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This paper describes the ultrastructural immunolocalization of the alpha 2 chain of collagen IV, laminin, and the amino terminal propeptide of collagen I (N-Pro I) in glomeruli of rapidly frozen, freeze-substituted, and low-temperature-embedded renal biopsy specimens from two cases of Alport disease and from normal kidneys. The alpha 2 chain of collagen IV is present in the whole thickness of the basement membrane in glomeruli of Alport patients, while it is limited to the subendothelial portion of the basement membrane of normal glomeruli. Laminin has the same distribution in both normal and Alport glomeruli, but is apparently more concentrated along the basement membrane of normal glomeruli. N-Pro I is localized in mesangial areas and in the basement membrane in Alport cases, while it is not detected in normal glomeruli. These data suggest complex rearrangements of major constituents of the glomerular basement membrane network and demonstrate early deposition of fibrillary collagen proteins in the matrix before the appearance of banded collagen fibres. This finding could be an indicator of early evolution towards glomerulosclerosis.

Absent or defective collagen VII at the dermo-epidermal junction is the hallmark of dystrophic recessive epidermolysis bullosa. Little is known of the alterations of other collagenous and non-collagenous components of the basement membrane; it is likely that their assembly may be disturbed by the lack of collagen VII molecules. The spatial relationship of collagen IV and laminin has been studied, both in bullous and in non-bullous areas. Skin biopsies from five patients affected by severe dystrophic recessive epidermolysis bullosa were rapidly frozen and freeze-dried. Collagen IV and laminin were labelled with specific monoclonal antibodies and FITC- or TRITC-conjugated secondary antibodies. Sections were observed with conventional light/fluorescence microscopy and with confocal laser scanning microscopy. Collagen IV and laminin were not co-localized: the former displayed a split image, being present at the floor and the roof of the blister, while the latter was confined to the roof. Confocal microscopy also allowed three-dimensional (3D) reconstruction of the dermo-epidermal junction from a series of optical sections, with viewing of the reconstructed specimen from a sequence of angles. By this procedure, laminin exhibits an irregular, coarsely granular distribution, both in affected and in apparently non-affected areas, while collagen IV appears as a homogeneous sheet. These results show that freeze-drying is the technique of choice for high-resolution immunofluorescence of skin samples and suggest that in dystrophic recessive epidermolysis bullosa, a complex disruption of the extracellular matrix assembly exists even before blister formation, probably due to the lack of collagen VII.

The histological picture of primary glomerulonephritis with glomerular IgA deposition (IgA nephropathy and Henoch-Schönlein disease) can vary from minimal mesangial involvement to severe endocapillary and/or extracapillary proliferation. Local activation of the complement cascade by glomerular IgA deposits and release of anaphylactoid factor are considered to be major triggers of inflammation, but clear-cut correlations between the severity of the histological findings and the intensity of glomerular deposition of immunoglobulin and complement fractions are still lacking. The purpose of this study was to investigate the spatial distribution of IgA and complement in mesangial deposits with confocal laser scanning microscopy (CLSM) and to correlate specific patterns of IgA-complement interaction with glomerular damage. Two groups of patients have been studied, one with mild to moderate diffuse mesangial proliferation and the other with diffuse proliferative endocapillary and/or extracapillary patterns. In milder forms of the disease, the majority of the immune deposits are composed of both IgA and C3, coated by an outer layer of IgA alone. Large C3 deposits, or deposits composed of IgA and C3 without an outer coat of IgA, were associated with more severe histological lesions. The results suggest that free access of active complement components to cell and/or mesangial matrix receptors could trigger a cytolytic reaction and that immunoglobulins seem to act as a protective layer on C3 components.