Tenascin is increased in epithelial lining fluid in fibrotic lung disorders.

Tenascin is an extracellular matrix glycoprotein increased immunohistochemically in tumorous and fibrotic lung tissues as demonstrated by immunohistochemistry. We hypothesized that in bronchoalveolar lavage (BAL) fluid also the tenascin concentration would be elevated in patients with various fibrotic lung disorders. The aim of our study was to investigate whether BAL fluid tenascin would be increased compared with serum tenascin in patients with usual interstitial pneumonia (UIP), sarcoidosis, and extrinsic allergic bronchioloalveolitis. For this purpose BAL fluid was collected from five patients with UIP, 12 patients with sarcoidosis, five patients with extrinsic allergic bronchioloalveolitis, and six patients in a control group. BAL fluid and serum tenascin concentrations were detected by the enzyme immunoassay method. The BAL fluid results were expressed as tenascin concentrations in the epithelial lining fluid (ELF), as estimated by the urea method. The ELF tenascin concentration was increased in the patients with fibrotic lung disorders relative to the control group (mean 0.12 microg/ml) and was highest in the UIP group (mean 5.72 microg/ml) and sarcoidosis group (mean 4.76 microg/ml). It is concluded that the tenascin concentration in the ELF is increased in patients with UIP, sarcoidosis, and extrinsic allergic bronchioloalveolitis, suggesting active synthesis of tenascin in the lower respiratory tract in such disorders.

Cellular and plasma fibronectin in the aqueous humour of primary open-angle glaucoma, exfoliative glaucoma and cataract patients.

PURPOSE: To compare the concentrations of cellular fibronectin (cFN), plasma fibronectin (pFN), tenascin and calcitonin gene-related peptide (CGRP) in the aqueous humour in primary open-angle glaucoma (POAG), exfoliative glaucoma (EXFG) or cataract (control group). METHODS: The concentrations were determined by enzyme-linked immunoassays in the aqueous humour of patients with EXFG (n = 26), POAG (n = 29) or cataract (control group, n = 13). RESULTS: The pFN concentrations of the three patient groups differed significantly from each other (p = 0.0004 in a non-parametric analysis of variance). In multiple comparisons EXFG patients showed significantly higher pFN levels than POAG patients (p < 0.05) or cataract patients (p < 0.01). the cFN level was also significantly higher in EXGF patients than in POAG patients (p < 0.05) or cataract patients (p < 0.05). The pFN or cFN concentrations of the POAG group did not differ from those of the control group. Neither tenascin nor CGRP was detected in the aqueous humour of any of our patients. CONCLUSIONS: The significantly higher aqueous humour pFN concentration in exfoliative glaucoma may be a consequence of disruption of the blood-aqueous barrier and may further add to an increased outflow resistance.

Cellular fibronectin concentration in the plasma of patients with malignant and benign diseases: a comparison with CA 19-9 and CEA.

EDAcFN enzyme immunoassay (EIA) is a new tumour marker assay measuring the extra domain A-containing isoform of cellular fibronectin (cFN), a component mainly found in extracellular matrices. The concentration cFN was measured in plasma and serum from 468 patients with malignant and benign diseases. The concentrations of cFN were higher in plasma than in serum. Using receiver operating characteristic (ROC) curve analysis, determination from plasma was superior to serum at specificity levels higher than 78% and was chosen for further analysis. The highest frequencies of elevated cFN values were seen in patients with hepato-pancreato-biliary malignancies (50-67%). In pancreatic and bile duct cancers, cFN provided little further information to that obtained by CA 19-9. The greatest advantage over CA 19-9 and CEA was seen in patients with local colorectal cancer and in hepatocellular carcinomas. Four out of nine patients with Dukes' stage B colorectal cancer had an elevated cFn level, but only one had an abnormal CEA level. In hepatocellular carcinomas, cFN was also compared with alpha-fetoprotein. The sensitivity of cFN (72%) was superior to that of AFP (61%), and concomitant use of cFN and AFP raised the sensitivity to 83%. The highest frequencies of elevated values in patients with benign diseases were observed in those with severe liver disease (32%) and biliary (17%) and pancreatic (24%) diseases. A combination of cFN and CA 19-9 showed the highest overall sensitivity of 47%, compared with 31% for cFN and 33% for CA 19-9. The corresponding specificities were 76% for cFN +/- CA 19-9, 85% for cFN and 83% for CA 19-9. The accuracy of a combination of cFN and CA 19-9 or CEA (60% respectively) was higher than that of cFN (55%), CA 19-9 (55%) or CEA (45%) alone. In conclusion, the results of the new cFN test are encouraging and further studies on larger patient materials have been started.

Increased release of tenascin in tear fluid after photorefractive keratectomy.

BACKGROUND: Extracellular matrix protein tenascin (TN) is expressed in the anterior stroma during corneal wound healing. In this study we analysed TN release in tear fluid after photorefractive keratectomy (PRK). METHODS: Tear fluid TN concentrations of ten PRK patients were measured with an immunoassay. Tear fluids were collected preoperatively and 1, 2 and 7 days after PRK. The tear fluid collection time and the volume of tears collected were registered. Because tear fluid flow was greatly increased postoperatively, tear fluid flow-corrected release (TN flux) was calculated. RESULTS: The tear fluid flow was 4.50 +/- 0.94 microliters/min (mean +/- SEM) preoperatively, 55.48 +/- 16.70 microliters/min (P < 0.01) on the 1st, 33.91 +/- 7.91 microliters/min (P < 0.01) on the 2nd, and 13.79 +/- 5.49 microliters/min (P > 0.05) on the 7th postoperative day. The preoperative TN concentration was 0.85 +/- 0.20 microgram/ml. On the 1st postoperative day it decreased to 0.37 +/- 0.17 microgram/ml (P > 0.05), most likely due to the dilution effect caused by hypersecretion after PRK. The TN concentration was 0.67 +/- 0.12 microgram/ml (P > 0.05) on the 2nd and 0.78 +/- 0.15 micrograms/ml (P > 0.05) on the 7th postoperative day. The preoperative TN flux was 5.23 +/- 1.88 ng/min. On the 1st and 2nd postoperative days the TN flux was 14.40 +/- 4.99 ng/min (P < 0.05) and 22.66 +/- 6.12 ng/min (P < 0.05), respectively. On the 7th postoperative day a tendency towards decreased flux (14.00 +/- 6.02 ng/min, P > 0.05) was observed. CONCLUSION: Although there is a minor decrease in TN concentration after PRK due to increased tear fluid flow, a significant increase in TN flux was observed. Complete reepithelialization of the ablated area was observed in all eyes at the follow-up visit on postoperative day 7.

Enzyme immunoassay for quantification of tenascin in biologic samples.

An enzyme immunoassay was developed for quantification of tenascin in biologic samples. An enzyme conjugate prepared by coupling peroxidase to a well-characterized, affinity-purified monoclonal antibody EB2 to human tenascin was used as principal reagent. The assay comprises 96-well microtitration strip plates with immobilized monoclonal antibody DB7 to human tenascin. By using a novel monoclonal antibody suppressing human-anti-mouse-factor, MAK33, in the sample buffer, the specificity of the test could be improved. The method has a minimum detectable sensitivity of 1.5 ng tenascin and permits determination of tenascin in various biologic samples. The coefficients of variation within run and between run ranged from 0.9% to 5.0%. The average tenascin concentration in normal plasma was 0.97 mg/L (n = 200) and in serum 0.73 mg/L (n = 200). The tenascin concentrations were also determined in samples of urine, bile, amniotic fluid, seminal fluid, cerebrospinal fluid, bronchoalveolar lavage (BAL) fluid, and pleural fluid showing general applicability of the assay. The method permits the determination of tenascin in samples of different body fluids from various diseases, including cancer, showing increased amounts of the protein at the tissue level.

Increased release of immunoreactive calcitonin gene-related peptide (CGRP) in tears after excimer laser keratectomy.

The purpose of the study was to quantify the neuropeptide calcitonin gene-related peptide (CGRP) in normal human tear fluid and to determine the effect of photorefractive excimer laser keratectomy (PRK) on its release in tears. CGRP was assayed in tear fluid samples using an enzyme immunoassay (detection limit 0.2 micrograms ml-1). Tear-fluid samples were collected preoperatively, 1, 2 and 7 days after PRK and analysed for CGRP. The changes in tear-fluid secretion were also monitored. The intra-assay variation was 3.0-7.0%. Despite the marked hypersecretion of tears, the concentration of CGRP did not decrease following PRK indicating a concomitant increase in CGRP release by sensory nerves and/or lacrimal gland(s). Consequently, the release of CGRP in tears increased from 197.9 +/- 36.6 ng min-1 (mean +/- S.E.M.) to 1723.0 +/- 402.4 ng min-1 (P < 0.01) on day 1, and to 2304.2 +/- 561.1 ng min-1 (P < 0.01) on day 2. On day 7, only minor elevation (377.02 +/- 83.24 ng min-1) was observed. It is concluded that CGRP is a component of normal human tear fluid. The ocular irritation response related to the photoablation induces an enhanced release of CGRP in tears. As a compound present in corneal sensory nerves CGRP may have a role in wound-healing.

Cellular fibronectin in serum and plasma: a potential new tumour marker?

The concentration of cellular fibronectin (cFN) containing the extra domain A (EDA) was measured in 479 plasma and 300 serum samples from healthy blood donors by a competitive enzyme immunoassay (EIA). Serum and plasma samples contained low concentrations of EDAcFN. The mean concentration of EDAcFN was higher in plasma (2.46 mg l-1) than in serum (0.30 mg l-1). No significant differences between sexes or age groups were found. The EDAcFN concentrations were also measured in 120 patients with various malignancies. The mean values in serum were 4.28 mg l-1, 2.01 mg l-1 and 5.18 mg l-1 in patients with digestive tract malignancies, breast cancer and a group of miscellaneous cancers respectively. In plasma, the corresponding values were 12.26 mg l-1, 4.38 mg l-1 and 11.12 mg l-1 respectively. The serum EDAcFN concentration was higher than the 97.5th percentile level of healthy blood donors in 86% of patients with digestive tract and in 76% with miscellaneous malignancies. In patients with breast cancer 60% had elevated levels of EDAcFN. The corresponding figures for plasma samples in patients with digestive tract and miscellaneous malignancies were 79% and 71% respectively. In patients with breast cancer only 30% had elevated plasma levels of EDAcFN. The mean values in serum and plasma of 20 patients with benign diseases were below the cut-off levels. Consistent with the EIA results, Western blotting revealed increased amounts of EDAcFN in blood samples from cancer patients. Pregnancy did not affect the EDAcFN concentration. The mean values in 20 pregnant women were below the cut-off levels.

Tear fluid cellular fibronectin levels after photorefractive keratectomy.

BACKGROUND: Fibronectin is supposed to have an important role in wound healing. The extradomain A-containing cellular fibronectin (EDAcFn) refers to fibronectin, which instead of being a hepatocyte derived component of blood plasma or body fluids, is produced locally. The present study was undertaken to clarify the possible changes in excretion of EDAcFn in tears following excimer laser photorefractive keratectomy (PRK). METHODS: An immunoassay was used to determine EDAcFn concentrations in human tear fluid samples of 11 eyes after PRK. Tear fluids were collected with scaled microcapillaries preoperatively as well as 1, 2, and 7 days after PRK. The time used to collect a known volume of tears was registered. This was done to estimate the dilution effect related to the hypersecretion of tears after PRK. RESULTS: The mean preoperative tear fluid EDAcFn concentration was 0.28 +/- 0.07 ng/microliter with a wide range (0.05 to 0.63). The tear fluid flow-corrected excretion of EDAcFn was 1.36 +/- 0.35 ng/min (range, 0.145 to 3.50). There was a significant increase in both postoperative tear fluid flow and excretion of EDAcFn on days 1 and 2. The elevation of the mean EDAcFn concentration did not decrease in spite of reflex tearing. The mean excretion of EDAcFn in tears was 28-fold on the first and 17-fold on the second postoperative day. Normal level was reached by day 7. CONCLUSION: There is a rapid increase in excretion of EDAcFn in tears following PRK. This seems to last only as long as an epithelial defect persists. The epithelium of all eyes healed in 3 to 4 days in spite of wide interindividual variations in both tear fluid flow and EDAcFn excretion.

Release of calcitonin gene-related peptide in tears after excimer laser photorefractive keratectomy.

BACKGROUND: Sensory nerves known to affect corneal healing are damaged to a variable degree after photorefractive keratectomy (PRK). To search for nerve-bound factors involved in corneal healing, we monitored tear fluid calcitonin gene-related peptide (CGRP) levels of six human eyes undergoing PRK. METHODS: CGRP concentrations were determined using an immunoassay. RESULTS: Normal human tear fluid contains CGRP. The mean CGRP concentration was slightly increased postoperatively, despite a marked tear fluid hypersecretion. Consequently, an almost ten-fold increase in release of CGRP in tears was observed on days 1 and 2 after PRK. Values measured on day 7 had returned to a normal level. CONCLUSION: The observed postoperative increase in release of CGRP in tears may have an impact on the healing of PRK wounds. Extensive neural damage following deep photoablations may impair healing and should probably be avoided.

An improved method for quantification of extra domain A-containing cellular fibronectin (EDAcFN) in different body fluids.

A quantitative direct enzyme immunoassay for the extra domain A-containing isoform of cellular fibronectin (EDAcFN) was established for screening of large series of blood samples and various body fluids of different pH and viscosity. The method is based on the monoclonal antibody DH1 recognizing the extra domain A in cellular fibronectin (EDAcFN). Studies on the effect of dilution of plasma and serum samples in this direct assay indicated that the measured concentration of cFN in the samples greatly depend on the ratio of sample dilution. The linearity of the assay was improved with sample dilution and the optimal dilution was 1:5. Stored diluted samples retained their cFN content at +4 degrees C, and -20 degrees C and -70 degrees C for months in contrast to samples stored undiluted. With this direct EIA the detection limit was 0.05 micrograms/ml and the linear portion of the standard curve could be extended above 30 micrograms/ml. Thus, the cFN concentration of blood samples could be measured reliably without inhibition also in samples with very high concentration of cFN. This is particularly important when measuring blood samples from cancer patients, since these samples may contain more than 20 micrograms/ml EDAcFN. The assay was standardized for blood samples but, due to the possibility of sample dilution, it also enabled reliable quantification of EDAcFN in various other body fluids. Undiluted some of the samples with non-neutral pH (urine, bile) or with high viscosity (seminal plasma) interfered with the assay. In addition to blood samples, the EDAcFN concentration was determined in samples of urine, bile, amniotic fluid, cervicovaginal secretions, seminal fluid, cerebrospinal fluid, bronchoalveolar lavage fluid, pleural fluid and saliva. Thereby, this modified method was shown to be applicable to various body fluids.