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BACKGROUND Chronic antibody-mediated rejection is an important cause of late graft failure. Developing an early marker of the disease may allow diagnosis and treatment before irreversible graft damage has occurred. The aim of this study was to assess whether, on electron microscopy examination, peritubular capillary (PTC) basement membrane multilayering precedes and predicts the development of transplant glomerulopathy (TG). METHODS We used a vintage matched case-control method. Sixteen case-control pairs were created among all renal transplant patients from October 2005. Cases were patients who developed TG, and controls were patients with a late (>36 months) posttransplant (indication or surveillance) biopsy without TG. Electron microscopy was carried out on a biopsy taken earlier in the posttransplantation period for both cases and controls. RESULTS For every additional PTC of 25 examined with three or more layers in the early biopsy, the risk of having TG in the later biopsy was increased by 1.4-fold (95% confidence interval, 1.1-1.9; P=0.015). For every PTC of 25 with five or more layers, the risk was increased by 1.6-fold (95% confidence interval, 1.0-2.7; P=0.063). Thus, the risk of future TG increased substantially with every additional PTC of 25 showing multilayering in the early biopsy. CONCLUSIONS Peritubular capillary basement membrane multilayering on electron microscopy is a useful marker of early chronic antibody-mediated damage, and information can be obtained by assessing PTC with three to four layers of basement membrane in addition to those with five or more layers. This finding must be validated in a prospective study.

A case of metastatic malignant melanoma exhibiting small cell morphology is described. The patient had had a previous primary nodular small cell melanoma. The metastatic tumor was examined by conventional histology, light microscope immunohistochemistry, conventional electron microscopy, and ultrastructural immunolabeling. It consisted of small cells, which, however, varied in size and were present in distinct but merging areas. Tumor cells were negative for S-100 protein and very focally positive for cytokeratin: these findings in combination with small cell morphology suggested the possibility of small cell carcinoma. However, other melanocytic markers were positive. Neuroendocrine markers were negative. By electron microscopy, tumor cells lacked unambiguous melanosomes but contained paranuclear aggregates of nondescript granules. Following ultrastructural immunolabeling, these were found to be decorated with gold-labeled HMB-45 antibodies, thereby confirming them as lattice-deficient melanosomes. This tumor is an uncommon example of malignant melanoma where immunoultrastructural analysis helped clarify the nature of otherwise nondescript granules as true but lattice-deficient melanosomes. This is also the first case of small cell melanoma to be studied by electron microscopy.

An investigation was undertaken on paraformaldehyde-fixed, Lowicryl resin-embedded renal biopsies from patients with AA amyloidosis to study the association of two small chondroitin sulphate/dermatan sulphate proteoglycans, decorin and biglycan, with amyloid fibrils using an ultrastructural immunogold technique. Biglycan was present in glomerular endothelial cells in both normal kidney and in amyloidosis, but little biglycan or decorin was present in the normal mesangial matrix. By contrast, conspicuous amounts of both biglycan and decorin were seen to be associated with amyloid fibrils in the glomerular matrix in cases of renal AA amyloidosis. The results further emphasise the close association between amyloid and extracellular matrix components which are now considered to be an integral part of the amyloid fibrils.

An ultrastructural investigation was undertaken on paraformaldehyde-fixed Lowicryl resin-embedded human kidneys of three patients with AA amyloidosis to investigate the association of various basement membrane components with amyloid fibrils. An immunogold technique was used and antibodies to serum amyloid A, heparan sulphate proteoglycan, type IV collagen, P component, and fibronectin were applied to human normal and amyloid glomeruli. The amyloid was identified as AA, and P component was shown to be intimately associated with the fibrils. In addition, heparan sulphate proteoglycan was associated with amyloid in all subendothelial, subepithelial and intramembranous glomerular basement membrane deposits, and those throughout the mesangial matrix. This contrasted with the distribution of the proteoglycan in the normal glomerulus where it was found predominantly on the epithelial aspect of the basement membrane and only in the more peripheral regions of the mesangium. The accumulation of heparan sulphate proteoglycan with amyloid resulted in a marked increase in its amount in the glomeruli. The amyloid deposits contained little or no type IV collagen or fibronectin. These findings demonstrate a strong association of heparan sulphate proteoglycan with amyloid and suggest different roles for the various glomerular basement membrane components in amyloidogenesis.

Goodpasture's, or anti-glomerular basement membrane (GBM), disease presents with rapidly progressive glomerulonephritis and lung haemorrhage, and is caused by autoimmunity to the NC1 domain of the alpha3 chain of type IV collagen (alpha3(IV)NC1). This study examines the development of crescentic nephritis and alveolar haemorrhage in a model of Goodpasture's disease, experimental autoimmune glomerulonephritis (EAG), induced in WKY rats by immunization with rat GBM in adjuvant. An increase in circulating anti-GBM antibodies and albuminuria was observed by week 2, which increased further by weeks 3 and 4, while a decrease in creatinine clearance was observed by week 2, which decreased further by weeks 3 and 4. The kidneys of animals with EAG showed linear deposits of IgG on the GBM and a transient glomerular infiltration by CD4+ T cells at week 2. By week 3 there were large deposits of fibrin in Bowman's space, and glomerular infiltration by CD8+ T cells and macrophages, accompanied by focal necrotizing glomerulonephritis with crescent formation. Ultrastructural studies showed glomerular endothelial cell swelling and epithelial cell foot process effacement at week 2. As the lesion progressed, capillary loops became occluded and the mesangium became expanded by mononuclear cells. By week 3 there was detachment of the endothelium from the GBM, and accumulation of fibrin beneath the disrupted endothelial cells and in Bowman's space. Occasional breaks were observed in the continuity of the basement membrane, and cytoplasmic projections from infiltrating mononuclear cells could be seen crossing the capillary wall between the lumen and the crescent. The lungs of animals with EAG showed patchy binding of IgG to the alveolar basement membrane (ABM) at week 2, and infiltration of the interstitium by CD8+ T cells and macrophages by weeks 3 and 4, accompanied by both interstitial and alveolar haemorrhage. Ultrastructural studies showed focal mononuclear cell infiltrates in alveolar walls at week 2. Occasional breaks were observed in the basement membrane and adjacent endothelium by weeks 3 and 4, together with accumulation of surfactant and erythrocytes within the alveolar spaces. This study defines for the first time the relationship between the immunological and pathological events during the evolution of EAG, and provides the basis for further work on the pathogenesis of Goodpasture's disease.

We have undertaken an ultrastructural immunogold investigation of the distribution of type IV collagen and heparan sulphate proteoglycan (HSPG) in glomeruli from the kidneys of one normal control and three patients with diabetes mellitus and proteinuria. The sample included both diffuse and nodular diabetic glomerulosclerosis. In the control and diabetic kidneys, the type IV collagen was present predominantly on the endothelial aspect of the glomerular basement membrane (GBM), and by contrast the HSPG was found mainly on the epithelial side. In the mesangium in both control and diabetic glomeruli, type IV collagen was found predominantly in the central regions, while HSPG was mostly restricted to the region beneath the epithelial cells. Consequently, where there is a marked increase in mesangial matrix with nodule formation in diabetics there is a corresponding increase in the amount of type IV collagen but not of HSPG. Although the three diabetic patients were proteinuric, the HSPG was not decreased in the thickened GBMs.

The distribution of heparan sulphate proteoglycans (HSPG) has been investigated in normal human glomeruli, membranous glomerulonephritis, mesangial IgA disease, and anti-glomerular basement membrane disease. HSPG was localized using anti-bovine HSPG antibody and 10 nm gold-labelled secondary antibody on paraformaldehyde-fixed, Lowicryl K4M resin-embedded kidneys. HSPG was present in all glomeruli and there was a zonation of its distribution in that it was predominantly on the epithelial aspect of the glomerular basement membrane (GBM) and mesangium with little in the central regions of the mesangial matrix. In the cases of immune complex glomerulonephritis, no HSPG was found in the electron-dense deposits. These findings contrast with our previous studies using the same technique in which type IV collagen and fibronectin were found predominantly on the endothelial aspect of the GBM.

The relationship between the immune complex deposits of mesangial IgA nephropathy and the basement membrane components, type IV collagen and fibronectin, has been investigated by an indirect immunogold technique in four cases of mesangial IgA disease. Using paraformaldehyde-fixed, Lowicryl K4M resin-embedded kidney, IgA, IgM and C3 were localized in the mesangial electron-dense deposits with 10 and 20 nm gold-labelled secondary antibodies. In the same glomeruli, type IV collagen and fibronectin were rarely present within the electron-dense deposits, although both were distributed throughout the remainder of the mesangial matrix with the exception of the subepithelial regions. These two components were also present within the glomerular basement membrane and localized mainly on the endothelial aspect. A similar distribution of the basement membrane components was seen in a control kidney processed in the same way. This technique gives reproducible results and has demonstrated for the first time the relationship between the mesangial immune complex deposits of mesangial IgA nephropathy and the basement membrane components of the matrix in which they are found.