Elevation of Anti-Cytokeratin 18 Antibody and Circulating Cytokeratin 18: Anti-Cytokeratin 18 Antibody Immune Complexes in Sera of Patients with Idiopathic Pulmonary Fibrosis

In this study, we hypothesize that anti-cytokeratin 18 (CK18) antibody and CK18:anti-CK18 immune complex increase in sera in patients with idiopathic pulmonary fibrosis (IPF). To prove the existence of anti-CK18 antibodies in patients' sera, bovine CK18 was stained with patients' sera using a Western blotting. In patients with IPF, anti-CK18 antibodies were clearly demonstrated in sera by Western blotting. Then, we tried to establish an enzyme-linked immunosorbent assay (ELISA) to quantify anti-CK18 antibodies and CK18:anti-CK18 immune complexes in sera of patients with IPF. Levels of anti-human CK18 antibodies in sera of patients with IPF (0.81 ± 0.31, mean ± SD) measured by ELISA were significantly high compared with that of normal volunteers (0.45 ± 0.06, p < 0.01). In addition, levels of CK18:anti-CK18 antibody complexes in patients' sera (0.64 ± 0.35, man ± SD) significantly increased compared with those of normal subjects (0.40 ± 0.10, p < 0.01). These results suggest that anti-CK18 antibody and its immune complex may have played a role in the process of lung injury in IPF. Accepted for publication 30 March 2000     

Elevation of Anti-cytokeratin 19 Antibody in Sera of the Patients With Idiopathic Pulmonary Fibrosis and Pulmonary Fibrosis Associated With Collagen Vascular Disorders

It has been suggested that cytokeratin 19 is expressed in regenerated bronchoepithelial cells in patients with pulmonary fibrosis, and serum cytokeratin 19 fragment is elevated in patients with pulmonary fibrosis. We hypothesized that serum antibodies to cytokeratin 19 may be formed in patients with pulmonary fibrosis. To prove the existence of anti-cytokeratin 19 antibodies in patients' sera, human recombinant cytokeratin 19 was stained with patients' sera by a Western immunoblot. Then, we tried to establish an enzyme-linked immunosorbent assay to quantitate anti-cytokeratin 19 antibody in the sera of patients with idiopathic pulmonary fibrosis (IPF) and pulmonary fibrosis associated with collagen vascular disorders (PF-CVD). We demonstrated the anti-cytokeratin 19 antibody in patient' sera by a Western immunoblot. In patients with IPF and PF-CVD, significantly high anti-cytokeratin 19 antibody was demonstrated compared with normal volunteers, patients with chronic bronchitis, and patients with pneumonia. These results suggest that anti-cytokeratin 19 antibody may have played a role in the process of lung injury in pulmonary fibrosis. Accepted for publication: 27 May 1999     

Elevated serum and BAL cytokeratin 19 fragment in pulmonary fibrosis and acute interstitial pneumonia.

Cytokeratin 19 fragment (CK19) levels in serum have already been documented as a useful tumour marker for lung cancer. In the present study, it was hypothesized that CK19 may be increased in the serum and epithelial lining fluid of the respiratory tract from patients with pulmonary fibrosis. CK19 was measured in the serum and bronchoalveolar lavage fluid (BALF) of patients with pulmonary fibrosis and the correlation between CK19 levels and clinical parameters evaluated. Nineteen patients diagnosed with idiopathic pulmonary fibrosis (IPF), eight with pulmonary fibrosis associated with a collagen vascular disorder (PF-CVD), seven patients with acute interstitial pneumonia (AIP), and 10 normal smokers as a control group were studied. CK19 levels in sera of patients with IPF and patients with PF-CVD were significantly increased compared to those of normal smokers. CK19 levels in sera of patients with AIP were significantly increased compared to those of other groups. CK19 values in the BALF of patients with pulmonary fibrosis were significantly elevated compared to those of normal smokers. CK19 values in sera charged according to the progression or improvement of the acute lung injury. Immunohistochemical study using pulmonary tissues obtained from patients with AIP demonstrated that the hyaline membrane and proliferating type II pneumocytes were stained by anti-human cytokeratin 19 antibody. These data demonstrated that the measurement of cytokeratin 19 fragment is a useful parameter to evaluate the activity of lung epithelial cell damage and repair.     

Prognostic role of eosinophils in pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) and pulmonary fibrosis associated with a collagen vascular disorder (PF-CVD) are chronic inflammatory lung disorders which may be characterized in various subgroups of patients by increased numbers of macrophages, neutrophils, lymphocytes, and/or eosinophils. Previous studies have suggested that the cell populations recovered with bronchoalveolar lavage (BAL) may be important in predicting disease progression and response to therapy. We evaluated this hypothesis in 27 patients by determining if the cell populations recovered with BAL differed between patients who improved, remained stable, or worsened in their pulmonary functions (as defined by at least a 15 percent change in forced vital capacity) over a six-month observation period. The findings suggested that BAL eosinophilia may be a marker of progressive lung disease in patients with IPF and PF-CVD.     

Role of neutrophilic leukocytes in pathogenesis of idiopathic fibrosing pulmonary alveolitis]

The study was undertaken to obtain additional data on the role of neutrophils in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Various functional parameters of neutrophilic functional activity, i.e. phagocytosis, chemotaxis, random migration, oxidative metabolism (3 types of chemiluminescence: spontaneous formylmethionyl peptide-stimulated, and prodigiosan-stimulated), and the level of circulating immune complexes of different size were determined in patients with IPF and healthy individuals. The patients showed a decrease in phagocytosis and an increase in neutrophilic chemotaxis and oxidative metabolism, which was related to the output of toxic oxygen forms. The changes revealed were found to be associated with the activity of an pathological process. The findings suggest that neutrophils can be involved both in the development of and in the occurrence of a pathological process in IPF.     

Role of carbohydrate antigens sialyl Lewis (a) (CA19-9) in bronchoalveolar lavage in patients with pulmonary fibrosis

BACKGROUND: It has been reported that carbohydrate antigen sialyl Lewis (a) (CA19-9) levels are elevated in serum as well as in bronchoalveolar lavage fluid (BALF) of patients with pulmonary fibrosis. However, the biological significance of CA19-9 is unclear. OBJECTIVE: The purpose of the present study was to evaluate correlations between CA19-9 levels in BALF and several biochemical as well as clinical parameters in patients with pulmonary fibrosis. In addition, biological functions of CA19-9 were also examined. METHODS: We studied 24 patients with a diagnosis of pulmonary fibrosis: 16 with idiopathic pulmonary fibrosis (IPF) and 8 with pulmonary fibrosis associated with a collagen vascular disorder (PF-CVD). In BALF, carbohydrate antigens sialyl Lewis (a) (CA19-9), elastase: alpha(1)-proteinase inhibitor complex (E-PI), hepatocyte growth factor (HGF), LDH, IgG, IgA, albumin, and cell differentiation were measured. We also evaluated the effects of CA19-9 on neutrophil functions. RESULTS: CA19-9/albumin levels in BALF significantly correlated with HGF/albumin, elastase/albumin, LDH/albumin, total number of alveolar macrophages, and total number of neutrophils. Purified CA19-9 had a chemotactic activity for neutrophils. In addition, neutrophil chemotactic activity to C5a, fMLP, and interleukin 8 was significantly stimulated after incubation with purified CA19-9. Furthermore, CA19-9 increased the expression of CD15s on neutrophils. CONCLUSIONS: Our data demonstrated (i) CA19-9 in BALF correlated with other markers of inflammation in pulmonary fibrosis, and (ii) CA19-9 can modify neutrophil functions. These results suggest that CA19-9 may play a role in the process of lung injury in patients with pulmonary fibrosis.     

Detection of anti-ADAM 10 antibody in serum of a patient with pulmonary fibrosis associated with dermatomyositis

OBJECTIVES: It has been suggested that the humoral immune system plays a part in the pathogenesis of pulmonary fibrosis. Although circulating autoantibodies to lung protein(s) have been suggested, few lung proteins have been characterised. The purpose of this study is to determine the antigen recognised by serum of a patient with pulmonary fibrosis associated with dermatomyositis. METHODS: To accomplish this, anti-small airway epithelial cell (SAEC) antibody in a patient's serum was evaluated using a western immunoblot. RESULTS: An autoantibody against SAEC was found, and the antigen had a molecular weight of 62 kDa. Using the patient's serum, clones from the normal lung cDNA library were screened and demonstrated that anti-SAEC antibody in the patient's serum was against ADAM (A disintegrin and metalloprotease) 10. CONCLUSION: This is the first report that demonstrates the existence of anti-ADAM 10 antibody in a patient with pulmonary fibrosis associated with dermatomyositis.     

Cells,collagen and idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a usually fatal disorder of lung with clearly defined clinical, roentgenographic, physiologic, morphologic, scintigraphic and bronchoalveolar lavage features. Current concepts of the pathogenesis of this disorder suggest a central role for a chronic alveolitis in causing changes in parenchymal cell populations and derangements in interstitial collagen. Of the many inflammatory and immune effector cells comprising the alveolitis of IPF, it is likely that the neutrophil is the most important mediator of parenchymal damage. To follow the status of lung neutrophils in patients with this disease, two methods have been utilized. Both gallium-67 scanning and bronchoalveolar lavage quantitate the extent of the alveolitis and can be used to stage and follow these patients. The treatment of IPF remains controversial, but it is likely that corticosteroids reduce the alveolitis and prolong the lifespan of these patients.     

Levels of cytokeratin 19 fragments in bronchoalveolar lavage fluid correlate to the intensity of neutrophil and eosinophil-alveolitis in patients with idiopathic pulmonary fibrosis

Cytokeratin 19 (CK19) is a specific cytoskeletal structure for simple epithelia, including bronchial and alveolar epithelial cells (BAEC). Since CK19 is abundant in alveolar epithelial cells, and could be released from injured alveolar epithelium in idiopathic pulmonary fibrosis (IPF), we investigated the levels of CK19 fragments in the bronchoalveolar lavage fluids (BALF) of 16 patients with idiopathic pulmonary fibrosis (IPF) and 12 patients with sarcoidosis using enzyme-linked immunoassay. There were also 19 control subjects (10 asymptomatic smokers and nine non-smokers). BALF from the non-smokers as well as the asymptomatic smokers contained few CK19 fragments (0.2+/-0.2, 1.3+/-0.5 pg ml(-1) respectively). There were significantly high levels of CK19 in the BALF from patients with IPF (7.3+/-1.4 pg/ml; P<0.01 vs. control non-smoker). Even if the levels of CK19 were expressed as relative to the albumin concentration, significantly increased levels of CK19 fragments were noted in BALF from patients with IPF. However, these levels were not found in BALF from patients with sarcoidosis. Importantly, levels of CK19 fragment in BALF were significantly correlated to the number of neutrophils (r = 0.791, P<0.001) and eosinophils (r = 0.771, P<0.001) but not to that of macrophages or lymphocytes in BALF from IPF patients. Our results suggest the usefulness of CK19 measurement in BALF for assessing the presence of bronchiolo-alveolar epithelial injuries in idiopathic pulmonary fibrosis.     

Filarial antigen in circulating immune complexes from patients with Wuchereria bancrofti filariasis

Detection of filarial antigen in circulating immune complexes from patient sera was performed by an enzyme immunoassay in which the immune complexes were precipitated in the cold with polyethylene glycol and then dissociated in an acid pH buffer before being added to an ELISA plate. The dissociated antigen bound to the plate where it could be detected by peroxidase-labeled polyclonal rabbit antifilarial antiserum. Control sera used for defining the specificity of the assay included sera with immune complexes not related to parasite infection with and without free parasite antigen added prior to polyethylene glycol precipitation as well as sera from normal individuals. Filarial antigen was detected in the circulating immune complexes from 10 of 28 patients with bancroftian filariasis residing in either the Cook Islands (subperiodic Wuchereria bancrofti) or India (periodic W. bancrofti). By immunoblotting, the most frequently identified filarial antigen in these complexes was an approximately equal to 200 kDa circulating antigen.     

Autoantibody to alanyl-tRNA synthetase in patients with idiopathic pulmonary fibrosis

BACKGROUND AND OBJECTIVES: The pathogenesis of IPF is unknown and it is hypothesized that immunological responses are involved. The purpose of this study was to detect autoantibodies in IPF patients and to identify the relevant antigens. METHODS: Sera from 37 healthy subjects and 22 IPF patients who had no clinical symptoms of collagen vascular disease were examined for immunostaining of A549 human type II cells and human lung tissue. Immunoprecipitation and proteome analysis were performed to identify the antigen. RESULTS: Fifty per cent of the patient sera and none of the control sera exhibited positive staining. Sera from 10 of the 22 IPF patients showed positive immunohistochemistry and immunoprecipitated a 110-kDa protein from the A549 cell lysate. Sera from only two of 41 patients with collagen vascular disease showed positive immunoreactivity. Proteome analysis using tandem mass spectrometry revealed that the protein was alanyl-tRNA synthetase. Transfection of cDNA of this enzyme into CHO-K1 cells conferred positive staining on these cells with the patients' IgG. The 135-kDa fusion protein consisting of 108-kDa enzyme protein and 27-kDa YFP from the cell lysate of the transfected cells was immunoprecipitated by the patient IgG. In addition, sera from IPF patients significantly inhibited the enzyme activity of alanyl-tRNA synthetase. CONCLUSION: A significant number of IPF patients possess circulating autoantibodies against alanyl-tRNA synthetase, suggesting the involvement of an autoimmune background in the pathogenesis of IPF.     

A simple method for detecting HIV antibodies hidden in circulating immune complexes

A method previously used for studying the specificity of antibody components of circulating immune complexes in different diseases has been applied to analyse circulating immune complexes in HIV-infected patients. Antibodies against HIV antigens hidden in circulating immune complexes were studied in 14 sera from 13 patients with asymptomatic HIV infection (group 1) and in 11 sera from seven patients with HIV symptoms (group 2). HIV antigen-coated wells from the Vironostika kit as well as core and envelope antigen-coated beads from the Abbott confirmatory kit were used as solid-phase antigen. Using the Vironostika plates, HIV antibodies were demonstrated in circulating immune complexes in three and five sera in groups 1 and 2, respectively. Anti-core antibodies hidden in circulating immune complexes were present in three out of eight and two out of nine sera, respectively, in groups 1 and 2, whereas anti-envelope antibodies were present in circulating immune complexes in one out of eight and six out of nine sera in the same groups. These findings demonstrate that not only core-anti-core but also envelope-anti-envelope immune complexes are present in the sera of HIV infected patients.     

己酮可可碱联合阿苯达唑治疗小鼠泡球蚴病的疗效观察

目的 观察己酮可可碱（PTX）和阿苯达唑（ABZ）单独及联合用药对小鼠继发性泡球蚴病的疗效。 方法 对小鼠继发性泡球蚴病进行药物治疗，各治疗组药物用量分别为：ABZ组 50 mg/（kg·d）；PTX高剂量组360 mg/（kg·d）；PTX低剂量组180 mg/（kg·d）；联合组 ABZ 50 mg/（kg·d） + PTX 180 mg/（kg·d）；感染对照组（未治疗组）和空白对照组均给予等体积生理盐水，用小鼠灌胃针经口每天灌胃给药1次，连续治疗100 d后 （其间14只死亡），检测各小鼠泡球蚴湿重、抑囊率及小鼠血清细胞因子转化生长因子?鄄β （TGF-β）、白细胞介素?鄄2 （IL-2）和IL?鄄10；并对泡球蚴组织进行病理组织学和超微结构观察。 结果 PTX在体外能有效地杀灭原头节（高剂量组为100%）, 在体内对泡球蚴抑制作用虽较弱（高剂量组为37%），但能增强小鼠的免疫力。联合用药对泡球蚴有明显的抑制作用，抑囊率为88 %，ABZ抑囊率为58 % （P<0.05）。 结论 PTX联合ABZ治疗小鼠继发性泡球蚴病疗效明显优于ABZ。     

Cytokeratin contents of basal cell carcinoma, epidermis overlying tumour, and associated stromal amyloidosis: an immunohistochemical study.

Cytokeratins (CKs) are expressed specifically in the cytoplasm of epithelial cells. We investigated the expression of CKs immunohistochemically in basal cell carcinomas (BCCs), epidermis overlying tumour, and skin tumor-associated amyloidosis (STA). Twenty cases of BCC, 11 of which had STA were included to the study. The primary antibodies of CK1-8 (AE3), CK10 (DEK-10), CK14 (LL002), CK17 (E3), CK18 (DC10), CK19 (KS19.1), CK 5/6/18 (LP34), CK8/18 (5D3) were applied to the section immunohistochemically. In BCCs without STA, CK1-8, CK14 and CK17 antibodies were expressed by tumour tissue in all biopsy specimens. In the BCCs with STA, tumour tissue was immunoreactive always with CK1-8 and CK17 antibodies, and commonly immunoreactive with anti-CK 14 antibody. In the epidermis overlying tumour tissue, there was positive immunoreactivity with anti-CK 1-8, CK 5/6/18, CK 10 and CK 14 antibodies in all biopsy specimens. Anti-CK 17 antibody was also positive in 17 biopsy specimens. STA is immunoreactive with anti-CK1-8 in all specimens. There was mild staining with anti-CK5/6/18 and with anti-CK19 whereas no immunoreactivity with anti-CK10 and CK18 antibodies was found. In conclusion, we could not find a significant CK expression difference between BCCs with and without STA. Weak positivity and a few number of CKs were shown in STA when compared with those of BCC and epidermis overlying tumour tissue expressing the more variable CKs. Interestingly, although CKs coexpressed in pairs consisting of one basic and one acidic CK, we detected predominantly basic CKs in STA.     

Clinical characterization of interleukin-8 in patients with idiopathic pulmonary fibrosis]

The levels of interleukin-8 (IL-8) in the serum, bronchoalveolar fluid (BALF) and epithelial lining fluid (ELF) were measured in patients with idiopathic pulmonary fibrosis. (IPF), in order to evaluate the clinical significance of IL-8. The serum levels were significantly higher in patients with active IPF (34.4 +/- 11.9 pg/ml, n = 8) than in those with stable IPF (mean: 14.6 +/- 10.9 pg/ml, n = 18), but neither correlated with the serum level of KL-6 or of SP-D, or with the intensity of chest Ga67-scintigraphy. There were no significant differences in BALF or ELF IL-8 levels between the active and stable IPF groups. These results suggest that the serum level of IL-8 is a useful marker for evaluating the disease activity in patients with IPF.     

Cytokeratin contents of basal cell carcinoma,epidermis overlying tumour,and associated stromal amyloidosis: An immunohistochemical study

Cytokeratins (CKs) are expressed specifically in the cytoplasm of epithelial cells. We investigated the expression of CKs immunohistochemically in basal cell carcinomas (BCCs), epidermis overlying tumour, and skin tumor-associated amyloidosis (STA). Twenty cases of BCC, 11 of which had STA were included to the study. The primary antibodies of CK1-8 (AE3), CK10 (DEK-10), CK14 (LL002), CK17 (E3), CK18 (DC10), CK19 (KS19.1), CK 5/6/18 (LP34), CK8/18 (5D3) were applied to the section immunohistochemically. In BCCs without STA, CK1–8, CK14 and CK17 antibodies were expressed by tumour tissue in all biopsy specimens. In the BCCs with STA, tumour tissue was immunoreactive always with CK1–8 and CK17 antibodies, and commonly immunoreactive with anti-CK 14 antibody. In the epidermis overlying tumour tissue, there was positive immunoreactivity with anti-CK 1–8, CK 5/6/18, CK 10 and CK 14 antibodies in all biopsy specimens. Anti-CK 17 antibody was also positive in 17 biopsy specimens. STA is immunoreactive with anti-CK1–8 in all specimens. There was mild staining with anti-CK5/6/18 and with anti-CK19 whereas no immunoreactivity with anti-CK10 and CK18 antibodies was found. In conclusion, we could not find a significant CK expression difference between BCCs with and without STA. Weak positivity and a few number of CKs were shown in STA when compared with those of BCC and epidermis overlying tumour tissue expressing the more variable CKs. Interestingly, although CKs coexpressed in pairs consisting of one basic and one acidic CK, we detected predominantly basic CKs in STA.     

Matrix metalloproteinases and tissue inhibitor of metalloproteinase-1 in sarcoidosis and IPF.

The purpose of this study was to examine the role of interstitial collagenases, members of the family of matrix metalloproteinases, in the development of pulmonary fibrosis. The activity, levels and molecular forms of collagenases (matrix metalloproteinases (MMP)-1, -8 and -13), gelatinase B (MM P-9) and its main endogenous inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1) were assessed in bronchoalveolar lavage fluid (BALF) from patients with idiopathic pulmonary fibrosis (IPF) and sarcoidosis patients with varying degrees of pulmonary parenchymal involvement. Collagenase activity was elevated in IPF and group 3 sarcoidosis patients. A positive correlation between BALF collagenase activity and MMP-8 levels was also observed. Western immunoblotting revealed the presence of two isoforms of MMP-8 in patient samples; an 80 kD form representing latent enzyme from polymorphonuclear neutrophils and a 55 kD form representing the fibroblast-type proform. MMP-9 levels were also elevated in both IPF and group 3 sarcoidosis patients, while TIMP-1 levels remained normal, indicating a shift in the balance between the enzyme and inhibitor, favouring MMP-9. Matrix metalloproteinase-8 is the major contributor to the bronchoalveolar lavage fluid collagenase activity in the airways of patients with idiopathic pulmonary fibrosis and sarcoidosis and may initiate collagen destruction and remodelling leading to the development of pulmonary fibrosis.     

Correlation between cholesterol content in circulating immune complexes and atherogenic properties of CHD patients' serum manifested in cell culture

Blood serum of most patients with coronary heart disease (CHD) caused a 2-5-fold increase in the total cholesterol content of smooth muscle cells cultured from unaffected human aortic intima, i.e. possessed an atherogenic potential manifested in culture. Removal of immunoglobulins G and M from an atherogenic serum brought about a fall in its atherogenic potential. The serum deficient in immunoglobulins A retained its ability to induce the cholesterol accumulation in cells. Treatment of the CHD patients' serum with 2.5% polyethylene glycol 6000 removed the circulating immune complexes. The serum subjected to this treatment lost its atherogenicity, i.e. failed to increase the cholesterol content in cultured cells. Incubation of smooth muscle cells derived from human aortic intima with circulating immune complexes isolated from an atherogenic patients' serum caused a 1.5-3-fold rise in the intracellular cholesterol. Blood sera of most (89%) CHD patients was characterized by a high cholesterol content in circulating immune complexes. More than 75% of healthy subjects and patients without stenosis of coronary arteries had low level of cholesterol in immune complexes. Blood sera atherogenicity manifested in culture directly correlated with the cholesterol level of circulating immune complexes (r = 0.90). These findings suggest that the atherogenicity of CHD patients blood serum is due to cholesterol-containing immune complexes.     

Newly developed assay measuring cytokeratins 8, 18 and 19 in serum is correlated to survival and tumor volume in patients with esophageal carcinoma

Esophageal carcinoma is the seventh most common cause of cancer-related death in the Western world. In Sweden, approximately 400 new esophageal carcinomas are diagnosed yearly. Cytokeratins (CK) are specific for epithelial cells and the expression profile usually remains unchanged even when the epithelium undergoes malignant transformation. In the present study, MonoTotal, a newly developed RIA-assay detecting circulating CK 8, 18 and 19 fragments, was investigated in sera from patients with esophageal carcinoma. Serum samples from 40 patients with esophageal carcinoma were collected. The median value of circulating CK 8, 18 and 19 measured with MonoTotal was 378 U/L (range 53-6843) and with regard to the defined cut-off (< 75 U/L), 39/40 (98%) patients were shown to have elevated levels of circulating CK 8, 18 and 19. Patients with localized disease had a median value of circulating CK 8, 18 and 19 of 305 U/L (mean: 500 U/L), whereas the corresponding value for metastatic disease was 771 U/L (mean: 1506 U/L). This difference was statistically significant (P = 0.016). Circulating CK 8, 18 and 19, according to cut-off, were not associated with survival in univariate analysis (P = 0.34). However, continuous values of circulating levels of CK 8, 18 and 19 were associated with survival (P = 0.000083) in univariate as well as in the multivariate analysis (P = 0.03). In conclusion, circulating CK 8, 18 and 19 correlates with increased tumor burden and might, in conjunction with other clinical parameters, aid the clinician in estimating the prognosis of the individual patient.