Transoesophageal echocardiography for the detection and quantification of pleural fluid in cardiac surgical patients.

BACKGROUND: Transoesophageal echocardiography (TOE) can image pleural fluid. Left pleural collections may be easier to detect than right, as the thoracic aorta serves as an acoustic window. Attempts to quantify pleural fluid using TOE are restricted to a case report in which volume was predicted by multiplying maximal cross-sectional area (CSA(max)) by axial length (AL). A computed tomography (CT) derived formula for quantifying pleural effusions is maximal effusion depth squared (d2) multiplied by maximal effusion length. METHODS: Eight patients were studied before chest closure following coronary bypass surgery. Fifty millilitre saline aliquots were instilled into the pleural space until detected by TOE. Saline was then instilled up to the next 200 ml increment and further 200 ml aliquots added until it spilled from the pleural space. CSA(max), d and AL were measured for each stage and used to calculate pleural fluid volume. RESULTS: Median detection volume (range) was 125 ml (50-200) on the left and 225 ml (150-300) on the right (P = 0.016). Volume calculated by CSA(max) x AL correlated strongly with actual volume (r2 = 0.93 left and 0.92 right) as did volume calculated by d2 x AL (r2 = 0.86 left and 0.89 right). Mean difference between volume calculated by CSA(max) x AL and actual volume was - 51 ml on the left and 45 ml on the right vs - 253 ml on the left and - 212 ml on the right for volume calculated by d2 x AL. CONCLUSIONS: TOE detects small volumes of pleural fluid on both sides of the chest. CSA(max) x AL provides a reasonably accurate measure of pleural fluid volume.

Up-regulation of matrix metalloproteinase expression and activation following cyclical compressive loading of articular cartilage in vitro.

Osteoarthritis (OA) results in articular cartilage degeneration and subchondral bone remodeling. Excessive or abnormal loading of the joint may contribute to matrix destruction by creating an imbalance between proteinases and their inhibitors. This study investigates whether cyclical loading regulates expression and/or activation of metalloproteinases 2 and 9 (MMPs) in articular cartilage explants. Gelatin zymography, reverse zymography, and MMP activity assays of mechanically loaded bovine cartilage explants (0.5 MPa, 1 Hz, 3 h) showed increased expression and activation of MMPs 2 and 9, whereas expression of the tissue inhibitors of MMPs was unaffected. This shows, for the first time that mechanical loading can influence tissue homeostasis generating an imbalance of proteinases and their inhibitors inducing turnover and/or catabolic events in cartilage.

Plasma levels of interleukin-1-alpha in rheumatoid arthritis.

Interleukin-1-beta (IL-1 beta) has been implicated as an inflammatory mediator in rheumatoid arthritis (RA) but little is known about the related cytokine, IL-1 alpha, in this disease. IL-1 alpha has biological properties similar to IL-1 beta but, unlike IL-1 beta, remains mostly cell-associated. In this study plasma IL-1 alpha was measured by radioimmunoassay in patients with RA and in healthy controls. Plasma levels were compared with conventional measures of disease activity. The mean levels in the two groups were not significantly different and, within the patient group (n = 53), the only significant cross-sectional correlation was between plasma IL-1 alpha and ESR. In longitudinal studies, some individual patients had plasma IL-1 alpha levels that correlated with different measures of disease activity. We conclude that while IL-1 alpha may be involved in the immunopathogenesis of RA, its measurement in plasma seems to offer little of clinical value.

Measurement of interleukin-6 in bronchoalveolar lavage fluid by radioimmunoassay: differences between patients with interstitial lung disease and control subjects.

Bronchoalveolar lavage fluid (BALF) from subjects with a variety of interstitial lung diseases (active sarcoidosis, pigeon breeders' disease (PBD), asymptomatic pigeon breeders, patients with idiopathic pulmonary fibrosis) and from control subjects were assayed for interleukin-6 (IL-6) using a novel radioimmunoassay system. IL-6 was detectable in BALF from all groups, with disease groups showing significantly increased IL-6 levels compared with controls (P less than 0.01 in all cases). When these results were standardized, using urea to compensate for dilution effects in the BALF, only the asymptomatic pigeon breeders had significantly higher IL-6 levels than the controls (P less than 0.025), with all other groups showing no difference. When albumin was used for standardization, both the PBD group (P less than 0.001) and the sarcoidosis patients (P less than 0.01) had considerably lower levels of IL-6 than the control subjects. Using either albumin or urea for standardization, the PBD patients had significantly lower levels of IL-6 than do their asymptomatic counterparts (P less than 0.001 in both cases). This is contrasted by the finding of greatly elevated levels of IgG in the BALF of the PBD patients compared with asymptomatics (P less than 0.001). There was, however, no relation between IL-6 and IgG in any patient group, although the PBD patients had the lowest IL-6 and highest IgG as a group. These findings may suggest a mechanism by which asymptomatic subjects remain free from clinical complaints.

Development and application of three radioimmunoassays of endothelin of varying specificities.

We have developed three radioimmunoassays (RIAs) of varying specificities toward the endothelin (ET) isoforms. The assays are called the endothelin-1,2[125I] assay system (RPA535), the endothelin 1-21 Specific [125I] assay system (RPA555), and the endothelin-1,2 high-sensitivity [125I] assay system (RPA545). We have fully characterized their cross-reactivities: RPA535-ED50 approximately 12 fmol/tube, ET-1 100%, ET-2 204%, ET-3 0.0024%, hBig ET 37.9%, pBig ET 32.9%; RPA545-ED50 approximately 4.8 fmol/tube, ET-1 100%, ET-2 1,300%, ET-3 less than 0.001%, hBig ET 189%, pBig ET 63%; RPA555-ED50 approximately 3.6 fmol/tube, ET-1 100%, ET-2 144%, ET-3 52%, hBig ET 0.4%, pBig ET 0.26%. These assays have also been applied to the measurement of ET immunoreactivity (ir) in normal human plasma following Amprep extraction. Further validation of the use of these assays has included the measurement of endothelin-1 (ET-1) derived from big ET by in vitro enzymatic conversion by cathepsin E, which has demonstrated that cathepsin E possesses the appropriate specificity of cleavage to be considered the endothelin-converting enzyme (ECE).

Generation of human endothelin by cathepsin E.

Highly-purified cathepsin D processed human big endothelin1-38 into endothelin-like fragments but did not appear to generate endothelin1-21 under the conditions employed. By contrast, human cathepsin E specifically cleaved human big endothelin into endothelin1-21 and the C-terminal fragment under identical conditions but did not degrade either product further.

Specific radioimmunoassays for IL 1 alpha and IL 1 beta in plasma at physiological and acidic pH: determination of immunoreactive forms by gel filtration and radioligand binding studies.

We have developed specific radioimmunoassays for interleukin 1 alpha (IL 1 alpha) and interleukin 1 beta (IL 1 beta) and applied these successfully to the measurement of interleukin 1 (IL 1) in neat plasma. Further characterization of the plasma immunoreactive forms of IL 1 was done using Sephadex G-75 chromatography and TSKG2000 high performance gel permeation chromatography. This revealed the immunoreactivity to be associated with a high molecular weight fraction for both IL 1 alpha and IL 1 beta. Incubation of plasma with iodinated IL 1 alpha and beta showed that there was a time-dependent association of tracer with the high molecular weight fraction and that this was predominantly with IL 1 beta. The activity was displaceable with unlabeled IL 1 beta, which together with the chromatography results, suggested that IL 1 beta is protein-bound in plasma. Furthermore, we have shown that under acid conditions both tracer and endogenous IL 1 beta immunoreactivity migrate as a low (17 kD) molecular weight fraction. This suggests that dissociation from a high molecular weight binder has occurred. Acid treatment of plasma raised the immunoreactive IL 1 beta level, but had no effect on IL 1 alpha levels, confirming the specificity of a binder to IL 1 beta, as shown by the tracer experiments. These results suggest that plasma contains high molecular weight binders of IL 1, particularly IL 1 beta, and that these may play a role in regulating the distribution, clearance and bioactivity of circulating IL 1.

Specificities compared for a radioreceptor assay and a radioimmunoassay of atrial natriuretic peptide.

The main active form of atrial natriuretic peptide (ANP) in the circulation is alpha ANP(1-28) [gamma-ANP(99-126)], although some active and inactive metabolites are also though to be present. Some immunoassays for alpha ANP reportedly detect inactive metabolites, as determined by their comparison with results by receptor assay, which would lead to misleading results for alpha ANP(1-28) measurements. Here we compare a radioreceptor assay (RRA) and a radioimmunoassay (RIA) for alpha ANP and show that their results correlate very well with regard to specificity and that the RIA appears to detect only the active circulating form of alpha ANP(1-28).

A sensitive radioimmunoassay measuring endothelin-like immunoreactivity in human plasma: comparison of levels in patients with essential hypertension and normotensive control subjects.

1. A radioimmunoassay has been developed for measuring endothelin-like immunoreactivity in human plasma using an antibody raised against endothelin-1 which also cross-reacts with big endothelin-1 and endothelin-2 but not endothelin-3. 2. The sensitivity of the assay was 1 fmol/tube with inter- and intra-assay coefficients of variation of 13% and 9%, respectively. Cross-reactivity with endothelin-3 and non-endothelin peptides was less than 1%. 3. Endothelin-like immunoreactivity was present in the plasma of hypertensive patients (n = 25) at a concentration of 5.7 +/- 0.5 pmol/l (mean +/- SEM), which was not significantly different from that of age-matched control subjects (5.1 +/- 0.5 pmol/l). At these levels, endothelin-1 is unlikely to function as a circulating hormone. 4. Within the normotensive group, the concentration of endothelin-like immunoreactivity in plasma was positively correlated with mean arterial blood pressure, but in hypertensive patients it showed a significant negative correlation.

Interleukin 1 in rheumatoid arthritis: potentiation of immune responses within the joint.

Specific immunoassays were used to measure IL-1 peptides in the serum and synovial fluid of patients with rheumatoid arthritis (RA) and in the serum of age-matched healthy controls. Patients with RA had raised levels of both IL-1 beta and IL-1 alpha in their sera compared to controls. Synovial fluid levels of IL-1 beta significantly correlated with immunoreactive IL-2 and soluble IL-2 receptor (sIL-2R). In addition, incubation of synovial fluid MNC with human recombinant (hr) IL-1 caused a dose-dependent increase in the level of sIL-2R in the cell supernatant. Finally, production of IL-1 beta and IL-6 from RA peripheral blood (PB) and synovial fluid (SF) MNC was examined. PBMNC spontaneously produced low levels of IL-1 beta and IL-6 that were augmented by the addition of hr IL-1 alpha. In contrast, SFMNC spontaneously produced high levels of IL-1 beta but only low levels of IL-6, again this production was augmented by the addition of hr IL-1 alpha. Taken together, the data suggests that IL-1 potentiates immune responses within the joint.

A specific radioimmunoassay for [Leu] and [Met]enkephalin applied to a HPLC separation of human CSF.

Highly specific antisera have been raised to [Leu]enkephalin and applied in a radioimmunoassay that is sufficiently sensitive and precise for the measurement of enkephalin pentapeptides in body fluids. Extraction of CSF by Sep-pak cartridges, followed by reverse-phase HPLC to separate [Leu] and [Met]enkephalin prior to assay, has assessed their concentrations as 59-170 pmol/l and 0.5-30 pmol/l, respectively, in three patients with chronic pain. The reproducibility of the HPLC separation step was checked by adding to the sample a synthetic analogue of very low cross-reactivity in UV detectable quantities. The assay procedure described is generally applicable to the assessment of [Leu] and [Met]enkephalin concentrations in CSF.

New methodology in the raising of antibodies to small peptides leading to a highly specific radioimmunoassay for enkephalins.

Temporary chemical 'protection' of the amino and/or carboxyl termini of small peptides in conjugation reactions with high molecular weight carriers has provided immunogens which give rise to antibodies with high N-terminal and/or C-terminal specificity. Applied to [Leu]enkephalin, the new methodology resulted in antisera of high affinity (K eff 0.1 x 10(12) M-1) which recognized only enkephalin-like pentapeptides. The detailed residue specificity of one enkephalin antiserum is described.

The effect of fenclofenac on thyroid function.

The non-steroidal anti-inflammatory agent fenclofenac competitively inhibits the binding of thyroxine(T4) and triiodothyronine (T3) by thyroxine-binding globulin(TBG). Eight male volunteers completed a 4-week study during which they took fenclofenac 600 mg twice daily. The concentration of fenclofenac in serum reached a plateau after 1 week of therapy after which the mean concentration(+/SEM) of the drug in serum was 78.6 o.2 mg/1. During the steady state period on treatment there were reductions of the mean serum concentrations of total T4 to 35% (P less than 0.001), total T3 to 55% (P less than 0.001), free T4 to 69% (P less than 0.001) and free T3 to 90% (NS) of the respective pretreatment values. There were also significant changes in the concentrations of thyrotropin and reverse T3 in serum. After starting treatment with fenclofenac serum concentrations of thyrotrophin fell to a nadir after 2-4 days at which time the mean concentration was 34% (P less than 0.01) of the pretreatment value, whilst reverse T3 values increased to a maximum of 136% (P less than 0.001) of the pretreatment values over 1-2 days. There was subsequently an increase of the thyrotrophin and a reduction in reverse T3 concentrations to normal by 2 weeks of pretreatment. Transient pituitary suppression was also suggested by the response to to thryotrophin-releasing hormone (TRH): 7 days after starting fenclofenac the mean thyrotrophin response was 62% (P less than 9.001) of the pretreatment value. After 4 weeks of fenclofenac the response of TRH had returned to normal. After discontinuing fenclofenac there was a transient increase in the mean concentration of thyrotrophin in serum, to 129% of the pretreatment value (P less than 0.001), with a subsequent return to normal. Four weeks after discontinuing fenclofenac the serum concentrations of thyroid hormones and thyrotrophin were normal.

Inhibition of thyroxine binding to serum proteins by fenclofenac and related compounds.

The anti-inflammatory drug fenclofenac lowers the serum concentrations of thyroxine (T4) and triidothyronine (T3) in subjects taking a regular dose of 600 mg twice daily. In eight male volunteers the concentration of T4 in serum was reduced to almost a third of the pre-treatment value while the concentration of T3 fell by about a half. The interaction of fenclofenac, and related compounds, while the thyroid hormone binding proteins of serum was studied in vitro by equilibrium dialysis with measurement of the concentrations of the free T4 and T3 in the dialysate by immunoassay. Fenclofenac, at a concentration of 100 mg/l (of the order achieved therapeutically), increased the concentrations of free T4 and by 131% and free T3 by 62%. The related compounds which increased the free hormone concentrations (although at higher concentrations than achieved during therapy) include diclofenac, monohydroxyfenclofenac and dihydroxyfenclofenac. A second in vitro method was used to study the interaction of these drugs with isolated thyroxine-binding globulin (TBG). An antiserum to TBG, coupled to Sephadex particles, was used to isolate TGB from other serum proteins. The effect of the different drugs on the binding of T4 by the immobilised TBG was then measured. These compounds competitively inhibit the binding of T4 by TBG, and for each an inhibitor constant (Ki) was determined.