Mesangial cell autoantigens in immunoglobulin A nephropathy and Henoch-Schönlein purpura.

The autoantigen(s) that we have previously described in human glomeruli, recognized in IgA nephropathy, has (have) been identified as mesangial cell in origin. Cultured mesangial cells expressed 48- and 55-kD components binding IgG isotype autoantibodies (IgG-MESCA) present in sera of patients with both IgA nephropathy and Henoch-Schönlein purpura (HSP). IgG-MESCA were not detected in sera of normals, or patients with other autoimmune-mediated glomerulonephritides: anti-glomerular basement membrane disease, Wegener's granulomatosis, or in IgM-mesangial proliferative disease. Binding specificity was proven by F(ab')2 studies in enzyme-linked immunosorbent assay (ELISA) and Western blotting, and there was no significant affinity of IgA or IgM immunoglobulins. Fluorescein isothiocyanate-conjugated IgG from ELISA-positive sera localized to the mesangium and peripheral capillary loops of glomeruli, supporting the belief that the antigen is expressed in normal human renal tissue. However, only about one third of mesangial cells in culture showed affinity for IgG from ELISA-positive sera, suggesting variable expression of the antigen(s) in vitro. The only autoantigen(s) present in glomeruli, and extractable from whole normal glomeruli by the techniques employed, localized on the mesangial cell. In both IgA nephropathy and HSP, autoimmunity was intermittently present, with fluctuating levels of IgG-MESCA detectable in sera. There were positive correlations with the degree of glomerular injury assessed by erythrocyturia and proteinuria in IgA nephropathy, but significance was reached with only the degree of hematuria in HSP. These findings suggest a contributing role in the pathogenesis of the mesangial proliferative lesions and demonstrate autoimmunity common to both IgA nephropathy and HSP.

Primary angiitis of the central nervous system: emerging variants.

BACKGROUND: Primary angiitis of the central nervous system (PACNS), a serious disease, has not featured prominently in the spectrum of multi-organ disease seen in vasculitis clinics. AIM: To evaluate the presentation, natural history and features of PACNS variants and compare to those of systemic vasculitides. DESIGN: Retrospective analysis. METHODS: Patients (n=105) presented during 1988-2003 to a tertiary regional vasculitis clinic receiving unselected disease types. Data were collected from a clinical database, patient and laboratory records. RESULTS: The frequency of PACNS presentation rose over the study period, compared with most of the other vasculitides. When PACNS was divided into small- and middle-sized vessel disease (SVD/MVD), their clinical courses differed substantially. SVD PACNS was responsive to immunosuppressive drugs, but relapsed during prolonged periods in all patients on maintenance immunosuppressives, or after withdrawal of treatment, causing recurrent, severe and irreversible CNS injury. MVD PACNS had isolated episodes at presentation, with a paucity of relapses during prolonged follow-up. DISCUSSION: Similarities between SVD PACNS and microscopic polyarteritis suggest the former may represent a limited form of the latter. MVD PACNS has a distinctly more benign relapse pattern than its multisystem counterpart polyarteritis nodosa. Acute-phase serology was useful in designating inflammatory processes at presentation of patients presenting with encephalopathy caused by SVD only, but were unhelpful in defining relapses in this form of PACNS, the definition of which in all cases rested on clinical assessment and MR scanning. Direct cerebral angiography was not diagnostic in any case of SVD PACNS; positive brain biopsy is diagnostically unequivocal, but the total clinical syndrome with imaging may establish a diagnosis with highest probability. In MVD PACNS, angiography with MR scan proved diagnostic. We suggest an algorithm for a rational, minimally invasive approach to investigation. In PACNS, SVD and MVD are important variants, and decisions about therapy should incorporate these distinctions.

Peritonitis induces the synthesis of 1 alpha,25-dihydroxyvitamin D3 in macrophages from CAPD patients.

Metabolism of 25-[3H]hydroxyvitamin D3 was studied in peritoneal macrophages from renal failure patients on continuous ambulatory peritoneal dialysis (CAPD). Cells from 5 out of 8 patients with a history of peritonitis produced significant amounts of a metabolite chromatographically identical to 1 alpha,25(OH)2D3; but none was produced by cells from non-infected patients. The evidence strongly suggests that peritoneal macrophages stimulated by infection can metabolise 25OHD3 to the active vitamin D3 metabolite, 1 alpha,25(OH)2D3, when maintained in short-term primary culture.

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.

Characterization of two corneal epithelium-derived antigens associated with vasculitis.

PURPOSE: In a previous investigation into corneal autoimmunity, it was demonstrated that a putative autoantigen, a protein of 66 kDa, present in bovine corneal epithelium, binds circulating autoantibodies in approximately 60% of patients with Wegener's granulomatosis (WG). The aim of the present study was to characterize and identify the 66-kDa protein. METHODS: A purification protocol was established for the 66-kDa protein using standard chromatography techniques. During the purification procedure it became clear that the 66-kDa protein detected in patients' sera was in fact two proteins, both running at 66 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, that eluted in different fractions on DE-52 chromatography columns. These two proteins have been labeled bovine corneal epithelial antigen-A and -B (BCEA-A and BCEA-B). Further investigations of antibody binding have demonstrated that patients' sera bind to either one or the other of these proteins with no cross-reactivity between them. Separated BCEA-A and BCEA-B protein extracts were immunoblotted with 27 WG patients' sera, 10 Churg-Strauss syndrome (CSS) patients' sera, 31 rheumatoid arthritis (RA) patients' sera, and 40 healthy control subjects' sera from the blood bank. RESULTS: Forty-six percent of WG patients' sera had antibodies to one of the 66-kDa antigens, whereas none of the healthy control subjects' sera had 66-kDa antibodies (P < 10(-5)). In the WG group, 31% were positive to BCEA-A (versus controls, P = 0.0023), and 15% were positive to BCEA-B. WG patients with peripheral ulcerative keratitis (PUK) had a significant association with anti-BCEA-A antibodies when compared with healthy control subjects (50%, P < 10(-6)). However, in the RA group with no eye disease there was an association with BCEA-A (25%, P = 0.011) but not in the RA group with PUK. The frequency of anti-BCEA-B antibodies was significantly increased in patients with CSS (60%, P < 10(-7)). CONCLUSIONS: In summary, it has been shown that vasculitis patients have antibodies to two 66-kDa corneal antigens and that autoantibodies to these antigens are mutually exclusive. It has also been shown that antibodies to BCEA-B are associated with CSS, whereas BCEA-A antibodies are associated with WG and RA.

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.

Effect of low-dose cyclosporin on plasma lipoproteins and markers of cholestasis in patients with psoriasis.

The benefits of using cyclosporin in organ transplantation to prevent graft rejection outweigh its potential disadvantages, but with the use of low-dose cyclosporin in relatively healthy individuals, such as those with psoriasis, the risk:benefit ratio is altered. The effects of low-dose cyclosporin (< 5 mg/kg body weight) on liver function and serum lipids and lipoproteins were examined in 40 normolipidaemic, normotensive psoriasis patients with normal renal function. After 3 months of treatment, serum cholesterol and bilirubin concentrations and alkaline phosphatase activity increased significantly (p = 0.001), and glomerular filtration rate (GFR) declined from 107 to 96 ml/min/1.73 m2 (p = 0.05). All these values returned to pretreatment levels 3 months after cessation of cyclosporin. In 15 patients in whom lipoproteins were isolated by ultracentrifugation, there was an increase in plasma low-density lipoprotein (LDL) cholesterol (p = 0.05), but very-low-density lipoprotein cholesterol, high-density lipoprotein (HDL) and HDL2 and HDL3 cholesterol concentrations did not change. The increases in serum bilirubin, alkaline phosphatase activity and LDL cholesterol, seen in individuals with normal baseline liver and renal function, which reverted to baseline following cessation of cyclosporin, suggest that cyclosporin-induced hypercholesterolaemia may be due to either decreased biliary excretion of cholesterol or impaired catabolism of LDL.

Serum amyloid P component in chronic renal failure and dialysis.

A normal reference interval for serum amyloid P component (SAP) concentration in the serum was established in 500 healthy adult individuals (274 women, 226 men), by electroimmunoassay calibrated with standards of highly purified, isolated SAP. The mass of SAP in these was determined from the extinction coefficient of SAP at 280 nm measured here precisely for the first time by spectrophotometry and cryogenic drying. The mean (SD, range) SAP concentration was significantly lower in women: 24 mg/l (8, 8-55), compared to 32 mg/l (7, 12-50) in men (P less than 0.001). In renal insufficiency patients, 38 with chronic renal failure, 79 on hemodialysis and 66 on continuous ambulatory peritoneal dialysis, the mean values for SAP concentration were all significantly higher than normal (range of means, 39-59 mg/l in men and 35-42 mg/l in women), but did not correlate with serum creatinine, duration of dialysis or the presence of an acute phase response. The metabolism of SAP is thus altered in renal failure and is not normalized by dialysis, but it is not clear whether this is relevant to the pathogenesis of dialysis related arthropathy and amyloidosis.

Sequential development of systemic vasculitis with anti-neutrophil cytoplasmic antibodies complicating anti-glomerular basement membrane disease.

Anti-neutrophil cytoplasmic antibodies (cANCA) were detected in 3 patients with anti-glomerular basement membrane (anti-GBM) disease. All 3 cases presented late in their clinical course, with severe renal involvement and alveolar hemorrhage. Anti-neutrophil cytoplasmic antibodies were associated with clinical features outwith the lungs and kidneys; in one case cANCA were initially absent but subsequently developed concurrently with the clinical appearance of systemic vasculitis as the anti-GBM antibody titer was falling. These findings confirm that cANCA can complicate anti-GBM disease and suggest that cANCA may identify a distinct subset of patients with anti-GBM disease, supporting a pathogenic role for cANCA in the development of systemic vasculitis.

Angiotensin II as a risk factor for cyclosporin nephrotoxicity in patients with psoriasis.

Abnormalities of the renin-angiotensin system after low-dose cyclosporin (5 mg/kg/day or less) have not been adequately defined in patients with normal kidneys. 27 patients with psoriasis were assessed before starting cyclosporin, after three months of cyclosporin (5 mg/kg/day or less) and then finally three months after finishing cyclosporin. On each occasion plasma renin activity (PRA), aldosterone, angiotensin II and atrial natriuretic peptide (ANP) were measured together with total renal blood flow (RBF), GFR and filtration fraction (FF) following an i.v. bolus injection of Tc-99m DTPA. Significant renal hemodynamic toxicity was defined as > 25% fall in RBF or > 20% fall in GFR. Using these criteria we identified 12 patients with hemodynamic toxicity (Group A) and 15 patients whose GFR and RBF did not fall significantly (Group B). In Group A a significant fall in GFR (p < 0.001) and reduction in renal blood flow (p < 0.04) were associated with significant rises in both ambulant and recumbent angiotensin II (p < 0.0005). PRA, aldosterone and ANP did not significantly alter. GFR partially recovered after withdrawal of cyclosporin although RBF remained significantly lower compared to initial values. In Group B there was no significant change in GFR or RBF although there was a reversible fall in FF (p < 0.02). There were no significant differences in angiotensin II, PRA, aldosterone or ANP. Circulating angiotensin II rises in patients who develop cyclosporin nephrotoxicity and may be responsible for mediating the hemodynamic effects.

Neighbouring acetamido-group participation in reactions of derivatives of 2-acetamido-2-deoxy-D-glucose.

Kinetic measurements suggest that neighbouring acetamido-group participation occurs in the spontaneous hydrolysis and methanolysis of o-carboxyphenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside and in the spontaneous hydrolysis of 2,4-dinitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside and 2-acetamido-2-deoxy-beta-D-glycopyranosyl fluoride. The methanolyses of these compounds proceed with predominant retention of configuration which is also consistent with neighbouring acetamido-group participation. The oxazoline intermediate which would arise from such a process was detected during methanolysis of 2-acetamido-2-deoxy-beta-D-glucopyranosyl fluoride in the presence of bases by n.m.r., i.r., and u.v. spectroscopy. Attempts to isolate the oxazoline were unsuccessful.

Prognostic indices and therapy in IgA nephropathy: toward a solution.

IgA nephropathy is the primary renal disease with the greatest impact on services. The paucity of trials with high evidence-based standards gives emphasis to issues of the rationalization of therapies. Now that certain treatments are increasingly accepted and others under evaluation, reliable discriminatory tests are essential to define and select patients at high risk of progression before irreversible loss of renal tissue, while avoiding drug exposure in others.

Partial reversal of carbamazepine-induced water intolerance by demeclocycline.

The anti-diuretic action of carbamazepine, before and after concurrent treatment with demeclocycline, has been studied in a single epileptic subject, in whom two episodes of status epilepticus had been associated with excessive fluid intake and hyponatraemia. After addition of demeclocyline, free water clearance, plasma arginine vasopressin concentration and serum osmolality (all appreciably reduced after carbamazepine alone) increased but did not revert to normal. The findings are consistent with direct antagonism by demeclocycline of the enhancing effect of carbamazepine on endogenous ADH activity.

Volumetric analysis of urinary erythrocytes: a standardized methodology to localize the source of haematuria.

Volumetric analysis of urinary red blood cells (RBCs) was performed in two groups of patients: (a) 91 with glomerulonephritis (GN), 158 with non-glomerular diseases (non-GN) and 53 controls without haematuria; (b) 97 with GN and 41 with non-GN diagnoses and greater than or equal to 2+ haematuria, analysed after modified sample preparation (isotonic dilution and haemolysis to eliminate non-RBC debris). In group A, diagnostic ranges were established for modal, mean and differential (urinary-peripheral blood modal) RBC volumes to differentiate GN from non-GN sources of blood loss according to RBC size. 54% (135/249) of modal volumes and 68% (124/183) with greater than or equal to 2+ haematuria were within a diagnostic range of values, i.e. 40-180 fl (sensitivity), and the source of haematuria was correctly identified in 85% (115/135) and 87% (108/124) of these, respectively (specificity). Of the remainder, with modal volumes less than 40 or greater than 180 fl, 45% (51/114) were from GN and 55% (63/114) from non-GN specimens, along with 94% (48/53) controls without haematuria (non-diagnostic analyses). In contrast, whilst 90% (74/82) of mean volumes greater than or equal to 110 fl and 94% (62/66) of differential volumes greater than or equal to 0 fl were from non-GN specimens, values below these were common to both diagnostic groups so that diagnostic sensitivities were reduced to 38% (82/219) and 34% (66/193), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Bone marrow transplantation: effects of conditioning and cyclosporin prophylaxis on microvascular permeability to a small solute (technetium 99m diethylene triamine penta-acetic acid).

Microvascular permeability to small diffusible solutes has rarely been measured at a clinical level. We have developed a simple non-invasive technique for measuring the permeability surface area (PS) product, which is suitable for clinical use. We illustrate its potential value in six subjects who underwent bone marrow transplantation for chronic myeloid leukaemia. These patients received high-dose cyclosporin A (CyA) for prevention of graft versus host disease (GVHD) and sustained an easily measurable increase in microvascular permeability to technetium 99m diethyltriamine penta-acetic acid (99mTc-DTPA). This was measured as the PS product, which increased from 1.1 (SD 0.3) to 2.2 (0.4) ml/min per 100 ml tissue between baseline and treatment with CyA for prevention of GVHD (P less than 0.01). The increase broadly correlated with nephrotoxicity which was measured, from the plasma DTPA clearance, as global glomerular filtration rate (GFR). This decreased from 106 (11.1) to 49 (6.7) ml/min (P less than 0.001). These abnormalities, both in PS product and GFR, were sustained for several months, after which they tended to return towards baseline levels. We conclude firstly that this technique has a potential clinical role and secondly that endothelial abnormalities due to CyA deserve further study.