The Discovery of Endoplasmic Reticulum Storage Disease. The Connection between an H&E Slide and the Brain

Abstract

The revolutionary evolution in science and technology over the last few decades has made it possible to face more adequately three main challenges of modern medicine: changes in old diseases, the appearance of new diseases, and diseases that are unknown (mostly genetic), despite research efforts. In this paper we review the road travelled by pathologists in search of a method based upon the use of routine instruments and techniques which once were available for research only. The application to tissue studies of techniques from immunology, molecular biology, and genetics has allowed dynamic interpretations of biological phenomena with special regard to gene regulation and expression. That implies stepwise investigations, including light microscopy, immunohistochemistry, in situ hybridization, electron microscopy, molecular histopathology, protein crystallography, and gene sequencing, in order to progress from suggestive features detectable in routinely stained preparations to more characteristic, specific, and finally, pathognomonic features. Hematoxylin and Eosin (H&E)-stained preparations and appropriate immunohistochemical stains have enabled the recognition of phenotypic changes which may reflect genotypic alterations. That has been the case with hepatocytic inclusions detected in H&E-stained preparations, which appeared to correspond to secretory proteins that, due to genetic mutations, were retained within the rough endoplasmic reticulum (RER) and were deficient in plasma. The identification of this phenomenon affecting the molecules alpha-1-antitrypsin and fibrinogen has led to the discovery of a new field of cell organelle pathology, endoplasmic reticulum storage disease(s) (ERSD). Over fifty years, pathologists have wandered through a dark forest of complicated molecules with strange conformations, and by detailed observations in simple histopathological sections, accompanied by a growing background of molecular techniques and revelations, have been able to recognize and identify arrays of grotesque polypeptide arrangements.

Keywords: alpha-1-antitrypsin deficiency; endoplasmic reticulum storage disease; hereditary hypofibrinogenemia hepatic storage.

Figure 1

Histological section from a Pi ZZ liver. Hepatocytes contain cytoplasmic, round eosinophilic inclusions in a periportal area (a), H&E × 40). The eosinophilic cytoplasmic inclusions are strongly positive on PAS staining after amylase digestion. (b), PAS-diastase (PASD) × 40). The inclusions were positively stained with a polyclonal alpha 1 anti-trypsin (AAT) antibody (c), immunostaining × 40). The electron microscopy (EM) picture shows amorphous fluffy semielectron dense material corresponding to AAT within dilated cisternae of the rough endoplasmic reticulum (RER). No material is observed within the smooth endoplasmic reticulum (SER) or Golgi apparatus (d), EM × 16,000). The chromatogram profile shows the sequence of SERPINA 1 Exon V flanking the p.Glu342Lys (ZAAT mutation) (e). The AAT inclusions were positively stained with the monoclonal ATZ11 antibody, specific for the Z variant of AAT (f), immunostaining × 40).

Figure 2

Histological section from a PiMmalton cirrhotic liver. Hepatocytes contain large numbers of cytoplasmic eosinophilic inclusions. A few inclusions are centered by a dark basophilic material (a), H&E × 60). The electron microphotograph shows electrondense crystal-like or granular precipitates inside the AAT intracisternal inclusions of three adjacent hepatocytes (c), EM × 20,000). X-ray microanalysis for metal detection shows two picks corresponding to calcium. The electron beams were centered on electron dense precipitates under the EM (b), X-ray).

Figure 3

Histological liver section from a hereditary hypofibrinogenemia with hepatic storage (HHHS) patient. Hepatocytes contain round or polygonal eosinophilic cytoplasmic inclusions. Inside the eosinophilic inclusions there are single or multiple lipid vacuoles (a), H&E × 100). Lipid vacuoles are better visualized (arrows) in sections stained with PASD, despite their negativity to the stain. (b), PASD × 100). The eosinophilic PASD negative inclusions react positively (red color) to an anti-fibrinogen antibody (c), ×100). The lipid droplets inside the fibrinogen inclusions (arrows) are immunoreactive (brown color) to an anti-APO-B-lipoprotein antibody (d), ×100). Double immunostaining on the same section shows the co-localization of the two proteins (fibrinogen in red, APO-B- in brown marked by arrows (e), ×100). The EM picture shows lipid droplets within fingerprint-like fibrinogen aggregates. (f), EM × 10,000).

Figure 4

The 3D modelling shows the locations of the eight fibrinogen gamma chain mutations in the globular D-domain of the monomers that hamper the dimerization.

Figure 5

(a) The upper part shows the dimer fragment D (gamma chains only), covalently linked. The lower part shows the interaction sites (D-domain) of each monomer where three mutations (brescia, aguadilla, and ankara) are located. The mutations prevent interaction and hamper the D:D formation. (b) The failure of a correct polymerization leaves exposed hydrophobic patches in each monomeric mutant gamma chains which give rise to undue interaction with lipids and with hydrophobic regions of APO-B-lipoproteins and other lipids.