Simcha Urieli-Shoval

Expression and function of serum amyloid A, a major acute-phase protein, in normal and disease states.

Serum amyloid A (SAA), the precursor protein in inflammation-associated reactive amyloidosis (AA-type), is an acute phase reactant whose level in the blood increases in response to various insults. It is expressed in the liver, but its physiological role is not well understood. Recently, a broader view of SAA expression and function has been emerging. Expression studies show local production of SAA proteins in histologically normal, atherosclerotic, Alzheimer, inflammatory, and tumor tissues. Binding sites in the SAA protein for high density lipoproteins, calcium, laminin, and heparin/heparan-sulfate were described. Adhesion motifs were identified and new functions, affecting cell adhesion, migration, proliferation and aggregation have been described. These findings emphasize the importance of SAA in various physiological and pathological processes, including inflammation, atherosclerosis, thrombosis, AA-amyloidosis, rheumatoid arthritis, and neoplasia. In addition, recent experiments suggest that SAA may play a “housekeeping” role in normal human tissues.

Widespread Expression of Serum Amyloid A in Histologically Normal Human Tissues: Predominant Localization to the Epithelium

Serum amyloid A (SAA) is an acute-phase reactant whose level in the blood is elevated to 1000-fold as part of the bodys responses to various injuries, including trauma, infection, inflammation, and neoplasia. As an acute-phase reactant, the liver has been considered to be the primary site of expression. However, limited extrahepatic SAA expression was described in mouse tissues and in cells of human atherosclerotic lesions. Here we describe nonradioactive in situ hybridization experiments revealing that the SAA mRNA is widely expressed in many histologically normal human tissues. Expression was localized predominantly to the epithelial components of a variety of tissues, including breast, stomach, small and large intestine, prostate, lung, pancreas, kidney, tonsil, thyroid, pituitary, placenta, skin epidermis, and brain neurons. Expression was also observed in lymphocytes, plasma cells, and endothelial cells. RT-PCR analysis of selected tissues revealed expression of the SAA1, SAA2, and SAA4 genes but not of SAA3, consistent with expression of these genes in the liver. Immunohistochemical staining revealed SAA protein expression that colocalized with SAA mRNA expression. These data indicate local production of the SAA proteins in histologically normal human extrahepatic tissues.

The absence of detectable methylated bases in Drosophila melanogaster DNA

Most of the eukaryotic organisms are methylated at specific cytosine residues in their DNA. For more than 2 decades efforts have been made to answer the question of whether the DNA of the fruit fly Drosophila melanogaster is methylated, but the results were inconclusive. The answer to this question is now attracting special interest in light of the fact that in recent years substantial evidence has accumulated, suggesting a correlation between vertebrate DNA methylation and gene expression [1]. Drosophila in particular is an interesting organism in this respect as it goes through severabdefined developmental stages and its genome organization has been extensively investigated. Here, a variety of highly sensitive methods have been used to analyze methylated bases in Drosophila melanogaster DNA. 5-Methyl-cytosine, which is the common methylated base in DNA of eukaryotic organisms, could not be detected in any of the developmental stages of this organism. There is no indication for other modifications of this DNA as well. The welldefined clonally inherited patterns of methylation of the genetic material in mammals [1] and the absence of such methylation patterns in Drosophila DNA suggested here, bring into focus the longstanding enigma of the biological role played by methylation patterns. 2.1. Preparation of DNA DNA of Drosophila melanogaster embryos, larvae, pupae and adults was prepared from isolated nuclei. The nuclei were lysed in 1 mM Tris (pH 8) and after centrifugation at 10 000 x g the chromatin pellet was suspended in a lysis mixture containing 0.5% (w/v) sodium lauryl sulphate; 2.5 mM EDTA; 0.5 M NaC1; 10 mM Tris (pH 8) and 100 ~g proteinase K/ml (Merck Co.). The mixture was incubated for 2 h at 37°C and RNase treated (300/~g pancreatic RNase/ml for l h at 37°C). Phenol extraction was followed by chloroform:isoamyl alcohol (24: 1, v/v) extraction and ethanol precipitation. The DNA was hydrolyzed to free bases for analysis as in [2].

Expression of serum amyloid A, in normal, dysplastic, and neoplastic human colonic mucosa: implication for a role in colonic tumorigenesis.

Serum amyloid A (SAA) is an acute phase reactant, whose level in the blood is elevated in response to trauma, infection, inflammation, and neoplasia. Elevated levels of SAA in the serum of cancer patients were suggested to be of liver origin rather than a tumor cell product. The role of SAA in human malignancies has not been elucidated. We investigated the expression of SAA at various stages of human colon carcinoma progression. Nonradioactive in situ hybridization applied on paraffin tissue sections from 26 colon cancer patients revealed barely detected SAA mRNA expression in normal looking colonic epithelium. Expression was increased gradually as epithelial cells progressed through dysplasia to neoplasia. Deeply invading colon carcinoma cells showed the highest levels of SAA. Expression was also found in colon carcinoma metastases. Cells of lymphoid follicles of the intestinal wall, inflammatory cells, ganglion cells, and endothelial cells, also expressed SAA mRNA. Immunohistochemical staining revealed SAA protein expression that colocalized with SAA mRNA expression. RT-PCR analysis confirmed the expression of the SAA1 and SAA4 genes in colon carcinomas, expression that was barely detectable in normal colon tissues. These findings indicate local and differential expression of SAA in human colon cancer tissues and suggest its role in colonic tumorigenesis.

Expression of Serum Amyloid A in Human Ovarian Epithelial Tumors: Implication for a Role in Ovarian Tumorigenesis:

Serum amyloid A (SAA) is an acute phase protein which is expressed primarily in the liver as a part of the systemic response to various injuries and inflammatory stimuli; its expression in ovarian tumors has not been described. Here, we investigated the expression of SAA in human benign and malignant ovarian epithelial tumors. Non-radioactive in situ hybridization applied on ovarian paraffin tissue sections revealed mostly negative SAA mRNA expression in normal surface epithelium. Expression was increased gradually as epithelial cells progressed through benign and borderline adenomas to primary and metastatic adenocarcinomas. Similar expression pattern of the SAA protein was observed by immunohistochemical staining. RT-PCR analysis confirmed the overexpression of the SAA1 and SAA4 genes in ovarian carcinomas compared with normal ovarian tissues. In addition, strong expression of SAA mRNA and protein was found in the ovarian carcinoma cell line OVCAR-3. Finally, patients with ovarian carcinoma had high SAA serum levels, which strongly correlated with high levels of CA-125 and C-reactive protein. Enhanced expression of SAA in ovarian carcinomas may play a role in ovarian tumorigenesis and may have therapeutic application.

Serum amyloid A enhances plasminogen activation : Implication for a role in colon cancer

We have recently reported that the acute phase protein serum amyloid A (SAA), is locally and differentially expressed in neoplastic tissues of human colon. In the present study, we demonstrate that SAA enhances the plasminogen activation (PA)-activity of HT-29 colon cancer cell line. Cell-associated PA-activity was measured following the plasminogen-dependent ability of the cells to cleave the chromogenic substrate S-2251. The SAA-enhanced PA-activity was inhibited by anti-SAA antibodies. These antibodies also decreased the basal PA-activity of HT-29 cells and neutralized their cytokines (Interleukin-1beta+Interleukin-6)-enhanced PA-activity. Using specific chromogenic substrates and the fibrin clot-lysis assay, we found that SAA enhances also the PA-activity mediated by purified urokinase- and tissue-type plasminogen activators. Together, the data indicate that SAA enhances plasminogen activation and suggest its possible role in plasmin(ogen)-mediated colon cancer progression.

Analysis of adenoviral attachment to human platelets

BackgroundSystemic adenoviral (Ad) vector administration is associated with thrombocytopenia. Recently, Ad interaction with mouse platelets emerged as a key player determining liver uptake and platelet clearance. However, whether Ad can activate platelets is controversial. Thus, in vitro analysis of Ad attachment to platelets is of interest.MethodsWe developed a direct flow cytometry assay to specifically detect Ad particles adherent to human platelets. The method was pre-validated in nucleated cells. Blocking assays were employed to specifically inhibit Ad attachment to platelets. Platelet activation was analyzed using annexin v flow cytometry.ResultsWe found in vitro that Ad binding to human platelets is synergistically enhanced by the combination of platelet activation by thrombin and MnCl2 supplementation. Of note, Ad binding could activate human platelets. Platelets bound Ad displaying an RGD ligand in the fiber knob more efficiently than unmodified Ad. In contrast to a previous report, CAR expression was not detected on human platelets. Integrins appear to mediate Ad binding to platelets, at least partially. Finally, αIIbβ3-deficient platelets from a patient with Glanzmann thrombasthenia could bind Ad 5-fold more efficiently than normal platelets.ConclusionThe flow cytometry methodology developed herein allows the quantitative measurement of Ad attachment to platelets and may provide a useful in vitro approach to investigate Ad interaction with platelets.

The Relationship of Plasminogen Activator Inhibitor-1 Levels to the ST Deviation Pattern of Acute Myocardial Infarction

Objectives: Myocardial infarction (MI) may be classified as ST elevation MI (STEMI) or non-ST elevation MI (NSTEMI). Procoagulants such as plasminogen activator inhibitor-1 (PAI-1) as well as markers of inflammation such as C-reactive protein (CRP), serum amyloid A (SAA) and interleukin-6 (IL-6) are elevated in acute coronary syndromes. However, no study has examined whether levels of these markers differ in patients with STEMI as opposed to NSTEMI. We sought to determine whether there are differences in plasma levels of PAI-1, CRP, SAA or IL-6 in patients with STEMI compared to patients with NSTEMI. Methods: Seventy-six consecutive patients presenting with acute MI (37 with STEMI and 39 with NSTEMI) were prospectively enrolled. Blood samples were obtained from patients within 6 h from presentation and plasma PAI-1, CRP, IL-6 and SAA concentrations were measured. Results: Plasma levels of PAI-1 were significantly higher in patients with STEMI compared to NSTEMI: 85.7 ± 5 vs. 61.3 ± 5 ng/ml (p < 0.001), while CRP, SAA and IL-6 levels were not significantly different between STEMI and NSTEMI patients. Conclusions: Higher plasma PAI-1 levels in STEMI patients may contribute to the predilection of these patients to occlusive thrombi and STEMI.

Serum Amyloid A: Expression Throughout Human Ovarian Folliculogenesis and Levels in Follicular Fluid of Women Undergoing Controlled Ovarian Stimulation

BACKGROUND Serum amyloid A (SAA) is an acute phase protein expressed primarily in the liver in response to various injuries and inflammatory stimuli and is recognized as a modulator of inflammation. Ovarian reproductive functions including folliculogenesis and ovulation use inflammatory processes; thus, studying SAA in this context is of interest. OBJECTIVES We investigated the expression and localization of SAA in ovarian developing follicles and its levels in follicular fluids. METHODS AND PARTICIPANTS Nonradioactive in situ hybridization and immunohistochemical staining were applied on ovarian paraffin tissue sections. ELISA and RT-PCR were applied on follicular aspirates and blood samples from women undergoing controlled ovarian stimulation for in vitro fertilization. RESULTS Expression of SAA mRNA and protein was found in follicular cells at all stages of follicular development, from primordial and primary follicles through antral follicles and corpora lutea. Expression was observed in granulosa, theca and luteal cells, and oocytes. Expression of SAA was also found in granulosa cells recovered from follicular aspirates. The SAA protein was detected in follicular fluids. Its levels were somewhat lower than in peripheral blood with strong correlation between the two compartments and with significant correlation with patients body mass index. High follicular fluid SAA levels were associated with reduced pregnancy rate. CONCLUSIONS SAA is locally produced in ovarian developing follicles and is a constituent of follicular fluids, suggesting its role within the follicular environment. Elevated follicular SAA levels are associated with decreased pregnancy rate and may signify lower reproductive performance.