Peter C.J. Tooten

Immunohistochemical investigation of the brain of aged dogs. I. Detection of neurofibrillary tangles and of 4-hydroxynonenal protein, an oxidative damage product, in senile plaques.

In the aging dog brain lesions develop spontaneously. They share some morphological characteristics with those of Alzheimers disease in man. Diffuse and primitive plaques are well known, whereas neuritic plaques rarely develop. Neurofibrillary tangles have not been seen in the canine. The aim of the present investigation was to study major age-related changes of the dogs brain using paraffin sections with respect to cross-immunoreactivity oftau, Aβ protein and other immunoreactive components including hydroxynonenal protein, which is a marker for oxidative damage. The occurrence of neurofibrillary tangles and of the protein tau therein was studied in serial brain sections of two dogs with the Gallyas stain and by immunohistochem-istry with three different antibodies against tau. Senile plaques were stained with a monoclonal anti-Aβ (residues 8-17), polyclonal anti-apolipoprotein E and a monoclonal antibody against 4-hydroxynonenal (HNE). Amyloid deposits and controls were screened by Congo red staining viewed in fluorescent light, followed by polarized light for green birefringence. With the Gallyas stain and one of the antisera against tau, neurofibrillary tangles were revealed in a similar dispersed pattern, whereas the other antitau antisera gave negative results. With the anti-HNE a positive reaction was found in cerebral amyloid deposits and in vascular wall areas where amyloid deposition was confirmed by Congo-red staining, and in perivascular cells and in some neurons. These results indicate that the canine with his tangles and plaques which show oxidative changes, forms a spontaneous model for understanding the early changes and their interrelationships in Alzheimers disease.

E2F8 is essential for polyploidization in mammalian cells

Polyploidization is observed in all mammalian species and is a characteristic feature of hepatocytes, but its molecular mechanism and biological significance are unknown. Hepatocyte polyploidization in rodents occurs through incomplete cytokinesis, starts after weaning and increases with age. Here, we show in mice that atypical E2F8 is induced after weaning and required for hepatocyte binucleation and polyploidization. A deficiency in E2f8 led to an increase in the expression level of E2F target genes promoting cytokinesis and thereby preventing polyploidization. In contrast, loss of E2f1 enhanced polyploidization and suppressed the polyploidization defect of hepatocytes deficient for atypical E2Fs. In addition, E2F8 and E2F1 were found on the same subset of target promoters. Contrary to the long-standing hypothesis that polyploidization indicates terminal differentiation and senescence, we show that prevention of polyploidization through inactivation of atypical E2Fs has, surprisingly, no impact on liver differentiation, zonation, metabolism and regeneration. Together, these results identify E2F8 as a repressor and E2F1 as an activator of a transcriptional network controlling polyploidization in mammalian cells.

Anti-inflammatory properties of heat shock protein 70 and butyrate on Salmonella-induced interleukin-8 secretion in enterocyte-like Caco-2 cells.

Intestinal epithelial cells secrete the chemokine interleukin (IL)‐8 in the course of inflammation. Because heat shock proteins (Hsps) and butyrate confer protection to enterocytes, we investigated whether they modulate Salmonella enterica serovar Enteritidis (S. serovar Enteritidis)‐induced secretion of IL‐8 in enterocyte‐like Caco‐2 cells. Caco‐2 cells incubated with or without butyrate (0–20 m M, 48 h) were infected with S. serovar Enteritidis after (1 h at 42°C, 6 h at 37°C) or without prior heat shock (37°C). Levels of Hsp70 production and IL‐8 secretion were analysed using immunostaining of Western blots and enzyme‐linked immunosorbent assay (ELISA), respectively. The cells secreted IL‐8 in response to S. serovar Enteritidis and produced Hsp70 after heat shock or incubation with butyrate. The IL‐8 secretion was inhibited by heat shock and butyrate concentrations as low as 0·2 m M for crypt‐like and 1 m M for villous‐like cells. In a dose‐dependent manner, higher butyrate concentrations enhanced IL‐8 secretion to maximal levels followed by a gradual but stable decline. This decline was associated with increasing production of Hsp70 and was more vivid in crypt‐like cells. In addition, the higher concentrations abolished the heat shock inhibitory effect. Instead, they promoted the IL‐8 production in heat‐shocked cells even in the absence of S. serovar Enteritidis. We conclude that heat shock and low concentrations of butyrate inhibit IL‐8 production by Caco‐2 cells exposed to S. serovar Enteritidis. Higher butyrate concentrations stimulate the chemokine production and override the inhibitory effect of the heat shock. The IL‐8 down‐regulation could in part be mediated via production of Hsp70.

Generalized AA-amyloidosis in Siamese and Oriental cats

During a 7 year period (1987-1994), 194 Siamese cats including a colour variant designated Oriental cat, were presented for post-mortem examination. Twelve of these animals (6.2%) were diagnosed with amyloidosis. Major gross pathological findings included enlarged pale livers with haemorrhages, pale and swollen spleens, and dilated intestines. Deposits of amyloid were found in these tissues. The amyloid was found to cross-react with anti dog AA-antiserum when examined with peroxidase antiperoxidase (PAP) staining (four cases). Amyloid fibrils were purified by the water extraction method and its major constituting protein (AA) was isolated by gel filtration. Amino acid sequence analysis of this protein from a Siamese cat and an Abyssinian cat revealed a significant difference between these breeds. In the Siamese protein AA two amino acid substitutions (46 R for Q and 52 V for A) were encountered. This finding indicates the existence of a new feline amyloid A protein occurring in the Siamese breed which differs from presently known (apoS)AA-proteins. Additionally, the pedigree analysis of affected cats suggests a familial trait.

First Evidence for the Existence of Multiple Isoforms of Bovine Serum Amyloid‐A (apoSAA)

Bovine serum amyloid‐A (SAA) was purified from acute‐phase high density iipoprotein (HDL) by affinity chromatography and subsequent gel filtration chromatography. The identity of the isolated protein was checked by Western blotting following SDS‐urea‐PAGE using antisera raised against the purified protein fraction (SAA) and Amyloid A (AA). The antiserum raised against the purified SAA stained Congo red positive regions in the kidney of an AA‐amyloidotic cow and reacted on Western blot with an AA‐related protein of approximately 14kDa. Moreover, it immunostained two to three bands, of approximately 14kDa, present in serum from diseased cows, proportionally to the serum SAA concentration as measured by ELISA.

Induction of amyloid arthropathy in chickens

The present report describes a novel chicken model in which reactive (AA) joint amyloidosis arises ajier a single injection of Enterococcus faecalis isolated from jield outbreaks of reactive amyloid arthropathy in domestic fowl. All six week old brown layer pullets injected intravenously with 109 or 109 colony forming units developed reactive amyloid arthropathy. Amyloid masses were also present in internal organs. The first articular amyloid deposits were observed 5 days after injection. in internal organs, deposits were encountered fiom day 13 onwards. on intra-articular injection, amyloid arthropathy developed particularly in Ihe target joint and in the internal organs.This model ofAA amyloid arthropathy may contribute in future studies to unravel the pathogenesis of the tissue predilection of amyloid deposition and 05 amyloid arthropathy in general.

Chicken joint amyloid protein is of the AA-type. I. Characterization of the amyloid protein.

Amyloid fibrils were extracted from deposits in joint tissue of heavy breed layers with spontaneous amyloid arthropathy and characterized as being of the AA‐type. Amino acid sequencing revealed a pattern quite similar to duck AA. Acute phase sera of chicken experimentally injected with Enterococcus faecalis showed a SAA‐protein like band cross reacting with anti‐chicken AA in immunoblot.

Casein related amyloid, characterization of a new and unique amyloid protein isolated from bovine corpora amylacea

Amyloid bodies can be found in mammary secretory tissue of various species. These corpora amylacea (CA) have a lamellated structure, contain amyloid fibrils and are predominantly located in the alveolar lumina. The nature of the amyloid was not known, but CA were suggested to originate either from milk casein or mammary alveolar epithelial keratin. In the present report, bovine CA were analyzed histochemically. Furthermore, CA were isolated, analyzed and the amyloid was purified and characterized by amino acid sequencing. CA amyloid appeared to be potassium permanganate sensitive and tryptophan positive, and in this respect different from most other amyloid types except for AA and beta-2 microglobulin amyloid. Gel filtration of purified amyloid fibrils showed a HMW peak and a major 4 kD peak. N-terminal amino acid sequencing showed the amyloid to consist of tryptic-like peptides with an unusually high content of amino acids with bulky side chains. The amyloid protein was identified as derived from alpha-S2-casein. The fragments are of varying length (32, 33 and 45 amino acids), but all start at position 81 of alpha-S2-casein. We have identified a new and unique amyloid protein, and we propose to designate it as A alpha-S2C according to the guidelines for amyloid nomenclature.

Leukocyte responses in two breeds of layer chicken that differ in susceptibility to induced amyloid arthropathy

Amyloid arthropathy in chicken can be induced by intravenous inoculation of an arthropathic and amyloidogenic Enterococcus faecalis in susceptible breeds. The commercial brown layer hybrids (BL) are more susceptible to the disease compared to their white counterparts (WL). The precursor of amyloid-A protein, which is serum amyloid-A (SAA), is identical in WL and BL. To investigate the factors involved in the breed-restricted susceptibility to amyloid arthropathy, we studied the type of leukocyte response and inflammatory reactions in E. faecalis-induced disease. In the BL, a significant dose dependent peripheral leukocytosis mainly by heterophils, and plasma cell infiltration in arthritic joints was found. In contrast, secondary lymphoid nodular aggregates in the synovial membrane were prominent in the WL. The aggregates consisted mainly of CD8+ T cells. The high number of circulating leukocyte and prolific plasma cell responses in the BL predict extensive humoral and acute phase reactions. This is in agreement with literature data on suppressed T-cell function in casein-induced amyloid-susceptible mice strains. The difference in leukocyte response and type of inflammation between WL and BL, when arthropathic and amyloidogenic bacteria induce infection, in conjunction with susceptibility to amyloid arthropathy, is discussed in view of the murine T-helper responses.

Characterization of proteoglycans and glycosaminoglycans in bovine renal AA-type amyloidosis.

SummaryHighly sulfated glycosaminoglycans (GAG) or proteoglycans (PG), especially heparan sulfate (HS) and heparan sulfate proteoglycan (HSPG), are considered to be intimately associated with amyloid deposits in different types of amyloidosis. Based on this relationship an important role for HS has been suggested in amyloidogenesis. The present immunohistological and ultrastructural study shows that in bovine renal AAamyloidosis, sulfated GAG/PG was not restricted to amyloid deposits proper and that areas without GAP/ PG were also present within the amyloid. Both glomerular and papillary amyloid contained HS (PG), and the latter also contained chondroitin sulfate (CS) and dermatan sulfate (DS), suggesting a correlation between the location of the amyloid and the type of GAG/PG deposited. Amyloid P component (AP) had a distribution similar to that of HSPG, confirming their affinity-based relationship.The GAG types found ultrastructurally in amyloid fibril preparations of glomerular and papillary amyloid isolated from the same kidney, reflected the immunohistological findings. HS was shown to be the predominant GAG in all papillary amyloid fibril extracts. Taking into account the chemico-physical properties of HS, it cannot be excluded that this predominance is introduced by the purification procedure. These results suggest that the association of GAG/PG and amyloid is not necessarily mutually obligatory and that the proposed importance of GAG in amyloidogenesis is disputable.