lung remodeling is a common end - stage sequela of idiopathic pulmonary fibrosis ( ipf ) 
 and systemic sclerosis ( ssc ) , resulting in disruption of lung architecture , leading 
 to progressive respiratory failure.(- ) 
 histologically , the remodeling process is characterized by diffuse chronic 
 interstitial inflammation and increased fibroblast proliferation , as well as by 
 increased extracellular matrix synthesis and collagen deposition .
( , , ) 
 therefore , modulation of inflammation , fibroblast proliferation , and collagen 
 synthesis by effector mediators in ipf and ssc is very important . despite the 
 characterization of a variety of key participants , the mediators and mechanisms 
 involved in the pathogenesis of ipf and ssc
have yet to be fully defined , which 
 might explain the limited number of therapeutic approaches , with little impact on 
 long - term survival .
( , ) it is known that cox is the key enzyme in the conversion of arachidonic acid to 
 prostaglandin e2 ( pge2 ) , the precursor of a diverse family of 
 bioactive lipid mediators including prostaglandins , thromboxane , and prostacyclin . 

the former is constitutively 
 expressed in most tissues and acts as a housekeeping enzyme regulating vascular 
 homeostasis , protecting the gastric mucosa , and maintaining renal integrity , ( , ) whereas the latter has lower levels of expression in 
 most tissues but is inducible in response to growth factors , cytokines , and other 
 proinflammatory molecules.(- ) regarding the proinflammatory and anti - inflammatory roles of cox-1 and cox-2 , 
 immunohistochemistry can be a useful tool to detect cox-1 and cox-2 in the remodeled 
 lung in patients with ssc and ipf .
data on the assessment of cox-1 and cox-2 in the 
 remodeled lung have previously been reported in serum ( , ) and bronchoalveolar lavage fluid ( ) from patients with ssc , as well as in fibroblast 
 cultures ( ) and biopsies ( , ) from patients with ipf . however , the roles of cox-1 
 and cox-2 in the mechanisms involved in the remodeled lung in ipf and ssc patients 
 are still unclear , and there has been uncertainty regarding the best way to detect 
 cox-2 .
the aim of the present study was to study the expression of cox-1 and cox-2 
 in lung biopsy specimens ( cox-1 and cox-2 expression being separately evaluated in 
 alveolar septa , bronchioles , and vessels ) and correlate it with patient survival . 

between january of 2002 and july of 2008 , 24 consecutive patients with ssc and 
 interstitial lung disease and 30 patients suspected of having ipf on the basis of 
 hrct findings were submitted to open lung biopsy at the university of so paulo 
 school of medicine hospital das clnicas , located in the city of 
 so paulo , brazil .
all patients fulfilled the diagnostic criteria for ssc ( ) and ipf ( ) open lung biopsy was performed by formal 
 thoracotomy , areas of honeycombing being avoided .
all patients gave written informed 
 consent , and the study was approved by the local research ethics committee ( protocol 
 no .
disease duration was determined on 
 the basis of the onset of the first symptom .
pulmonary function testing and hrct 
 were performed within up to 3 months before the biopsy .
pulmonary function testing 
 included vc , fev1 , fvc , fev1/fvc , tlc , rv , and dlco . 

physiological assessment was performed before open lung biopsy and before the 
 initiation of treatment .
all pulmonary function tests , including spirometry , 
 determination of lung volumes , and measurement of dlco , were performed on the same 
 day .
all spirometric tests were performed with a calibrated pneumotachograph 
 ( medical graphics co. , st .
paul , mn , usa ) , all values being expressed as a 
 percentage of their respective predicted value , the reference values having been 
 established by pereira et al .
( ) 
 lung volumes were measured with a whole - body plethysmograph ( medical graphics co. ) , 
 all values being expressed as a percentage of the predicted values .
( ) all 
 patients were followed regularly after treatment until death , blood and lung 
 function tests being regularly performed .
the primary endpoint was to evaluate the 
 impact of cox-1 and cox-2 changes on survival and analyze differences between ssc 
 and ipf .
, normal lung tissue was obtained from 10 individuals ( 6 males and 4 
 females ) whose median age was 46.6  5.8 years and who had died suddenly of 
 nonpulmonary causes .  

table 1 clinical data of the patients with systemic sclerosis and of those 
 with idiopathic pulmonary fibrosis.a 
  regarding open lung biopsy findings , usual interstitial pneumonia ( uip ) , the 
 histological pattern of ipf , was characterized by patchy subpleural and paraseptal 
 distribution of parenchymal injury .
temporal heterogeneity was seen at low 
 magnification , areas of normal lung parenchyma alternating with alveolar collapse , 
 interstitial mononuclear infiltrates , septal fibromyxoid tissue ( fibroblastic foci ) , 
 and honeycomb lung .
( ) all of the 
 patients with ssc had histological patterns consistent with fibrotic nonspecific 
 interstitial pneumonia ( nsip ) , as defined by temporally homogeneous septal 
 thickening and interstitial fibrosis .
( ) for immunohistochemistry analysis , a standard peroxidase technique was used ( harris 's 
 hematoxylin being used as the counterstain ) in order to identify cox-1 and cox-2 
 expression in alveolar septa , bronchiolar walls , and vascular walls in normal lung 
 tissue ( the control tissue ) , in lung tissue showing the uip pattern ( the uip 
 tissue ) , and in lung tissue showing the nsip pattern ( the nsip tissue ) .
anti - cox-1 and 
 anti - cox-2 antibodies ( santa cruz biotechnology , inc . , santa cruz , ca , usa ) were 
 incubated with tissue sections at dilutions of 1:50 and 1:100 , respectively . the 
 novolink max polymer amplification kit ( leica biosystems
newcastle ltd , newcastle 
 upon tyne , uk ) was used for signal amplification , and 3,3'-diaminobenzidine 
 tetrahydrochloride ( 0.25 mg dissolved in 1 ml of 0.02% hydrogen peroxide ) was used 
 as a precipitating substrate for signal detection .
the specificity of primary 
 antibodies was confirmed by appropriate reagent controls ( the primary antibody being 
 omitted or nonimmune serum being substituted for the primary antibody in the 
 staining protocol ) , which revealed no staining .  
regarding histomorphometry , cox-1 expression and cox-2 expression were assessed by a 
 point - counting technique in 50 and 30 fields in alveolar septa , bronchiolar walls , 
 and vascular walls in the control tissue , in the uip tissue , and in the nsip tissue . 

the technique was performed with a 100-point grid ( area , 187,500 m ( ) ; magnification , 400 ) attached 
 to the microscope eyepiece .
( ) at 
 a magnification of 400 , the septal , bronchiolar , and vascular areas in each field 
 were calculated on the basis of the number of points overlying connective tissue , as 
 a proportion of the total grid area .
subsequently , the number of immunostained cells 
 within the septal , bronchiolar , and vascular areas was counted .
the areal fraction 
 of immunostained cells represents the percentage ratio of the area of labeled cells 
 in relationship to the total area covered by the grid in the eyepiece .   in order to assess interobserver variability , we compared the results obtained by two 
 observers in 20% of the slides .
the coefficient of variation for the interobserver 
 error of the cell count was 5% .  
the student 's t - test for independent 
 samples was used in order to test the relationship between continuous variables , and 
 the residuals were examined to ensure that they were approximately normally 
 distributed . the relationship between cellularity ( as determined by immunostaining ) 
 and pulmonary function test results was evaluated by pearson 's correlation 
 coefficient . for all cases ,
measured variable values were arranged in ascending 
 order and divided into two groups on the basis of the median value of each variable . 
 for each variable , the groups were designated low degree and high degree , as 
 follows : alveolar septal cox-1 ( low degree , < 2.35% ; high degree , 2.35% ) ; 
 vascular cox-1 ( low degree , < 2.91% ; high degree , 2.91% ) ; bronchiolar cox-1 
 ( low degree , < 2.88% ; high degree , 2.88% ) ; total cox-1 ( low degree , < 
 2.77% ; high degree , 2.77% ) ; alveolar septal cox-2 ( low degree , < 2.04% ; high 
 degree , 2.04% ) ; vascular cox-2 ( low degree , < 2.34% ; high degree , 2.34% ) ; 
 bronchiolar cox-2 ( low degree ,
< 2.34% ; high degree , 2.34% ) ; and total cox-2 
 ( low degree , < 2.16% ; high degree , 2.16% ) .  
first , we performed a 
 univariate analysis relating overall follow - up to each of the measured variables by 
 means of the kaplan - meier method and then analyzed survival using the log - rank test . 

the variables that were found to be significant in the univariate analysis were 
 included in the multivariate analysis based on the cox proportional hazards 
 regression model .
deaths from causes other than ipf or ssc and living patients were included in the 
 models as censored cases .  
all statistical procedures were performed with the statistical package for the social 
 sciences , version 18.0 ( spss inc .

 table 1 summarizes the clinical features of 
 the patients with ssc ( n = 24 ) and those of those with ipf ( n = 30 ) .
six of 17 ssc 
 patients ( 35.29% ) and 13 of 19 ipf patients ( 68.42% ) had restrictive lung disease . 

respiratory function test results were as follows : fvc < 80% in 18 ( 75% ) of the 
 24 ssc patients and in 19 of 22 ipf patients ( 86.36% ) ; tlc < 80% in 6 of 17 ssc 
 patients ( 35.9% ) and in 13 of 19 ipf patients ( 68.42% ) ; dlco < 80% in 12 of 15 
 ssc patients ( 80% ) and in 8 of 9 ipf patients ( 88.88% ) ; and dlco / alveolar volume 
 < 80% in 11 of 18 ssc patients ( 61.11% ) and in 11 of 14 ipf patients ( 78.57% ) . 

a significant negative correlation was found between cox-2 expression in vessels and 
 fvc ( r= 0.28 ; p = 0.05 ) , as well as between cox-2 expression in alveolar septa and 
 dlco ( r = 0.80 ; p = 0.009 ) .  

figure 1 shows alveolar septa , vessels , and 
 bronchioles in the control tissue , in the nsip tissue , and in the uip tissue 
 immunostained for cox-1 ( in a , c , e , g , i , k , m , o , and q ) and cox-2 ( in b , d , f , h , 
 j , l , n , p , and r ) .
the nsip and uip tissues differed from the control tissue in 
 terms of the immunostaining intensity of epithelial cells , endothelial cells , 
 myofibroblasts , and smooth muscle cells in the alveolar septa , vessels , and 
 bronchioles .  

figure 1 cellular expression of cox-1 and cox-2 in alveolar septa , 
 intrapulmonary vessels , and bronchioles in normal lung tissue ( control 
 tissue ) ; in lung tissue obtained from patients with systemic sclerosis 
 ( ssc ) and showing fibrotic nonspecific interstitial pneumonia ( nsip ) ; 
 and in lung tissue obtained from patients with idiopathic pulmonary 
 fibrosis ( ipf ) and showing usual interstitial pneumonia ( uip ) .
the 
 intensity of cox-1 immunostaining of epithelial cells , endothelial 
 cells , myofibroblasts , and smooth muscle cells in ssc - nsip and ipf - uip 
 tissue alveolar septa ( g and m , respectively ) , vessels ( i and o , 
 respectively ) , and bronchioles ( k and q , respectively ) was higher than 
 was that of cox-1 immunostaining of those cells in control tissue 
 alveolar septa ( a ) , vessels ( c ) , and bronchioles ( e ) .
likewise , the 
 intensity of cox-2 immunostaining of those cells in ssc - nsip and ipf - uip 
 tissue alveolar septa ( h and n , respectively ) , vessels ( j and p , 
 respectively ) , and bronchioles ( l and r , respectively ) was higher than 
 was that of cox-2 immunostaining of those cells in control tissue 
 alveolar septa ( b ) , vessels ( d ) , and bronchioles ( f ) .
the bar plots show 
 the quantification of cox-1 and cox-2 immunostaining of cells in 
 alveolar septa ( s ) , total lung parenchyma ( t ) , and bronchioles ( w ) in 
 the control tissue , in the ssc - nsip tissue , and in the ipf - uip tissue 
 ( immunohistochemical staining ; magnification , 400 ) . 
 

the proportion of alveolar septal cells immunostained for cox-1 and cox-2 was 
 significantly higher in the uip and nsip tissues than in the control tissue . in 
 other words ,
high proportions of alveolar septal cells staining for cox-1 and cox-2 
 were associated with the uip and nsip patterns . as can be seen in the bar plots in 
 figure 1 ( s and t ) the relationship of 
 cox-1 and cox-2 with ipf ( the uip pattern ) was stronger than was that of cox-1 and 
 cox-2 with ssc ( the nsip pattern ) . although the proportion of bronchiolar cells 
 immunostained for cox-2 was lower in the nsip and uip tissues than in the control 
 tissue ( figure 1w ) , the difference was not 
 statistically significant .
in addition , although the proportion of bronchiolar cells 
 immunostained for cox-1 was higher in the uip and nsip tissues than in the control 
 tissue ( figure 1w ) , the difference was not 
 significant .
no differences were found among the tissues in terms of the cox-1 or 
 cox-2 immunostaining , for vessels or for the total parenchyma ( table 2 ) .  

table 2 morphometric results in normal lung tissue ( control tissue ) , in lung 
 tissue showing the usual interstitial pneumonia pattern ( from patients 
 with idiopathic pulmonary fibrosis ) , and in lung tissue showing the 
 nonspecific interstitial pneumonia pattern ( from patients with systemic 
 sclerosis).a 
  a preliminary analysis of the kaplan - meier survival curves showed that survival was 
 better in the patients with ssc ( the fibrotic nsip pattern ) and cox-2 expression 
 > 2.25% ( median survival , 70.75 months ) than in those with ipf ( the uip 
 pattern ) and cox-2 expression < 2.25% ( median survival , 46.32 months ; figure 2 ) . therefore , we coded the fibrotic nsip 
 pattern as a single dummy variable with a value of 1 and the uip pattern with a 
 value of 2 .
the results of the multivariate analysis based on the cox proportional 
 hazards regression model are shown in table 
 3 .
after controlling for age , pulmonary function test results , the uip 
 pattern , and the fibrotic nsip pattern , we found that only two variables were 
 significantly associated with survival time : the fibrotic nsip pattern and alveolar 
 septal cox-2 ( p = 0.02 ) .
once these two variables were accounted for , none of the 
 others were related to survival .
the multivariate analysis showed a low risk of 
 death for young patients with low fev1/fvc , fibrotic nsip pattern , and 
 high - degree alveolar septal cox-2 .  

figure 2 cox regression plots for risk of death risk versus duration of 
 follow - up ( in months ) in young patients with low dlco / alveolar volume , 
 systemic sclerosis ( and a histological pattern of cellular nonspecific 
 interstitial pneumonia ) , high - degree total cox-1 , and low - degree 
 alveolar septal cox-2 .
the top curve represents the group of patients 
 with systemic sclerosis and cellular nonspecific interstitial pneumonia . 

the bottom curve represents two groups of patients : those with systemic 
 sclerosis and fibrotic nonspecific interstitial pneumonia ; and those 
 with idiopathic pulmonary fibrosis and the usual interstitial pneumonia 
 pattern . 
 

table 3 cox proportional hazards regression to ascertain the individual 
 contribution of the histological pattern and morphological factors 
 associated with survival and to compare adjusted survival between the 
 two groups . 

the limited number of therapeutic approaches that have any impact on long - term 
 survival in patients with ipf - uip and in those with ssc and fibrotic nsip is due to 
 the lack of definition regarding the mediators and mechanisms involved in the 
 pathogenesis of ipf and ssc .
therefore , the question of interest is whether 
 additional mediators can provide a better understanding of the pathogenesis of these 
 diseases .
the repair process involves two distinct stages : a regenerative , 
 inflammatory phase , in which the microenvironment attempts to replace injured cells ; 
 and a fibrotic phase , in which connective tissue replaces normal parenchymal 
 tissue.(- ) in the repair process , 
 pge2 production by fibroblasts is increased , ( , ) which constitutes further evidence of the 
 antiproliferative , anti - inflammatory and antifibrotic properties of 
 cox-2/pge2 .
( ) 
 therefore , our finding that immunohistochemistry staining for cox provides important 
 information on the repair processes in pulmonary fibrosis is not surprising , and our 
 results confirm that the expression of cox-2 is increased in ipf and ssc , with 
 improved outcome in a group of patients .
we found that the proportion of alveolar 
 septal cells immunostained for cox-1 and cox-2 was significantly higher in lung 
 tissue showing the uip pattern and the fibrotic nsip pattern than in normal lung 
 tissue . increased cox-1 expression
was expected because cox-1 is constitutively 
 expressed in most cells and tissues , whereas cox-2 is induced by inflammatory or 
 mitogenic stimuli .
( , , ) 
 those studies showed reduced cox-2 expression in pulmonary fibroblasts secondary to 
 decreased cox-2 production . however , in those studies , cox-2 expression was measured 
 only in fibroblasts , whereas in our study it was measured in the alveolar septa , 
 including epithelial cells and fibroblasts in normal areas , collapsed areas , and 
 fibroblast foci .
( ) found increased expression of 
 cox-2 in metaplastic epithelium and fibroblasts from fibrotic areas in ipf - uip . 

first , 
 reduced cox-2 expression in normal areas , collapsed areas , and fibroblastic foci 
 suggests an anti - inflammatory role for cox-2 in early - stage ipf - uip .
second , the 
 presence of progressive fibrosis even in the presence of increased cox-2 expression 
 suggests that fibroblasts are unable to respond to stimulation by cox-2 and its main 
 product ( pge2 ) so as to inhibit fibroblast proliferation , myofibroblastic 
 transformation , and increased production of collagen and other extracellular matrix 
 molecules .   in the present study , the proportion of alveolar septal cells immunostained for cox-1 
 and cox-2 was found to be lower in fibrotic nsip tissue ( from ssc patients ) than in 
 uip tissue ( from ipf patients ) .
this finding contrasts with those of previous 
 studies showing that cox-2 levels are higher in ssc patients.(- ) in addition , cox-2 production has been shown to be much 
 greater in the inflammatory resolution phase than in the early phase .
( ) these contradictory findings 
 suggest that cox-2 has a dual role in a normal inflammatory process , playing a 
 proinflammatory role in the early phase and an anti - inflammatory role in the 
 resolution phase .
( ) therefore , 
 in view of the abovementioned evidence and of the latent inflammation in patients 
 with ssc and lung involvement , our results emphasize the idea that cox-2 does not 
 exert its anti - inflammatory effect properly , because there is inflammation even when 
 cox-2 expression is increased in patients with ssc and fibrotic nsip .
however , 
 further studies are needed in order to clarify the real reason why the cox-2 
 mechanism is deficient .
we hypothesize that this is due to an inability of cox-2 to 
 stimulate the production of pge2 or other anti - inflammatory mediators in 
 opposition to its own proinflammatory effects or an inability of the cells to 
 respond appropriately to cox-2 .  
we sought to establish a correlation 
 between cox-2 and patient survival controlled for age , pulmonary function test 
 results , the uip pattern ( in patients with ipf ) and the nsip pattern ( in patients 
 with ssc ) .
our multivariate analysis showed a low risk of death for younger patients 
 with low dlco / alveolar volume , ssc ( and the nsip histological pattern ) , high - degree 
 total cox-2 , and high - degree alveolar septal cox-1 .   in conclusion ,
the expression of cox-1 and cox-2 in the lung parenchyma offers us the 
 potential to control repair processes involved in the progression of ssc - nsip and 
 ipf - uip , suggesting that strategies aimed at preventing low cox-1 synthesis will 
 have a greater impact on ssc , whereas those aimed at preventing high cox-2 synthesis 
 will have a greater impact on ipf .
  estudar a expresso de cox-1 e cox-2 em reas pulmonares remodeladas em 
 pacientes com esclerose sistmica ( es ) ou fibrose pulmonar idioptica ( fpi ) 
 e correlacion - la com a sobrevida desses pacientes .
  examinamos espcimes de bipsia pulmonar a cu aberto de 24 pacientes com es 
 e de 30 pacientes com fpi , utilizando - se tecido pulmonar normal como 
 controle .
os padres histolgicos incluram pneumonia intersticial no 
 especfica ( pine ) fibrtica em pacientes com es e pneumonia intersticial 
 usual ( piu ) nos pacientes com fpi .
imuno - histoqumica e histomorfometria 
 foram usadas para avaliar a expresso celular de cox-1 e cox-2 em septos 
 alveolares , vasos e bronquolos , sua correlao com provas de funo 
 pulmonar e seu impacto na sobrevida .
  a expresso de cox-1 e cox-2 em septos alveolares foi significativamente 
 maior em fpi - piu e es - pine do que no tecido controle .
no houve diferena 
 entre fpi - piu e es - pine quanto  expresso de cox-1 e cox-2 . a anlise 
 multivariada baseada no modelo de regresso de cox mostrou que os fatores 
 associados a baixo risco de morte foram ter idade menor , valores elevados de 
 dlco / volume alveolar , fpi , e alta expresso de cox-1 em septos alveolares , 
 ao passo que os fatores associados a alto risco de morte foram ter idade 
 maior , valores baixos de dlco / volume alveolar , es ( com pine ) e baixa 
 expresso de cox-1 em septos alveolares
  nossos resultados sugerem que estratgias de preveno de baixa sntese de 
 cox-1 tero maior impacto sobre a es , ao passo que as de preveno de alta 
 sntese de cox-2 tero maior impacto sobre a fpi .
o remodelamento pulmonar  uma sequela terminal comum da fibrose pulmonar 
 idioptica ( fpi ) e da esclerose sistmica ( es ) ; resulta em desorganizao da 
 arquitetura pulmonar e , consequentemente , insuficincia respiratria 
 progressiva.(- ) histologicamente , o processo 
 de remodelamento  caracterizado por inflamao intersticial crnica difusa e 
 aumento da proliferao de fibroblastos , da sntese de matriz extracelular e da 
 deposio de colgeno .
( , , ) portanto , a modulao da inflamao , da 
 proliferao de fibroblastos e da sntese de colgeno por mediadores efetores na 
 fpi e na es  muito importante .
no obstante a caracterizao de vrios 
 participantes cruciais , os mediadores e mecanismos envolvidos na patognese da 
 fpi e da es ainda no foram totalmente definidos , o que pode explicar o nmero 
 limitado de opes teraputicas , com pouco impacto na sobrevida em longo 
 prazo .
( , ) sabe - se que a cox  a enzima essencial para a converso de cido araquidnico em 
 prostaglandina e2 ( pge2 ) , que  o precursor de uma famlia 
 de mediadores lipdicos bioativos diversos , como as prostaglandinas , o 
 tromboxano e a prostaciclina
. a cox existe em duas isoformas : cox-1 e cox-2 . a 
 cox-1  expressa constitutivamente na maioria dos tecidos e atua como uma enzima 
 " que mantm a casa em ordem " , isto  , que regula a homeostase vascular , protege 
 a mucosa gstrica e mantm a integridade renal , ( , ) ao passo que a cox-2 tem nveis mais baixos de 
 expresso na maioria dos tecidos , embora possa ser induzida em resposta a 
 fatores de crescimento , citocinas e outras molculas pr - inflamatrias.(- ) no tocante ao papel pr - inflamatrio e anti - inflamatrio
da cox-1 e da cox-2 , a 
 imuno - histoqumica pode ser til para detectar essas enzimas no pulmo 
 remodelado de pacientes com es e fpi .
dados relativos  avaliao de cox-1 e 
 cox-2 no pulmo remodelado foram anteriormente relatados no soro ( , ) e lavado broncoalveolar ( ) de pacientes com es , bem 
 como em culturas de fibroblastos ( ) e bipsias ( , ) de pacientes com fpi .
no entanto , o papel da cox-1 e 
 da cox-2 nos mecanismos envolvidos no remodelamento pulmonar em pacientes com 
 fpi e es ainda no est claro , e h incerteza sobre a melhor maneira de detectar 
 a cox-2 .
o objetivo do presente estudo foi estudar a expresso de cox-1 e cox-2 
 ( avaliada separadamente em septos alveolares , bronquolos e vasos ) em espcimes 
 obtidos por meio de bipsia pulmonar e correlacion - la com a sobrevida dos 
 pacientes .
entre janeiro de 2002 e julho de 2008 , 24 pacientes consecutivos com es e doena 
 pulmonar intersticial e 30 pacientes com suspeita de fpi baseada em achados de 
 tcar foram submetidos a bipsia pulmonar a cu aberto no hospital das clnicas 
 da faculdade de medicina da universidade de so paulo , na cidade de so paulo 
 ( sp ) .
todos os pacientes preencheram os critrios para o diagnstico de es ( ) e fpi .
( ) a bipsia pulmonar a cu aberto foi realizada 
 por meio de toracotomia formal , evitando - se reas de faveolamento
. todos os 
 pacientes assinaram um termo de consentimento livre e esclarecido , e o estudo 
 foi aprovado pelo comit de tica em pesquisa da instituio ( protocolo n 
 0960/08 ) .  
foram analisados os pronturios clnicos de todos os pacientes . a durao da 
 doena foi determinada com base no incio do primeiro sintoma .
os testes de 
 funo pulmonar e a tcar foram realizados em um prazo de at 3 meses antes da 
 bipsia .
os testes de funo pulmonar incluram cv , vef1 , cvf , 
 vef1/cvf , cpt , vr e dlco
. a avaliao fisiolgica foi realizada 
 antes da bipsia pulmonar a cu aberto e antes do incio do tratamento . todos os 
 testes de funo pulmonar , incluindo espirometria , determinao dos volumes 
 pulmonares e medio da dlco , foram realizados no mesmo dia . todos os testes 
 espiromtricos foram realizados com um pneumotacgrafo calibrado ( medical 
 graphics co. , st .
paul , mn , eua ) ; todos os valores foram expressos em 
 porcentagem de seus respectivos valores previstos , os valores de referncia 
 tendo sido estabelecidos por pereira et al .
( ) os volumes pulmonares foram medidos com um 
 pletismgrafo de corpo inteiro ( medical graphics co. ) ; todos os valores foram 
 expressos em porcentagem dos valores previstos .
( ) todos os pacientes foram acompanhados 
 regularmente aps o tratamento at o bito ; exames de sangue e testes de funo 
 pulmonar foram realizados regularmente .
o desfecho primrio foi avaliar o 
 impacto de alteraes em cox-1 e cox-2 na sobrevida e analisar as diferenas 
 entre es e fpi . a tabela 1 mostra os 
 dados demogrficos .
como controle , obtivemos tecido pulmonar normal de 10 
 indivduos ( 6 homens e 4 mulheres ) cuja mediana de idade foi de 46,6  5,8 anos 
 e que morreram subitamente de causas no pulmonares .  
 tabela 1 dados clnicos dos pacientes com esclerose sistmica e daqueles 
 com fibrose pulmonar idioptica.a 
  quanto aos achados da bipsia pulmonar a cu aberto , a pneumonia intersticial 
 usual ( piu ) , o padro histolgico da fpi , foi caracterizada por distribuio 
 subpleural e parasseptal segmentada de leses parenquimatosas .
com um aumento 
 pequeno , observou - se heterogeneidade temporal , com reas de parnquima pulmonar 
 normal se alternando com colapso alveolar , infiltrado mononuclear intersticial , 
 tecido fibromixoide septal ( focos fibroblsticos ) e faveolamento .
( ) todos os pacientes com es 
 apresentavam padres histolgicos consistentes com pneumonia intersticial no 
 especfica ( pine ) fibrtica , definida por espessamento septal temporalmente 
 homogneo e fibrose intersticial .
( ) para a anlise imuno - histoqumica , foi usada uma tcnica - padro de peroxidase - 
 com a hematoxilina de harris como contracorante - a fim de identificar a 
 expresso de cox-1 e cox-2 em septos alveolares , paredes bronquiolares e paredes 
 vasculares em tecido pulmonar normal ( o tecido controle ) , em tecido pulmonar com 
 padro histolgico de piu ( o tecido piu ) e em tecido pulmonar com padro 
 histolgico de pine ( o tecido pine ) .
anticorpos anti - cox-1 e anti - cox-2 ( santa 
 cruz biotechnology , inc . , santa cruz , ca , eua ) foram incubados com cortes 
 histolgicos em diluies de 1:50 e 1:100 , respectivamente .
o kit de 
 amplificao novolink max polymer ( leica biosystems newcastle ltd , newcastle 
 upon tyne , reino unido ) foi usado para a amplificao do sinal , e 
 3,3'-diaminobenzidina tetra - hidrocloreto ( 0,25 mg dissolvidos em 1 ml de 
 perxido de hidrognio a 0,02% ) foi usado como substrato precipitante para a 
 deteco do sinal . a especificidade dos anticorpos primrios foi confirmada por 
 controles de reagentes apropriados - o anticorpo primrio sendo omitido ou soro 
 no imune sendo usado em vez do anticorpo primrio no protocolo de colorao - 
 os quais no revelaram nenhuma colorao .  
quanto  histomorfometria , avaliamos a expresso de cox-1 e cox-2 por meio de uma 
 tcnica de contagem de pontos em 50 e 30 campos em septos alveolares , paredes 
 bronquiolares , e paredes vasculares no tecido controle , no tecido piu e no 
 tecido pine .
a tcnica foi executada com um retculo de 100 pontos ( rea : 
 187.500 m ( ) ; aumento : 400 ) 
 ligado  ocular do microscpio .
( ) com um aumento de 400 , as reas septais , 
 bronquiolares e vasculares em cada campo foram calculadas com base no nmero de 
 pontos sobrepostos ao tecido conjuntivo , na forma de porcentagem da rea total 
 do retculo .
posteriormente , contou - se o nmero de clulas imunomarcadas nas 
 reas septais , bronquiolares e vasculares .
a frao de rea de clulas 
 imunomarcadas representa a relao percentual entre a rea de clulas marcadas e 
 a rea total coberta pelo retculo na ocular .  
para avaliar a variabilidade interobservador , comparamos os resultados obtidos 
 por dois observadores em 20% das lminas . o coeficiente de variao para o erro 
 interobservador da contagem de clulas foi de 5% .  
os dados so apresentados na forma de mdia  dp e ic95% . o teste t de student 
 para amostras independentes foi usado a fim de testar a relao entre variveis 
 contnuas , e os resduos foram examinados para certificar que sua distribuio 
 era aproximadamente normal .
a relao entre celularidade ( determinada pela 
 imunomarcao ) e os resultados dos testes de funo pulmonar foi avaliada pelo 
 coeficiente de correlao de pearson .
para todos os casos , os valores das 
 variveis medidas foram dispostos em ordem crescente e divididos em dois grupos 
 com base na mediana de cada varivel .
para cada varivel , os grupos foram 
 denominados baixo grau e alto grau , a saber : cox-1 em septos alveolares ( baixo 
 grau : < 2,35% ; alto grau : 2,35% ) ; cox-1 em vasos ( baixo grau : < 2,91% ; 
 alto grau : 2,91% ) ; cox-1 em bronquolos ( baixo grau : < 2,88% ; alto grau : 
 2,88% ) ; cox-1 total ( baixo grau : < 2,77% ; alto grau : 2,77% ) ; cox-2 em 
 septos alveolares ( baixo grau : < 2,04% ; alto grau : 2,04% ) ; cox-2 em vasos 
 ( baixo grau : < 2,34% ; alto grau : 2,34% ) ; cox-2 em bronquolos ( baixo grau : 
 < 2,34% ; alto grau : 2,34% ) e cox-2 total ( baixo grau : < 2,16% ; alto 
 grau : 2,16% ) .  
primeiro , realizamos 
 uma anlise univariada relacionando o acompanhamento global com cada uma das 
 variveis medidas por meio do mtodo de kaplan - meier e ento analisamos a 
 sobrevida por meio do teste de log - rank .
as variveis que se 
 mostraram significantes na anlise univariada foram includas na anlise 
 multivariada baseada no modelo de regresso de riscos proporcionais de cox . 

mortes 
 cujas causas foram outras que no fpi ou es e pacientes vivos foram includos 
 nos modelos como casos censurados .   todos os procedimentos estatsticos foram realizados com o programa 
 statistical package for the social sciences , verso 18.0 
 ( spss inc . ,
a tabela 1 resume as caractersticas 
 clnicas dos pacientes com es ( n = 24 ) e dos pacientes com fpi ( n = 30 ) .
seis de 
 17 pacientes com es ( 35,29% ) e 13 de 19 pacientes com fpi ( 68,42% ) apresentaram 
 distrbio ventilatrio restritivo .
os resultados dos testes de funo 
 respiratria foram os seguintes : cvf < 80% em 18 ( 75% ) dos 24 pacientes com 
 es e em 19 de 22 pacientes com fpi ( 86,36% ) ; cpt < 80% em 6 de 17 pacientes 
 com es ( 35,9% ) e em 13 de 19 pacientes com fpi ( 68,42% ) ; dlco
< 80% em 12 
 de 15 pacientes com es ( 80% ) e em 8 de 9 pacientes com fpi ( 88,88% ) ; dlco / volume 
 alveolar < 80% em 11 de 18 pacientes com es ( 61,11% ) e em 11 de 14 
 pacientes com fpi ( 78,57% ) .
houve correlao negativa significativa entre a 
 expresso de cox-2 em vasos e a cvf ( r = 0,28 ; p = 0,05 ) e entre a expresso de 
 cox-2 em septos alveolares e a dlco ( r = 0,80 ; p = 0,009 ) .  
a figura 1 mostra septos alveolares , vasos 
 e bronquolos no tecido controle , no tecido pine e no tecido piu com 
 imunomarcao positiva para cox-1 ( em a , c , e , g , i , k , m , o e q ) e cox-2 ( em b , 
 d , f , h , j , l , n , p e r ) .
os tecidos pine e piu diferiram do tecido controle 
 quanto  intensidade da imunomarcao de clulas epiteliais , clulas 
 endoteliais , miofibroblastos e clulas musculares lisas nos septos alveolares , 
 vasos , e bronquolos .  

figura 1 expresso celular de cox-1 e cox-2 em septos alveolares , vasos 
 intrapulmonares e bronquolos em tecido pulmonar normal ( tecido 
 controle ) ; em tecido pulmonar de pacientes com esclerose sistmica 
 ( es ) e pneumonia intersticial no especfica ( pine ) fibrtica ; e em 
 tecido pulmonar de pacientes com fibrose pulmonar idioptica ( fpi ) e 
 pneumonia intersticial usual ( piu ) . a intensidade da imunomarcao 
 positiva para cox-1 em clulas epiteliais , clulas endoteliais , 
 miofibroblastos e clulas musculares lisas em septos alveolares de 
 tecido es - pine e fpi - piu ( g e m , respectivamente ) , em
vasos de 
 tecido es - pine e fpi - piu ( i e o , respectivamente ) e em bronquolos 
 de tecido es - pine e fpi - piu ( k e q , respectivamente ) foi maior que a 
 da imunomarcao positiva para cox-1 nessas clulas em septos 
 alveolares do tecido controle ( a ) , em vasos do tecido controle ( c ) e 
 em bronquolos do tecido controle ( e ) .
do mesmo modo , a intensidade 
 da imunomarcao positiva para cox-2 nessas clulas em septos 
 alveolares de tecido es - pine e fpi - piu ( h e n , respectivamente ) , em 
 vasos de tecido es - pine e fpi - piu ( j e p , respectivamente ) e em 
 bronquolos de tecido es - pine e fpi - piu ( l e r , respectivamente ) foi 
 maior que a da imunomarcao positiva para cox-2 nessas clulas em 
 septos alveolares do tecido controle ( b ) , em vasos do tecido 
 controle ( d ) e em bronquolos do tecido controle ( f )
. os grficos de 
 barras mostram a quantificao da imunomarcao positiva para cox-1 
 e cox-2 em clulas em septos alveolares ( s ) , parnquima pulmonar 
 total ( t ) e bronquolos ( w ) no tecido controle , no tecido es - pine e 
 no tecido fpi - piu ( marcao imuno - histoqumica ; aumento : 
 400 ) . 
 
a proporo de clulas em septos alveolares com imunomarcao 
 positiva para cox-1 e cox-2 foi significativamente maior nos tecidos piu e pine 
 do que no tecido controle .
de clulas em 
 septos alveolares marcadas positivamente para cox-1 e cox-2 associaram - se aos 
 padres histolgicos de piu e pine . como se pode observar nos grficos de barras 
 na figura 1 ( s e t ) , a relao de cox-1 e 
 cox-2 com a fpi ( o padro histolgico de piu ) foi mais forte do que a de cox-1 e 
 cox-2 com a es ( o padro histolgico de pine ) .
embora a proporo de clulas 
 bronquiolares com imunomarcao positiva para cox-2 tenha sido menor nos tecidos 
 pine e piu do que no tecido controle ( figura 
 1w ) , a diferena no foi estatisticamente significante .
alm disso , 
 embora a proporo de clulas bronquiolares com imunomarcao positiva para 
 cox-1 tenha sido maior nos tecidos piu e pine do que no tecido controle ( figura 1w ) , a diferena no foi 
 significante .
no foram encontradas diferenas entre os tecidos no tocante  
 imunomarcao de cox-1 e cox-2 em vasos e no parnquima total ( tabela 2 ) .  

tabela 2 resultados morfomtricos em tecido pulmonar normal ( tecido 
 controle ) , em tecido pulmonar com o padro histolgico de pneumonia 
 intersticial usual ( proveniente de pacientes com fibrose pulmonar 
 idioptica ) e em tecido pulmonar com o padro histolgico de 
 pneumonia intersticial no especfica ( proveniente de pacientes com 
 esclerose sistmica).a 
  uma anlise preliminar das curvas de sobrevida de kaplan - meier mostrou que a 
 sobrevida foi melhor nos pacientes com es ( o padro histolgico de pine 
 fibrtica ) e expresso de cox-2 > 2,25% ( mediana da sobrevida : 70,75 meses ) 
 do que naqueles com fpi ( o padro histolgico de piu ) e expresso de cox-2 
 < 2,25% ( mediana da sobrevida : 46,32 meses ; figura 2 ) .
nica varivel dummy com valor = 1 e o 
 padro histolgico de piu com valor = 2 .
os resultados da anlise multivariada 
 baseada no modelo de regresso de riscos proporcionais de cox so apresentados 
 na tabela 3 .
aps termos controlado a 
 idade , os resultados dos testes de funo pulmonar , o padro histolgico de piu 
 e o padro histolgico de pine fibrtica , constatamos que apenas duas variveis 
 se associaram de maneira significativa ao tempo de sobrevida : o padro 
 histolgico de pine fibrtica e cox-2 em septos alveolares ( p = 0,02 ) .
uma vez 
 que essas duas variveis foram contabilizadas , nenhuma das demais se relacionou 
  sobrevida . a anlise multivariada revelou baixo risco de morte para pacientes 
 jovens com vef1/cvf baixa , padro histolgico de pine fibrtica e 
 cox-2 de alto grau em septos alveolares .  

figura 2 curvas de regresso de cox para risco de bito versus tempo de 
 acompanhamento ( em meses ) em pacientes jovens com dlco / volume 
 alveolar baixa , esclerose sistmica ( e padro histolgico de 
 pneumonia intersticial no especfica celular ) , cox-1 total de alto 
 grau e cox-2 de baixo grau em septos alveolares .
a curva superior 
 representa o grupo de pacientes com esclerose sistmica e pneumonia 
 intersticial no especfica celular .
a curva inferior representa 
 dois grupos de pacientes : aqueles com esclerose sistmica e 
 pneumonia intersticial no especfica fibrtica e aqueles com 
 fibrose pulmonar idioptica e padro histolgico de pneumonia 
 intersticial usual . 
 

tabela 3 regresso de riscos proporcionais de cox para verificar a 
 contribuio individual do padro histolgico e fatores morfolgicos 
 associados  sobrevida e comparar os dois grupos quanto  sobrevida 
 ajustada . 

o nmero limitado de abordagens teraputicas que tenham qualquer impacto na 
 sobrevida em longo prazo em pacientes com fpi - piu e naqueles com es e pine 
 fibrtica deve - se  falta de definio a respeito dos mediadores e mecanismos 
 envolvidos na patognese da fpi e da es .
portanto , a questo de interesse  se 
 mediadores adicionais podem ajudar a compreender melhor a patognese dessas 
 doenas . o processo de reparo envolve duas fases distintas : uma fase 
 inflamatria regenerativa , em que o microambiente tenta substituir as clulas 
 lesadas , e uma fase fibrtica , em que o tecido conjuntivo substitui o tecido 
 parenquimatoso normal.(- ) 
 no processo de reparo , aumenta a produo de pge2 por 
 fibroblastos , ( , ) o que constitui evidncia 
 adicional das propriedades antiproliferativas , anti - inflamatrias e 
 antifibrticas da cox-2/pge2 .
( ) portanto , nosso achado de que a marcao 
 imuno - histoqumica para cox fornece informaes importantes sobre os processos 
 de reparo na fibrose pulmonar no  surpreendente , e nossos resultados confirmam 
 que a expresso de cox-2  maior na fpi e na es , com desfecho melhor em um grupo 
 de pacientes
. constatamos que a proporo de clulas em septos alveolares com 
 imunomarcao positiva para cox-1 e cox-2 foi significativamente maior em tecido 
 pulmonar com padro histolgico de piu e pine fibrtica do que em tecido 
 pulmonar normal . a maior expresso de
cox-1 era esperada , pois a cox-1  
 expressa constitutivamente na maioria das clulas e tecidos , ao passo que a 
 cox-2  induzida por estmulos inflamatrios ou mitognicos .
( ) esses resultados contrastam 
 com os de estudos anteriores nos quais se investigou a fpi .
( , , ) esses estudos mostraram uma reduo da expresso 
 de cox-2 em fibroblastos pulmonares em consequncia de uma diminuio da 
 produo de cox-2 .
no entanto , nesses estudos , a expresso de cox-2 foi medida 
 apenas em fibroblastos , ao passo que em nosso estudo ela foi medida nos septos 
 alveolares , incluindo clulas epiteliais e fibroblastos em reas normais , reas 
 em colapso , e focos de fibroblastos .
outros estudos , inclusive um estudo 
 realizado por lappi - blanco et al . ,
( ) encontraram um aumento da expresso de cox-2 em 
 epitlio metaplsico e fibroblastos provenientes de reas fibrticas em fpi - piu . 

esses resultados divergentes sugerem que a cox-2 desempenha um papel duplo na 
 fpi - piu .
primeiro , a reduo da expresso da cox-2 em reas normais , reas em 
 colapso e focos fibroblsticos sugere que a cox-2 desempenha um papel 
 anti - inflamatrio na fpi - piu em fase inicial .
segundo , a presena de fibrose 
 progressiva mesmo na presena de aumento da expresso de cox-2 sugere que os 
 fibroblastos so incapazes de responder ao estmulo da cox-2 e seu produto 
 principal ( pge2 ) de modo a inibir a proliferao de fibroblastos , a 
 transformao miofibroblstica e o aumento da produo de colgeno e outras 
 molculas da matriz extracelular .  
no presente estudo , a proporo de clulas em septos alveolares com imunomarcao 
 positiva para cox-1 e cox-2 foi menor em tecido fibrtico pine ( proveniente de 
 pacientes com es ) do que em tecido piu ( de pacientes com fpi ) .
esse achado 
 contrasta com os de estudos anteriores em que se mostrou que os nveis de cox-2 
 so mais elevados em pacientes com es.(- ) alm disso , relatou - se que a produo de cox-2  
 muito maior na fase de resoluo inflamatria do que na fase inicial .
( ) esses achados divergentes 
 sugerem que a cox-2 tem um papel duplo em um processo inflamatrio normal , 
 desempenhando um papel pr - inflamatrio na fase inicial e um papel 
 anti - inflamatrio na fase de resoluo .
( ) portanto , diante das evidncias mencionadas acima 
 e da inflamao latente em pacientes com es e envolvimento pulmonar , nossos 
 resultados reforam a ideia de que a cox-2 no exerce seu efeito 
 anti - inflamatrio de maneira apropriada , pois h inflamao mesmo quando a 
 expresso de cox-2 est aumentada em pacientes com es e pine fibrtica .
no 
 entanto , mais estudos so necessrios para esclarecer o verdadeiro motivo pelo 
 qual o mecanismo da cox-2  deficiente .
supomos que isso se deva  incapacidade 
 da cox-2 de estimular a produo de pge2 ou de outros mediadores 
 anti - inflamatrios em oposio a seus prprios efeitos pr - inflamatrios ou  
 incapacidade das clulas
procuramos estabelecer uma 
 correlao entre a cox-2 e a sobrevida dos pacientes controlada quanto  idade , 
 aos resultados dos testes de funo pulmonar , ao padro histolgico de piu ( em 
 pacientes com fpi ) e ao padro histolgico de pine ( em pacientes com es ) .
nossa 
 anlise multivariada revelou baixo risco de morte para pacientes mais jovens com 
 dlco / volume alveolar baixa , es ( e o padro histolgico de pine ) cox-2 total de 
 alto grau e cox-1 de alto grau em septos alveolares .  
em suma , a expresso de cox-1 e cox-2 no parnquima pulmonar nos oferece o 
 potencial de controlar processos de reparo envolvidos na progresso da es - pine e 
 da fpi - piu , sugerindo que estratgias destinadas a prevenir a baixa sntese de 
 cox-1 tero maior impacto na es , ao passo que as destinadas a prevenir a elevada 
 sntese de cox-2 tero maior impacto na fpi . ensaios randomizados prospectivos 
 so necessrios para confirmar isso .
 objective : to study the expression of cox-1 and cox-2 in the remodeled lung in systemic 
 sclerosis ( ssc ) and idiopathic pulmonary fibrosis ( ipf ) patients , 
 correlating that expression with patient survival . 
 methods : we examined open lung biopsy specimens from 24 ssc patients and 30 ipf 
 patients , using normal lung tissue as a control . 
 the histological patterns 
 included fibrotic nonspecific interstitial pneumonia ( nsip ) in ssc patients 
 and usual interstitial pneumonia ( uip ) in ipf patients . 
 we used 
 immunohistochemistry and histomorphometry to evaluate the expression of 
 cox-1 and cox-2 in alveolar septa , vessels , and bronchioles . 
 we then 
 correlated that expression with pulmonary function test results and 
 evaluated its impact on patient survival . 
 results : the expression of cox-1 and cox-2 in alveolar septa was significantly higher 
 in ipf - uip and ssc - nsip lung tissue than in the control tissue . no 
 difference was found between ipf - uip and ssc - nsip tissue regarding cox-1 and 
 cox-2 expression . 
 multivariate analysis based on the cox regression model 
 showed that the factors associated with a low risk of death were younger 
 age , high dlco / alveolar volume , ipf , and high cox-1 expression in alveolar 
 septa , whereas those associated with a high risk of death were advanced age , 
 low dlco / alveolar volume , ssc ( with nsip ) , and low cox-1 expression in 
 alveolar septa . 
 conclusions : our findings suggest that strategies aimed at preventing low cox-1 synthesis 
 will have a greater impact on ssc , whereas those aimed at preventing high 
 cox-2 synthesis will have a greater impact on ipf . 
 however , prospective 
 randomized clinical trials are needed in order to confirm that . 
Introduction
Methods
Results
Discussion
OBJETIVO:
MTODOS:
RESULTADOS:
CONCLUSES:
Introduo
Mtodos
Resultados
Discusso