl)' took the fornt of concentric proliferative fibroe]astosis occurring on the luminal side, i.e., intimal, of intact inner elastic lamina (grade II) (fig. 6). Muscular hypertrophy (grade 1), although seen in most vessels, was disproportionate to the considerable intintal change. Cholesterol clefts and erosion of the arterial wall, especially the elastic lamina, were rarely seen. "I'ite concentric pattern o f fibroelastosis was unlike age-related eccentricall)' located atherosclerotic plaques and was far more severe tilth expected tbr 18-, 19-, 21-, 22-, or 32-year-old donor vessels. It coincided witlt the severe proliferative intimal thickening observed in the coronary arteries in cases 1, 4, and 5 (fig. 7). Venous clmnges, seen in four of the five patients, consisted of waxy, sclerotic intimal tlfickening of the 9renules and some veins (figs. 8 and 9). Again, the elastica appeared intact, with the thickening confined to the intinta. While the intitnal thickening was sitnilar to tlmt seen in the arterial systent, the concentric plaqttes were more pancicelhtlar and sclerotic than those seen in associated arteries. Diffilse increases in interstitial fibrosis were seen in all lfiops)' specimens, although extensive l)ulmo nar)' scarring and honeycombing were not seen. ha ._cases 1 and 2 focal stellate scars were usually centered on the interlobttlar septa or in the subpletn'al regions. The plettra was focally fibrotic and thickened in the patients who survived the longest. Formed over

HEART-LUNG TRANSPLANTATION (u

et al.)

FIGURE 2 (top). Case 1. Bronchiolitis obliterans. The earliest change observed is acute bronchitis and bronchiolitis, with denuding of the mucosa by an exudate of fibrin and neutrophils. A concentric peribronchial infiltrate of mononuclear cells is aTso present. (Hematoxyiin-eosin stain, x 100.) FIGURE 3 (botlom left and righl~. Case t. Left,, an organizing plug of granulation tissue containing mononuclear cells and foamy histiocytes extends from the submucosa to occlude the lumen of the bronchiole. The smooth muscle wall appears to have been interrupted and replaced by scar tissue. [Hematoxylin-eosin stain, x 200.) Right, elastic tissue stains demonstrate interruption of the basal elastica and expansion of the submucosa by mucopolysaccharide-rich granulation tissue. A peribronchial infiltrate is still present. [Hematoxylin-eosin stain. x 200.]

915

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Volume 16, No. 9 [September 1985]

FIGURE 4. Case 4. Bronchiolitisobliterans. Chronic airway injury resulted in dense, hyalinized submucosal scarring and attenuation of the respiratory epithelium. The inflammatory infiltrate is diminished. [Hematoxylin-eosin stain, x 200.]

an intact elastica were plaque-like and thready tibrovascular scars and adhesions (fig. I0). Frequently, pleural scars extended down interlobular septa and radiated into the lobule. No evidence of acute pleural inflammation was observed. In two patients (cases 2 and 3), marked perivascular, particularly perivenular, cuffs of pyroninophilic lymphoid cells and rare eosinopltils were arranged concentrically in edematous adventitial connective tissue (figs. 11 a n d 12). Endothelial cell swelling, llypertl'oi)ily, and mitotic activity were seen as well. Mononuclear cells had percolated out into interstitial tissues in tltese two cases, ahhough this feature was not seen in other cases. In both of these cases (one retrosl)ective ), the pel'iva~cular cuffs were b(.~lieved to suggest acute rejection. Additional findings included focal broncltopneumonia in cases 1, 3, and ,t, with Aspergillus identiffed in the latter. Viral cuhures from the biopsy in case 3 grew CMV; however, viral inclusions were not identified, despite CMV titers suggesting recent infection. A cluster of four Pneuntocystis cysts, identified in only one of muhil)le sections in case 3, were considered incidental and not tile primary cause of the perivascular mono~mclear aggregates. One patient (case 5) showed disseminated coccidioidomycosis at aUtOl)Sy, with involvement o f the 9t6

hmgs, pericardiuln, heart, liver, kidneys, pancreas, spleen, and colon. Nonetheless while the lungs showed acute suppurative consolidation of the alveoli by Coccidioides immitis, the bronchioles demonstrated severe dense submucosal fibrosis, with compromise or obliteration o f the bronchiolar lumina by scar tissue, in addition to earlier stages o f obliterative bronchiolitis. Given the apparent age of tile bronchial damage and the clinical suspicion of bronchiolitis obliterans, we believe that this finding preceded the acute pneumonic process and reflects pathopltysiologic alterations similar to those seen in the other cases presented. Electron microscopic exantination in cases 1 and 4 showed normal types I and II alveolar pneumocytes, with an intact basement membrane. No immune complex-type deposits were observed. Mild focal increases ill interstitial collagen were also observed. Pulmonary capillaries showed intact, wellpreserved endothelial and supportive cells without basement ntembrane duplication or immune complex deposits. Scanning electron microscopy yielded similar nonspecific findings. I m m u n o f l u o r e s c e n c e studies showed no imnaunoglobulin, coml)lement, or fibrin deposits in cases 1 and 4. h n m u n o p e r o x i d a s e studies of representative lung tissue in cases I and ,1 showed adntixtures of cytotoxic/suppressor and helper T cells, witlt no predilection o f either type for perivascular, peribronchial, or interstitial locations:Scattered B cells and macrophages were also seen. DISCUSSION

Since the late 1960s, more than 300 cardiac transplantations have been p e r f o r m e d at Stanford University Medical Center. The procedure has become routine, with more than 80 per cent of patients survMng for one year. As a result of this experience, the pathologic features of both acute and chronic cardiac rejection have been well described. 15,16 With the advent of combined h e a r t - h m g allograft transplantation, new observations of pulmonary rejection have been made to s u p p l e m e n t those o f Veith and others, 6-12 i)articularly with respect to long-term lristopathologic changes. While acute puhnonary rejection is best recognized by perivascular lynq)ilocytic cuffing and a diffuse ah'eolar damage pattern (alveolar rejection), bronchiolitis obliterans appears to be the major problem in the chronic course of these patients (occurring in up to 30 per cent of transplant recipients). Accelerated intimal thickening in both arteries and veins may also pose a future problem. Each of these issues is discussed. Bronchiolitis Obliterans

Bronchiolitis obliterans is a nonspecific finding in airway injury. Recognizing this, Epler and Colby 17 recently proposed that bronchiolitis obliterans be divided into tire clinical classifications: 1) lesions resnhing from toxic fume inhalation; 2) postinfectious

HEART-LUNG TRANSPLANTATION(Yousem et al.)

FIGURE 5. Case 4. BronchiolitisobIiterans. Left, occasional bronchioles had been completely obliterated by scar tissue and were identified only by their location adjacent to arteries, residual smooth muscle wall, and elastic tissue stains. [Hematoxylin-eosin stain, x'lO0.) Right, elastic van Gieson stain highlights the obliterated bronchiole. [Hematox~in-eosin stain, x 100.)

lesions; 3) lesions associated with connective tissue disorders; ,t) localized lesions; and 5) idiopathic, patchy or diffuse infihrative lesions. Bronchiolitis obliterans represents a fibrosing inflammatory l)rocess involving the terminal and respiratory bronchioles, occasionally leading to progressive small airway obstruction if it persists. 18 In the presumed sequence of injury, tire initial insult resuhs in denuding and ulceration of the ciliated respiratory epithelium and slougifing of necrotic debris, macrophages, and inflammatory cells into the lumen. With time, ingl'owth of fibroblasts fl'om the exposed submucosa results in intraluminal l)lugs of acid mucopolysaccimride-rich myxoid tissue (Masson bodies). As this tissue organizes, concentric sheets of more ntature collagen envelop the central core of necrotic debris, lymphocytes, l)lasma cells, and macrophages, l'l'-ogressive scarring resuhs in a thickened fibrotic submucosa lined by attenuated respiratory el)ithelium or squamous metaplasia, functionally creating a rigid broncitiole. With time, the only residua of injury ntay be an eccentric submucosal scar, as well as a disrupted elastica. For tiffs reason elastic tissue stains and, sometinaes, serial sectioning provide the only clue of l)ast bronchiolitis. In these five l)atients, extensive, althougll I)atclly, bronchiolitis obliterans was l)resent at various stages of 0rganizatim!. FrequeL~tly, dense submucosal and peribronchiolar fil)rous scarring had rel)laced tim smooth muscle wall, resuhing in reduced lunfinal di917

ameters. Still other areas showed coml)lete occlusion by concentric fibrous plugs. In these regions bronchioles were identified only by 1) their location adjacent to puhnonary arterioles and 2) elastic tissue stains. Why should lleart-lung transplantation result in bronchiolitis obliterans? Several reasons, some not specific to the allograft recit)ients, are al)parent (table 2). First, these five patients experienced repeated upper respiratory tract infections, both bacterial and viral. 19 Resl)iratory syncytial virus, adenovirus, and parainfluenza virus are fi'e(luent causes of I)ronchiolitis in children, while ltemophilus infhrenzae, Legionella, and Mycoplasma are common aduh pathogens. ''~ These i m m u n o s u p p r e s s e d hosts have a greater suscel)tibility to these common l)atliogens, as well as to more unusual ones. 15,16 In addition, an impaired immunologic resl)onse to these bronchial infections may result in continued progressive bronclfiolitis obliterans. Secondary phenomena may exacerbate thi,s l)ropensity to bronclfiolitis. Chronic inflammation can result in squamous metaplasia with loss of surface cilia, which leads to mucus stasis and i)lugging, fiu'ther increasing the risk of infection. Inflammation may also impair the secretion of IgA, an important opsonin in tim l)rotection o f lung and bowel. Obliterative bronchiolitis in transplant recipients may be a result of imntune phenomena. It is well

HUMAN PATHOLOGY

Volume 16, No. 9 (September 1985]

FIGURE 6 (top]. Case 1. Arteriolar fibroelastosis. The small pulmonary arteriole shows preservation of the internal and external elastic lamina, with fibrointimal thickening, unexpected in this 18-year-old donor. [Elastic van Gieson stain. x 400.] FIGURE 7 (bottom left and righl]. Case 1. Left, arteriolar fibroelastosis. This muscular arteriole shows muscle wall hypertrophy a n d concentric intimal thickening, with preservation of its elastica, unlike typical age-related atherosclerosis. (Elastic van Gieson stain, x 200.] Right, coronary arterial fibroelastosis. The left anterior descending artery shows concentric intimal fibroelastosis similar to, although more marked than, that of the elastic pulmonary arteries and arterioles. {Hematoxyiin-eosin stain, x 20.)

918

HEART-LUNGTR,N~SPLANTATION[Yousem et al.)

FIGURE 8 (left. Case 1. Venous intimal thickening. This vein within the interlobular septa shows waxy intimal sclerosii, similar to age-related phlebosclerosis; however, it is more marked than expected in a 21-year-old donor. [Hematoxylin-eosin stain, x 400.] FIGURE 9 (right). Case 4. Venous intimal thickening. The intimal atherosclerosis was particularly marked in the small venules. [Elastic van Gieson stain, x 200.]

docuinented in patients with collagen vascular disease, and Geddes et al., '-'3 Epler et al., al and others 24 have shown an association with rheumatoid arthritis, Sj6gren's syndrome, eosinophilic fasciitis, and ltq)us erythenmtosus. In each o f these conditions an imm u n o l o g i c mechanism has been proposed as the cause o f the bronchiolar disease. In the cases of these investigators, as well as in the two patients studied in this report, no significant amounts of immunoglobulin or immune complexes were fotmd. Nonetheless, a cellular, i.e., T-cell-mediated, process may be the true cause, as observed in both cardiac and renal graft rc~jection, z5-27 In a recent report Ralph et al. 78 described four patients with n m r r o w transplants in wholn obstructive lung disease ' developed one to two years atier surgery. Bronchiolitis obliterans was demonstrated by biopsy in one patient. They speculated that a graftversus-l]ost reaction in the lung was responsible for bronchiolitis obliterans in these patients. The opposite, i.e., host-versus-graft disease, may occur in our patients, although bronchial inflammation has not been described in classic lmlmanar) ' rejection in animal or humal~ models. T h i r d , although the cilia examined ultrastructurally were morphologically normal, no in vivo measurements of their inherent motility were performed. 949

The effect of severing tile lmlmonary nerve supply on ciliar)' motility is also unknown. Furthernlore, chronic inflanunation that persists for any reason frequently results in morphologicall), abnormal cilia, which would worsen the clearance problems. 29 Ligation of the bronchial circulation may alter repair of injured bronchi and bronchioles. This, as well as repeated infection, ma)' explain the diffilse bronchiectasis on specimen bronchograms, which appears paradoxical to the distal obliterative bronchiolitis. Evaluation of tile bronchial arteries was inadequate, as injection studies were not performed. Drugs, especiall)' penicillamine, are a well-known cause of bronchiolitis obliterans.30. 31 Cyclosporine has been t'Iscd routinel)' as part of the immunostq)pressire regimen in these patients. It has a well-recognized fibroproliferative effect, which, if affecting tile bronchioles, would result in progressive narrowing and obliteration, i.e., bronchiolitis oblitcrans. This, however, is speculation and has certainly not been documented in heart transplant recipients receiving this imnmnosuppressive drug. Finall)', bronchiolitis obliterans may be a result ofde!~ervation of the recipient bronchi. T h e cough reflex beyond the tracheal anastomosis is lost, and self-mediated pulmonar)' toilet is thus limited. Inadequate mucociliary clearance may result in muco-

HUMANPATHOLOGY Volume'16,No. 9 (September1985]

in the media of the arterioles. Occasionally, concentric fibroelastosis had obliterated the arteriolar lumina. Plexiform and angionmtoid lesions were not seen. The causes of the arterial disease may be nmltiple. As indicated previously and seen in two of our patients, acute graft rejection was reflected in perivascular lymphoid aggregates, frequently associated witlt endothelial cell injury. It is possible that subclinical rejection episodes result in repeated vascular trauma and accelerated arteriosclerosis. The two patients who showed the most prominent perivascular mononuclear clusters also showed intimal proliferation, wlfich would support this theory. Renal pathologists have fotmd tint endothelial cell injury and fibrin deposition resuh in the peculiar concentric inritual fibrosis found in tire arter'ies of the transplanted kidney and consider this lesion a manifestation of chronic rejection. 36-3s Humoral mechanisms, i.e., immtme complex deposits, may have been the cause of the vascular disease in our study. We did not observe immunoglobulin in the walls of the puhnonary arterioles in immtmofluorescence studies; however, tiffs may have been due to low levels of immune complexes, the stage of injur)' at the time of biopsy, or immnnosuppression. Venous Changes

The puhnonary veins showed intimal tlfickening similar to that seen in the arteries and arterioles. Unlike the arterial lesions, the venous tlfickening had a waxy sclerotic appearance, sinfilar to the changes of aging described by Wagenvoort and Wagenvoort 39 and Harris and I-leath. 4~ Recanalized veins were rare, althougll the lumina were occasionally compromised to 50 per cent of the original diameters. Veith and others have shown the early vascular clmnges of acute rejection to be periventdar. Again, it can be speculated tint subclinical rejection episodes cause the venous damage. Corrin et al. 41 showed immunoglobulin deposits in ptflmonary veno-occlusive disease; such deposits were not observed in o u r studies of transplanted lungs. Venous intimal thickening may l'epresent an accelerated aging phenomenon, with the degree of plflebosclerosis being disprol)ortionate to the age of the transplanted organs.

FIGURE 10. Case 4. Marked pleural thickening and diffusely increased interstitial fibrosis were consistent findings in all five patients. [Elastic van Gieson stain, x 40.}

stasis, a fertile soil for infectious agents. Similarl)', chronic aspiration may pose a problem with irritation and inflammation of the bronchial tree, as described previously by Gosink et al. 33 Arterial Changes

Early in the era of heart transplantation, an accelm,'ated tbrm of arteriosclerosis was observed in the coronary arteries of transplant recipients, vs,16.3J It invoh'ed both large arteries and arterioles and consisted of concentric intimal fibrous proliferations. It was observed in recipients whether or not they experienced episodes of cardiac rejection and was presumed to be a inanifestation of chronic l'ejection. Several heart transp'lant recipients lmve either died, received new grafts, or experienced myocardial infarcts as a resuh. A sinfilar clmnge is seen in renal transplantation, in which endothelial injury from p r e s u m e d immunologic mechanisnls restflts in progressive endarteritis obliterans and nephrosclerosis. 35 In all five patients in this study, accelerated intimal Ityperlflasia was seen in both elastic and musctdar arteries and arterioles in tim lungs. Tiffs tinding was especially tmexpected in light of the youth of tim donor hemts and hfflgs (18, 19, 21, 22, and 32 years). In addition, mild nmscular Iwpertropl W was present

Interstitial Changes

All patients showed diffuse increases in peribronclfial and interstitial fibrosis, particularly in the subpleural and septal regions. Focal scarring was also o b s e r , ~ d , althougl~ it was not associated with increased numbers of imerstitial mononuclear cells, as in idiopatlfic puhnonary fibrosis. In animal models and hntg allografts tlfis delicate fibrosis has been described as a manifestation of chronic rejection. r-' The finding of acute rejection (perivascular 1)'ntplmcytic cuffing) in two of the five patients lemls credence to Otis interpretation; however, its presence in the remaining three suggests its nonspecificity. Interstitial 920

HEART-LUNGTRANSPLANTATION(Yousem et al.)

t

FIGURE 11 [left. Case 3. Acute pulmonary rejection. A marked perivascular, particularly perivenular infiltrate [arrows] was observed at low power. The air spaces contained foamy histiocytes, suggesting proximal obstruction and secondary atelectasis. [Hematoxylin-eosin stain, x 40.] FIGURE 12 [right). Case 3. Acute pulmonary rejection. A marked perivenuTar infiltrate of small, round and transformed lymphocytes, and eosinophils, was associated with perivascular edema. [Hematoxylin-eosin stain, x 40.}

fibrosis may result fl'om organized diffilse alveolar damage (aduh hyaline membrane disease) secondary to hypoxic injury or infection, but tiffs iwpothesis has not been proved. Other causes of fibrosing processes intrinsic to h e a r t - b r a g recipients were discussed previously.

Pleural Changes Aii of tire lung specimens showed m a r k e d pleural fibrosis, wificll was contirmed by chest radiography, tomography, and thoracic CT scanning. Morphologically, the fibrosis consisted of dense lamellar bands of collagen coating.an intact pleurai TABLE 2.

Causes of Bronchiolitis Obliterans in Heart-Lung Transplantation

Recurrent/persistent infection hnmutle reaction to transplanted lung--host-versus-graft disease, i.e., rejection Ahered mucociliar) clearance Impaired ciliary flmction Abnormal mucus chemistr)" and viscosity Bronchial artery ligature C)closporine and other drugs Chronic aspiration and loss of cough reflex

921

elastica. It was, Ilowever, focal in distribution and particularly prominent in tile posterior aspects of tile lungs. Fibrosis may be related to surger)', especially postoperative bleeding or mecilanicai factors, i.e., size mismatcil of hmgs and thoracic cavity. Chronic irritation would result in progressive tifickening. It may also represent a chronic rejection pllenomenon, 42 altilough its distribution is not supportive of this interpretation, since it diffuse distribution o f fibrosis, rather than the focal process described earlier, would be expected. A possible role for cyclosporine A must be considered in any discussion of fibrosing processes. Recent '~'eports of interstitial fibrosis in i~eart biopsies and in kidneys fi'om animals and hunmns raise the possibilit)' that l)ronchiolar, vascular, pleural, and interstitial fibrosis is related to drugs, especially cyclosporine. 31 h ~ b e a r t - h m g transplantation the data are inadcquate to exciude cyclosporine as the cause of pulmonary fibrosis (or arterial or bronchiolar injury) because there are no noncyclosporine-treated human control subjects. I Iowever, heart transplant recipients ilave..not silown a propensity for bronchiolitis obliterans or interstitial fibrosis. In addition, tile accelerated arteriosclerosis observed in tile coronary arteries of heart transplant recipients had been de-

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Volume '16. No. 9 [September 5985)

scribed prior to the advent of cyclosporine usage. Resolution of these problems reqtfires studies of appropriate animal models. Consideration must also be given to the role of ligation of bronchial arteries and lyml)hatics. Ill tire major bronchi in case~ 1 and ,t, a diffilse cylindrical type of broncltiectasis was seen on specimen bronchograms. This may have resulted from impaired Ire;fling of bronchi injured by repeated infection. It is difficult to assess tlte effect of ligation of the lympltatic circulation, since no injection studies were perforn]ed. We did not see evidence o f lymphatic neovascularization or significant endothelial injury with fibroelastosis, despite diffuse pleural fibrosis. Future injection studies might address tiffs issue. T h e difference between transplant recipients with and without obliter,ttive broncbiolitis is prinmrily clinical, in tttat all autopsies and open hmg biopsies perfornted beyond the intntediate perioperative period showed histologic evidence of bronchiolitis obliterans and were perfornted in symptomatic recipients. Thus, we lmve no baseline patltologic findings for asymptomatic h e a r t - h m g transplant recipients. The development of chronic postnasal drainage, cough, dyspnea, purulent sputtnn, and recurrent pulmonary infections was observed in all of tire recipients in whom bronchiolitis oblkerans developed. Tire most severe bronMfiolar damage occurred in the patients in whom clinical symptoms persisted tire longest. Tire bronchiolar and vascular clmnges do not appear to be related to tire cause of pulmonary hypertension (printary or secondary to congenital heart defects), age o f d o n o r or recipient, mismatclles in lung size, duration o f survival period, or number of cardiac rejection episodes. 18 In ligitt of these findings, what are the practical diagnostic implications for the surgical patltologist? First, the interpretation of acute rejection in an intntunocontpromised host ltas proved to be extremely difficult. Interstitial inflammation and alveolar exudates are almost uniforntly accompanied by perivascular inflanmtatory cell infiltrates. Infection must be excluded, first by culturing the tissue for bacteria, viruses, attd fungi and then by staining paraffin-embedded tissue witlt stains for specific organisms. The characteristic distribution of tire periwtscular infiltrate, especially along the venous routes, disportionate to the interstitial or alveolar infihrate, should be sought, althougll it is not an absolute criterion. The tntiformity of tire ntonon{lclear lympllocytic ihfiltrate is also helpfid, althouglt eosinophils were prominent in our two cases, ht one of our cases a rare Pneumocystis organism was fi)untl, yet the predominantly perivascular chlstel'ing of iymphocytes and tire paucity of organisms led us to favor a diagnosis of rejection on tire basis of tlmt biol)sy. A drantatic intmediate response to sterokls in cases 3 and ,t provided the best confirmation of the l)athologic interl)retation of i)uhnonary rejection. Tire distinction between infection and rc~jcctimt is very difficult and warrants f u r t h e r attention in futtlre animal 922

studies. T h e ramifications of increased immunosuppression, as o p p o s e d to c o n t i n u e d antibiotic therapy for an infectious agent, are great, since the majority o f transplant recipients die o f infectious causes.

Second, the presence of bronchiolitis obliterans in h e a r t - l u n g transplant recipients is an ominous sign. Although bronchiolitis obliterans may appear at varying times after transplantation and is not related to duration of survival after transplantation, once it develops it runs a progressively unremitting course. The response of bronchiolitis obliterans to corticosteroids has been inconsistent historically, -09 and in the four patients treated witit prednisone, no sustained response was seen. However, it is possible that corticosteroids or otherwise a u g m e n t e d immunosuppression may prove useful in the acute phase of the airway disease; tlte clinical response in case 3 in tire present study provides some evidence to support tltis. Treatment may be ineffective by the time airflow obstruction is manifested, and a more sensitive indicator of broncttial disease is thus an obvious priority to facilitate tire m a n a g e m e n t of h e a r t - h m g transplant recipients. Indeed, in tltis era of transplant surgery, bronchiolitis obliterans may prove to be the limiting factor in the success of h e a r t - h m g transplantation.

Acknowledgments. The authors acknowledge the photographic assistance of Richard Coffin and the editorial contributions of Thomas V. Colby, MD, Stuart Jamieson, MB, FRCS, Keith Dawkins, MRCP, Edward B. Stinson, MD, Norman E. Shumway, MD, James Theodore, MD, Adrian Morris, MRCI', and Charles B. Carrington, MD. AI)I)ENI)UM

Since the st,bmission of this report, one patient (case 3) died of l)rogressive airway obstruction. Autopsy showed extensive pan-lobar bronchiolitis obliterans. REFERENCES I. Bieber C, Stinson E, Shumway N, et al: Cardiac transplantation in Ill:)*). Circulation 41:753, 1970 2. Jamieson S, Baldwin J, Stinson E, et al: Cliuical heart-brag transplantation. Tr,mspl:mtation 37:8 i, 1984 3. Reitz B: l t e a r t - l u n g transplantation: a review, lleart Transplant 1:291, 1982 4. Reitz B, Burton N, Jamicson s, ct al: lleart and lung transplantation: autotranlsl)lantatioxl and ,dlotransl)lantation in pri,nates with extended survival. J Thorac Cardiovasc Surg 80:360, 1980 5. Veith F, llagstrom J: Alveolar manifestations of rejection: an important cause of the poor resuhs with human hmg Iransplal~tion. Ann Surg 175:336, 1972 6. Veith F, Koerner S. Siegelman S, et al: Diagnosis and reversal of rejection in expcrinaental and clinical lung allografts. A,m Thorac Surg 16:172, 1973 7. Veith F, Sildla S, Bhmlcke S, et al: Nature and evoh,tion of lung allograft rejection with and without immunosul)pression. J Thorac Cardiovasc Surg 63:51)9, 1972 8. Joseph W, Morton D: Morphologic alterations in the trimsplanted primate hmg. Surg G)necol Obstet 133:821, 1971 9. Baker R, Sabanayagan P, Zarins C, et ah Functional and mor-

HEART-LUNG TRANSPLANTA11ON[Yousem et al.]

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26. Stein-StrerleinJ, l.ipscomb hi, llart D, et al: Graft-versus-host reaction in the hmg. Transplantation 32:38, 1981 27. Grebe S, StreileinJ: Graft versus host reactions: a review. Adv Immunol 22:119, 1976 28. Ralph D, Springmeyer S, Sullivan K, et al: Rapid progressive airflow obstruction in marrow transplant recipients. Am Rev Respir Dis 129:641, 1984 29. McI)owell E, Barrett L, ttarris C, et al: Abnormal cilia in h u n m n bronchial epitllelium. Arch Pathol Lab Med 100:429, 1976 30. Murph)' K, Alkins C, Offer R, et al: Obliterative bronchiolitis in two rheumatoid arthritics treated with penicillamine. Arthritis P,heum 24:557, 1981 31. Epler G, Snider G, Gaensler E, et ah Bronchiolitis and bronchitis in connective tissue disease. JAMA 242:528, 1979 32. Myers B, Ross J, Newton L, et al: Cyclosporine A associated chronic nephropathy: a potentially irreversible renal injury. N EnglJ Med 311:699, 1984 33. Gosink B, Friedman P, Liebow A: Bronchiolitis obliterans. AJR 117:816, 1973 3-t. Griepp R, Stinson E, Bieber C, et al: Control of graft arteriosclerosis in h n n m n heart transplant recipients. Surger)" 81:262, 1977 35. l'ortcr K: Renal transplantation. In tleptinstall RH (ed): Patholog)' of the Kidney. Boston, Little, Brown, 1974 36. Corson J: The pathologist and the kidney transplant. Pathol Annu 7:251, 1972 37. Busch G, Galvanek EG, Reynolds ES: l-tunlan renal allografts: analysis of lesions in long term snrvivals, ttuxl I:DATIIOL 9"953 1[171 38. Crosnier B: The transplanted kidney. Contrib Nephrol 7:177, 1977 39. Wagenvoort CA, Wagenvoort N: Pathology of l'uhnnnary llypertension. New York, John Wiley and Sons, 1977 40. Harris 1', lleath D: The l l n m a n Puh!lotiary Circtdation. Its Form and Function in I leahh and Disease, ed 2. New York, Churchill Livingstone, 1977 41. Corrin B, Spencer l'l, Turner-Warwick M, et al: Puhnonary veno-occlnsion--an immune complex disease? Virchows Arclx [A] 36.t:81, 1974 42. llaverich A, Dawkins K, Baldwin J, et al: Long term cardiac and puhnonary histolog)' in primates fifllowing combined heart and lung transplantation. In press, Transplantation