Intestinal Epithelial Serum Amyloid A Modulates Bacterial Growth In Vitro and Pro-Inflammatory Responses in Mouse Experimental Colitis

<p>Abstract</p> <p>Background</p> <p>Serum Amyloid A (SAA) is a major acute phase protein of unknown function. SAA is mostly expressed in the liver, but also in other tissues including the intestinal epithelium. SAA reportedly has anti-bacterial effects, and because inflammatory bowel diseases (IBD) result from a breakdown in homeostatic interactions between intestinal epithelia and bacteria, we hypothesized that SAA is protective during experimental colitis.</p> <p>Methods</p> <p>Intestinal SAA expression was measured in mouse and human samples. Dextran sodium sulfate (DSS) colitis was induced in SAA 1/2 double knockout (DKO) mice and in wildtype controls. Anti-bacterial effects of SAA1/2 were tested in intestinal epithelial cell lines transduced with adenoviral vectors encoding the CE/J SAA isoform or control vectors prior to exposure to live <it>Escherichia coli</it>.</p> <p>Results</p> <p>Significant levels of SAA1/SAA2 RNA and SAA protein were detected by in situ hybridization and immunohistochemistry in mouse colonic epithelium. SAA3 expression was weaker, but similarly distributed. SAA1/2 RNA was present in the ileum and colon of conventional mice and in the colon of germfree mice. Expression of SAA3 was strongly regulated by bacterial lipopolysaccharides in cultured epithelial cell lines, whereas SAA1/2 expression was constitutive and not LPS inducible. Overexpression of SAA1/2 in cultured epithelial cell lines reduced the viability of co-cultured <it>E. coli</it>. This might partially explain the observed increase in susceptibility of DKO mice to DSS colitis. SAA1/2 expression was increased in colon samples obtained from Crohn's Disease patients compared to controls.</p> <p>Conclusions</p> <p>Intestinal epithelial SAA displays bactericidal properties in vitro and could play a protective role in experimental mouse colitis. Altered expression of SAA in intestinal biopsies from Crohn's Disease patients suggests that SAA is involved in the disease process..</p

The Contribution of SAA1 Polymorphisms to Familial Mediterranean Fever Susceptibility in the Japanese Population

Background/Aims: Familial Mediterranean Fever (FMF) has traditionally been considered to be an autosomal-recessive disease, however, it has been observed that substantial numbers of patients with FMF possess only 1 demonstrable MEFV mutation. The clinical profile of familial Mediterranean fever (FMF) may be influenced by MEFV allelic heterogeneity and other genetic and/or environmental factors. Methodology/Principal Findings: In view of the inflammatory nature of FMF, we investigated whether serum amyloid A (SAA) and interleukin-1 beta (IL-1β) gene polymorphisms may affect the susceptibility of Japanese patients with FMF. The genotypes of the -13C/T SNP in the 5′-flanking region of the SAA1 gene and the two SNPs within exon 3 of SAA1 (2995C/T and 3010C/T polymorphisms) were determined in 83 Japanese patients with FMF and 200 healthy controls. The same samples were genotyped for IL-1β-511 (C/T) and IL-1 receptor antagonist (IL-1Ra) variable number of tandem repeat (VNTR) polymorphisms. There were no significant differences between FMF patients and healthy subjects in the genotypic distribution of IL-1β -511 (C/T), IL-1Ra VNTR and SAA2 polymorphisms. The frequencies of SAA1.1 allele were significantly lower (21.7% versus 34.0%), and inversely the frequencies of SAA1.3 allele were higher (48.8% versus 37.5%) in FMF patients compared with healthy subjects. The frequency of -13T alleles, associated with the SAA1.3 allele in the Japanese population, was significantly higher (56.0% versus 41.0%, p = 0.001) in FMF patients compared with healthy subjects. Conclusions/Significance: Our data indicate that SAA1 gene polymorphisms, consisting of -13T/C SNP in the 5′-flanking region and SNPs within exon 3 (2995C/T and 3010C/T polymorphisms) of SAA1 gene, are associated with susceptibility to FMF in the Japanese population

Monoclonal hybridoma antibodies to human amyloid related protein SAA.

Problems concerning isolation and characterization of the amyloid related serum protein SAA in a pure form prompted us to make monoclonal antibodies to the protein. Protein SAA isolated by gel filtration under dissociating conditions was used for immunization of BALB/c mice, and spleen cells from a mouse producing high titred antiserum to SAA were fused with cells from the mouse plasmacytoma line P3U1. Antibody specificity to various preparations of protein SAA was tested using an indirect enzyme-linked immunosorbent assay. Monoclonal antibodies with specificity for SAA were obtained in addition to antibodies which reacted with both SAA and the related amyloid protein AA. Antibodies specific for one of the apoC proteins of the lipoprotein fraction were also produced showing that the SAA preparation used for immunization was contaminated with apoC proteins

Serum amyloid A gene expression in rabbit, mink and mouse

The expression of serum amyloid A (SAA) protein, a major acute-phase reactant in most species, was examined by in situ hybridization in multiple organs of rabbit, mink and mouse. In livers of unstimulated mice and rabbits a heterogeneous pattern of SAA expression in hepatocytes was observed. In all three species, lipopolysaccharide (LPS) administration resulted in extensive uniform hybridization of SAA probes to hepatocytes and in the rabbit SAA transcripts were detected in cells in the white pulp of the spleen, the adrenal cortex and ovary as well as in the mucosa and lymphatic vessels of the small intestine. Examination of hybridizing SAA signals in the rabbit myocardium showed a speckled distribution in myocytes. The rabbit endocardium was strongly positive, and in the kidney rabbit SAA mRNA was mainly confined to epithelial cells of the proximal and distal convoluted tubules. In the unstimulated mouse, SAA mRNA was detected in the liver and epithelial cells of the small and large intestine. After stimulation of an acute-phase response with LPS a strong response was seen in these organs as well as in the convoluted tubules of the kidney. In extrahepatic organs of the mink, no SAA mRNA was detectable in unstimulated animals, while the convoluted tubules of the kidney and uterine endometrium were strongly positive after systemic LPS injection

Mink serum amyloid-a protein - expression and primary structure of amyloidogenic and nonamyloidogenic isotypes

When Northern blot analysis of LPS stimulated mink liver RNA was performed using a SAA specific probe, several signals were found. Nucleotide sequence analysis of two clones from a cDNA library was performed. Deduced amino acid sequence of the longer one, SAA1 (776 bp), indicates that it represents a SAA isotype found only in serum, while the shorter one, SAA2 (552 bp), corresponds to an isotype found both in serum and degraded to protein AA in secondary amyloid. These results show that the situation in mink is the same as in mice, where only one out of at least two SAA isotypes has been shown to be amyloidogenic. Northern blot analysis also showed that almost all SAA mRNA was found in the liver, while only minor amounts of SAA1 and SAA2 mRNA was found in the brain and the lungs. No signals could be detected in amyloid prone organs like intestine and spleen. In the liver a SAA specific signal was found corresponding to 2.2 kb, and after in vitro translation a protein SAA band corresponding to 28,000 Daltons was detected, indicating that a SAA isotype with high molecular weight exists in this animal.4 page(s