Hiroo Kawano

Ultrastructural evidence for intracellular formation of amyloid fibrils in macrophages

Early amyloid deposition in the spleen was studied by immunoelectron microscopy following the administration of rapid amyloid-inducing agents to mice. Two days after the injection of an amyloidenhancing factor and casein solution, a small amount of amyloid material was observed at the border of the white pulp and the marginal zone (perifollicular area) and also within the white pulp. At this stage, amyloid fibrils were seen mainly in an extracellular distribution along the cytoplasmic processes of reticular cells and also in the cytoplasmic invaginations of macrophages. By immunoelectron microscopy, gold particles labelled fibrillar structures in lysosome-derived organelles in some macrophages as well as dense bodies consisted of a homogeneous, granular matrix not having any recognizable fibrillar structures. Similar immunolabelled organelles were also observed in the amyloid resorption stage, although, at that stage, they commonly contained other phagocytized materials as well. From these findings, we suggest that at least some amyloid fibrils are polymerized in the cytoplasm of the macrophages by the proteolytic cleavage of previously pinocytized serum amyloid A protein (SAA).

Useful polyclonal antibodies against synthetic peptides corresponding to immunoglobulin light chain constant region for immunohistochemical detection of immunoglobulin light chain amyloidosis

For the immunohistochemical detection of immunoglobulin (Ig) light chain amyloidosis on formalin‐fixed, paraffin‐embedded tissue sections, we prepared polyclonal antibodies against synthetic peptides corresponding to positions 118–134 of Ig λ light chain and positions 116–133 of Ig κ light chain. Nineteen cases of systemic Ig λ light chain amyloidosis (Aλ amyloidosis), 10 cases of systemic Ig κ light chain amyloidosis (Aκ amyloidosis), one case of immunohistochemically unclassified systemic amyloidosis and five cases of localized Aλ amyloidosis were tested with these antibodies. Anti‐λ (118–134) antiserum and the affinity‐purified antibody both reacted with 18 of the 19 cases of systemic Aλ amyloidosis and all cases of localized Aλ amyloidosis, although the immunoexpression was somewhat variable in intensity in different areas within the same specimen in both systemic and localized amyloidosis. The signal intensities in plasma cells and serum reacted for anti‐λ (118–134) antiserum were weaker than signals obtained with commercially available anti‐Ig λ light chain antibodies. Anti‐κ (116–133) antiserum and the affinity‐purified antibody reacted with nine of the 10 cases of systemic Aκ amyloidosis. We conclude that these antibodies against synthetic peptides corresponding to the Ig light chain constant region are useful for the classification of amyloidosis on formalin‐fixed, paraffin‐embedded tissue sections.

Acceleration of murine AA amyloidosis by oral administration of amyloid fibrils extracted from different species.

We herein report that experimental murine amyloid A (AA) deposition is accelerated by oral administration of semipurified amyloid fibrils extracted from different species. Three groups of mice were treated with semipurified murine AA amyloid fibrils, semipurified bovine AA amyloid fibrils or semipurified human light chain‐derived (Aλ) amyloid fibrils for 10 days. After 3 weeks, each mouse was subjected to inflammatory stimulation by subcutaneous injection with a mixture of complete Freund’s adjuvant supplemented with Mycobacterium butyricum. The mice were killed on the third day after the inflammatory stimulation, and the spleen, liver, kidney and gastrointestinal tract were examined for amyloid deposits. Amyloid deposits were detected in 14 out of 15 mice treated with murine AA amyloid fibrils, 12 out of 15 mice treated with bovine AA amyloid fibrils and 11 out of 15 mice treated with human Aλ amyloid fibrils. No amyloid deposits were detected in control mice receiving the inflammatory stimulant alone or in amyloid fibril‐treated mice without inflammatory stimulation. Our results suggest that AA amyloid deposition is accelerated by oral administration of semipurified amyloid fibrils when there is a concurrent inflammatory stimulation.

Induction of Murine AA Amyloidosis by Various Homogeneous Amyloid Fibrils and Amyloid-like Synthetic Peptides

We investigated amyloid‐enhancing factor (AEF) activity of amyloid fibrils extracted from amyloid‐laden livers of mice, cow, cheetah, cat and swan. All amyloid fibrils were confirmed to be amyloid protein A (AA) by an immunohistochemical analysis. We found that these fibrils accelerated the deposition of amyloid in an experimental mouse model of AA amyloidosis. Furthermore, the degree of deposition was dependent on the concentration of fibrils. When we compared the minimal concentration of amyloid fibrils needed to induce deposition, we found that these fibrils showed different efficiencies. Murine amyloid fibril induced amyloid deposition more efficiently than cow, cat, cheetah or swan amyloid fibrils. These data suggest that amyloid deposition is preferentially induced by amyloid fibrils with the same primary sequence as the endogenous amyloid protein. We then analysed the AEF activity of synthetic peptides, synthesized corresponding to amino acids 1–15 of mouse SAA (mSAA), 2–15 of cow SAA (bSAA), 1–15 of cat SAA (cSAA), which was the same as cheetah, and the common amino acids 33–45 of these four SAA (aSAA). We found that mSAA, bSAA and cSAA formed amyloid‐like fibrils in morphology and showed similar AEF properties to those of native amyloid fibrils. Although aSAA also formed highly ordered amyloid‐like fibrils, it showed weaker AEF activity than the other synthetic fibrils. Our results indicate that amyloidosis is transmissible between species under certain conditions; however, the efficiency of amyloid deposition is species‐specific and appears to be related to the primary amino acid sequence, especially the N‐terminal segment of the amyloid protein.

Acceleration of murine AA amyloid deposition by bovine amyloid fibrils and tissue homogenates.

Acceleration of amyloid deposition by administration of amyloid fibrils and transmissibility of disease have been reported for several types of amyloidosis. Reactive amyloidosis (AA) occurs in a wide variety of domestic animal species and is characterized by amyloid deposition mainly in spleen, liver, and kidneys. Because the visceral organs of domestic animals have traditionally been used in Asian cuisines, it is important to examine whether dietary ingestion of the organs themselves (rather than purified amyloid fibrils) accelerates AA amyloid deposition. Herein, we show that murine AA amyloidosis develops rapidly after intraperitoneal or oral administration of purified amyloid fibrils or homogenates of amyloid-laden bovine liver. The amyloidosis development in mice was dependent on the concentration of amyloid fibrils or amyloidotic liver homogenates. We found that experimental murine AA amyloidosis was accelerated by dietary ingestion of both purified amyloid fibrils and tissue homogenates that contain amyloid fibrils. We also investigated livers of beef cattle and food chickens to examine whether they contain amyloid-enhancing factor activity. By microscopic examination of hematoxylin and eosin- and Congo red-stained sections, no amyloid deposition was detected in these livers, and no effective activity for experimental induction of AA amyloidosis in mice was detected in homogenates of these livers.

Hepatic amyloidosis in Japan: Histological and morphometric analysis based on amyloid proteins

To investigate the relationship between the tissue distribution and the types of amyloid proteins, the detailed histopathologic features of the available liver in 284 cases of amyloidosis were examined. We classified hepatic amyloidosis into three types, namely, the vascular pattern, parenchymal pattern, and stromal pattern according to the topographic distribution pattern of amyloid. Of the 152 amyloid A (AA) cases, all but one exhibited the vascular pattern; the single exception had the parenchymal pattern. Among 117 amyloid light chain (AL) cases, 51.3% exhibited the vascular pattern and 43.6% the parenchymal pattern. The stromal pattern was observed in 5.1% of the cases but was found only in AL amyloidosis. The parenchymal and stromal patterns in the liver seemed to be characteristic morphological distributions of AL amyloidosis. Routine histochemical study is useful to distinguish AL from AA, although some ethnic differences were apparent. Morphometric results showed that the walls of the hepatic arteries with amyloid deposition were significantly thicker than walls in arteries from the control group. The arterial walls in AA amyloidosis, especially, were significantly thicker than walls in AL amyloidosis of any pattern.

ADENOCARCINOMA DEVELOPING IN AN ILEAL CONDUIT

We report a case of adenocarcinoma that developed in an ileal conduit 18 years after cystectomy for transitional cell carcinoma of the bladder. The only presenting sign was gross hematuria. Retrograde ileal loopography and endoscopy of the ileal loop were useful in the diagnosis. To our knowledge adenocarcinoma arising in an ileal conduit after a radical procedure for transitional cell carcinoma of the bladder has not been reported previously. The literature regarding malignancies that arise in portions of intestinal segments used as conduits is reviewed and carcinogenesis is discussed.

Carcinosarcoma of the gallbladder. A case report with immunohistochemical and ultrastructural studies

The authors present the histologic features, immunohistochemical findings, and ultrastructure of a carcinosarcoma of the gallbladder containing rhabdomyosarcoma as a mesenchymal element. A pedunculated polypoid tumor protruded into the lumen from the fundus of the gallbladder. The neoplasm contained two divergent components. One was malignant mesenchymal tissue with rhabdomyoblastic differentiation; the other was ordinary adenocarcinoma which was observed predominantly at the base of the polyp. Immunohistochemically, the cytoplasm of the rhabdomyoblasts stained with anti‐myoglobin, myosin, and muscle actin antibodies. Ultrastructurally, there were a large number of malignant mesenchymal tissues in which various stages of differentiated rhabdomyoblasts were noted. Ultrastructural study was particularly valuable for the identification of sarcomatous elements.

Immunohistochemical study of immunoglobulin light chain amyloidosis with antibodies to the immunoglobulin light chain variable region

To detect immunoglobulin (Ig) light chain amyloidosis (AL amyloidosis) in formalin‐fixed, paraffin‐embedded tissue sections by immunohistochemistry, polyclonal antibodies were generated against synthetic peptides corresponding to amino acids 1–19 of the Ig λ light chain V λ VI subgroup (anti‐V λ VI (1–19)) and the Ig κ light chain Vκ I subgroup (anti‐Vκ I (1–19)). Anti‐V λ VI (1–19) antibody reacted with amyloid deposits in 21 of 22 Aλ amyloidosis cases, and anti‐Vκ I (1–19) antibody reacted with amyloid deposits in 10 of 11 Aκ amyloidosis cases. Immunoreactivity varied in intensity by case and within specimens. Surprisingly, amyloid deposits were positive for anti‐V κ I (1–19) staining in one case of Aλ amyloidosis. Analysis of anti‐V λ VI (1–19) and anti‐Vκ I (1–19) antibody reactivity by ELISA showed some cross‐reactivity with peptides other than antigen peptides. The antibodies were not reactive in all cases of AL amyloidosis examined but may be useful, together with anti‐Ig constant region antibodies, for immunohistochemical diagnosis of AL amyloidosis.

IMMUNOLOGICAL HOMOGENEITY OF LAFORA BODY, CORPORA AMYLACEA, BASOPHILIC DEGENERATION IN HEART, and INTRACYTOPLASMIC INCLUSIONS OF LIVER and HEART IN TYPE IV GLYCOGENOSIS

Antisera against Lafora bodies were made in rabbits by subcutaneous injection of the myocardium from a patient died of Lafora disease. Using this antisera, immunohistochemistry revealed that corpora amylacea, the basophilic degeneration of myocardium and deposits of type IV glycogenosis contained the materials which were antigenically common to Lafora bodies.