The detection of intracytoplasmic interleukin-1 alpha, interleukin-1 beta and tumour necrosis factor alpha expression in human monocytes using two colour immunofluorescence flow cytometry.

Two colour flow cytometry was used to analyse in situ cytokine expression by human monocytes. Whole blood was cultured in siliconised glass bottles, with or without E. coli lipopolysaccharide (LPS), for various times, and the mononuclear cells (MNCs) then exposed to a variety of permeabilisation procedures prior to flow cytometric analysis. Paraformaldehyde (PF)/saponin fixation preserved cellular morphology, and caused a reproducible degree of permeabilisation (estimated by propidium iodide inclusion: mean 94%, range 86-99% (n = 33)). After fixation with 4% PF and permeabilisation with 1% saponin at 0 degrees C in PBS containing 20% human serum, MNCs were incubated with phycoerythrin(PE)-conjugated mouse anti-CD14 (monocyte phenotype) and polyclonal rabbit anti-human interleukin-1 alpha (IL-1 alpha), IL-1 beta, tumour necrosis factor alpha (TNF-alpha), or control rabbit IgG. Binding of rabbit antibodies was detected using goat anti-rabbit IgG fluorescein isothiocyanate (FITC). FITC fluorescence was increased in CD14 PE positive cells with the three anti-cytokine antibodies following LPS stimulation, compared with controls. There was a reproducible dose related response in monocyte IL-1 beta and TNF-alpha expression following LPS stimulation, with early peaks in TNF-alpha (2 h), compared with IL-1 beta (4 h), and IL-1 alpha (12 h). Specificity of this cytokine detection system was confirmed by inhibition studies using the corresponding recombinant human cytokines, by an absence of staining in CD14 negative or unpermeabilised MNCs, and by the characteristic cytoplasmic localisation of the different cytokines visualised with UV immunochemistry. Hence, the methods described here provide a reproducible, semiquantitative and specific assay for the detection of cell associated monokines. The technique may be applicable to the analysis of a variety of different cytokines in other phenotypically defined cell populations.     

Changes in numbers of epidermal cell adhesion molecules caused by oral cyclosporin in psoriasis.

AIM--To determine the effects of a three month course of low dose cyclosporin on the expression of epidermal cell adhesion molecules. METHODS--Eighteen patients with psoriasis were treated for 12 weeks with either 2.5 or 5 mg/kg/day of oral cyclosporin. Biopsy specimens taken from skin before, during, and after cyclosporin treatment were stained immunohistochemically for CD 54 (ICAM-1), CD 29 (beta-1 integrins), and CD18 (beta-2 integrins). RESULTS--There was a highly significant (p < 0.01) clinical response after 12 weeks of cyclosporin as assessed by the Psoriasis Area and Severity Index (PASI) score. The staining of CD 29 on keratinocytes of affected and unaffected psoriatic skin was not affected by cyclosporin. Epidermal CD54 was variably expressed in active psoriatic plaques and changed unpredictably after cyclosporin (p = NS). Staining for CD18 on large epidermal dendritic cells was reduced after cyclosporin (p < 0.02). The expression of CD18 by large epidermal dendritic cells during treatment correlated strongly with the PASI score at that time and one month after stopping cyclosporin (p < 0.02). CONCLUSIONS--Persistence of epidermal staining for CD 54 in psoriasis is compatible with a good clinical response to cyclosporin. Residual staining for CD 18 on large epidermal dendritic cells may be a useful marker for early clinical relapse.     

Goodpasture's syndrome in a patient with two endocrine tumours

We describe the occurrence of two anatomically separate and histologically distinct endocrine tumours of bronchus and pancreas in a 64 year old woman with biopsy and serology proven Goodpasture's syndrome. The association between anti-glomerular basement membrane antibody disease and multiple endocrine tumours has not been reported previously; possible pathogenetic mechanisms are discussed.     

Modulation of abnormalities in renal haemodynamics and vasoactive mediators by nifedipine in patients with psoriasis on low-dose cyclosporin

Ten patients with psoriasis received a 3-month course of cyclosporin(2.5 mg/kg/day) followed by a 3-month washout period, beforecommencing a 3-month course of cyclosporin and nifedipine SR20 mg b.d. Serial haemodynamic and biochemical measurementswere performed before, during, and after treatment. Total renalblood flow (RBF) was measured following an intravenous injectionof [^99mTc]-DTPA based on a renographic analysis of the first-passeffect in the kidneys, and GFR was estimated from the subsequentclearance of this radiotracer. A significant individual changein RBF or GFR was taken as 25/ and 20/ respectively. Simultaneousassays of the circulating vasoactive mediators renin, aldosterone,angiotensin II, and atrial natriuretic peptide were performed.Two patients withdrew from the study because they could nottolerate nifedipine, leaving eight for complete analysis. Thesignificant reductions in RBF and GFR which occurred on cyclosporinalone (P<0.05; ANOVA) did not occur with added nifedipine.Four months after this second course, RBF and GFR had recovered.The response to nifedipine was, however, variable and unpredictable.Of the four patients to show a significant decline in GFR oncyclosporin alone, only two showed a significant improvementon the combined therapy. Of the six patients who showed a significantdecline in RBF on cyclosporin alone, only four showed benefitfrom the added nifedipine. Nifedipine suppresses the increasein blood pressure which occurred on cyclosporin alone. The circulatingconcentration of angiotensin II was significantly less on cyclosporinand nifedipine than on cyclosporin alone (P<0.05; Student'st test). There was a significant reduction in serum aldosteroneafter cyclosporin administration (P<0.05; ANOVA). Plasmarenin activity did not change. There were statistically significantincreases in ANP on cyclosporin and nifedipine (P<0.05; ANOVA). The interaction between nifedipine and cyclosporin on the glomerularmicrocirculation and on the release of angiotensin II by thenormal innervated kidney can either reduce or increase haemodynamictoxicity. Regardless of haemodynamic improvement, nifedipinedoes not correct all the hormonal changes.     

Computer prediction of the need for dialysis and transplantation using calculated creatinine clearance

A computerised system capable of prospective assessment of the course of chronic renal failure has been devised. Rapid analysis is possible of the progress of a large cohort of patients and therefore of the prospective load on renal unit facilities. Calculated creatinine clearance values provide a more accurate and earlier prediction of end stage failure than the reciprocals of serum creatinine values.     

Urinary C3dg and C5b-9 indicate active immune disease in human membranous nephropathy.

We have measured complement activation markers, C3dg and C5b-9 in plasma and urine from patients with idiopathic membranous nephropathy and IgA nephropathy. There was no significant difference in levels of plasma C5b-9 between the patient groups. However, high plasma concentrations of C3dg were associated significantly with IgA nephropathy with 45% of patients having levels over 25 U/ml (P less than 0.001). High concentrations of urinary C3dg and C5b-9 were associated significantly with membranous nephropathy (43% and 43% of the patient group, respectively) compared to patients with IgA nephropathy (10% and 0%, respectively, P less than 0.001). In a retrospective analysis of 31 patients with membranous nephropathy, 66% of patients with high initial urinary C5b-9 showed an unstable clinical course compared to 18% of patients with initially absent or low C5b-9 (P less than 0.001). We suggest that high urinary C5b-9 identifies those patients with a membranous lesion which retains an active immunological component contributing to the pathology of progressive glomerular damage.