Sari Ylätupa

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.

Increased release of tenascin in tear fluid after photorefractive keratectomy

Abstract• 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 μl/min (mean±SEM) preoperatively, 55.48±16.70 μl/min (P<0.01) on the 1st, 33.91±7.91 μl/min (P<0.01) on the 2nd, and 13.79±5.49 μl/min (P>0.05) on the 7th postoperative day. The preoperative TN concentration was 0.85±0.20 μg/ml. On the 1st postoperative day it decreased to 0.37±0.17 μg/ml (P>0.05), most likely due to the dilution effect caused by hypersecretion after PRK. The TN concentration was 0.67±0.12 μg/ml (P>0.05) on the 2nd and 0.78±0.15 μg/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.I2 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.

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.

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.

Competitive enzyme immunoassay for quantification of the cellular form of fibronectin (EDAcFN) in blood samples

A competitive enzyme immunoassay based on the monoclonal antibody DH1 was developed for the quantification of extradomain A (EDA)-containing isoform of cellular fibronectin (cFN). The average EDAcFN concentration in normal plasma was 2.46 micrograms/ml and in serum 0.68 micrograms/ml. Similar results were obtained by gelatin-Sepharose binding and immunoblotting. Studies on the effect of storage of plasma and serum as frozen samples indicated that the amount of EDAcFN decreased rapidly when stored at -20 degrees C. Storage at -70 degrees C resulted in less of a decrease in the concentration of EDAcFN. The method permits the determination of EDAcFN in blood samples from diseases showing increased amounts of the protein at the tissue level.