Wayne A. Gonnerman

Direct binding enzyme-linked immunosorbent assay (ELISA) for serum amyloid A (SAA)

A solid-phase, direct binding ELISA for serum amyloid A (SAA) proteins is described, in which noncovalent interactions of SAA with other plasma constituents are disrupted to permit direct coating of the wells of flexible polyvinyl chloride microtitration plates with an amount of SAA antigen proportional to its concentration in plasma. The wells are coated overnight at 60 degrees C with plasma diluted in 3 M potassium bromide and 0.1 M sodium bicarbonate. pH 9.6. The next day, any remaining sites on the wells are blocked by incubation for 1 h at ambient temperature with a 5% solution of dry milk solids and 0.05% Tween 20 in 0.02 M phosphate buffer, pH 7.4. The wells are rinsed and incubated for 90 min at 37 degrees C with polyclonal rabbit or rat anti-human SAA antiserum. Then, the wells are rinsed and incubated with goat anti-rabbit or rat IgG antiserum to which has been conjugated horseradish peroxidase. o-phenylenediamine and hydrogen peroxide substrates are added to the wells, color is allowed to develop, and sulfuric acid is added to stop the enzyme-catalyzed reaction. The amount of SAA coated to wells is quantified by absorbance at 490 nm. Four or more serial three-fold dilutions of plasma samples are assayed simultaneously on separate plates. Each plate contains a set of wells with identical concentrations of SAA standard protein diluted in decreasing concentrations of plasma proteins corresponding to the dilution of sample. The method can detect SAA concentrations in plasma samples ranging from 1 microgram/ml to greater than 1000 micrograms/ml. The method is suited to serial monitoring of SAA concentration in patients undergoing treatment for inflammatory conditions and to the quantitative analysis of large numbers of samples.

Effect of varying amounts of ascorbate on collagen, elastin and lysyl oxidase synthesis in aortic smooth muscle cell cultures

In the presence of ascorbate, there is an increase in collagen synthesis with a concomitant decrease in insoluble elastin and lysyl oxidase activity in cultured rabbit aortic smooth muscle cells. While the addition of 0.5 micrograms ascorbate per ml of medium enhances collagen synthesis and accumulation, detectable insoluble elastin and lysyl oxidase activity remain essentially unchanged. However, at 2 micrograms ascorbate per ml, the integrity of the insoluble elastin is lost and lysyl oxidase activity is decreased. These studies suggest that by modifying the levels of ascorbate in the culture medium one can alter the nature of the extracellular matrix produced by the smooth muscle cells.

The acute phase response in Syrian hamsters elevates apolipoprotein serum amyloid A (apoSAA) and disrupts lipoprotein metabolism

Lipoprotein metabolism was assessed in hamsters following subcutaneous injection of AgNO3. Apolipoprotein serum amyloid A (apoSAA) peaked at 36 h, followed by elevations in plasma cholesterol at 56 h and triglycerides at 96 h. There was a striking increase in LDL and a de-crease in HDL. Migration of all acute phase (AP) lipoproteins was retarded compared to controls and SDS-PAGE electrophoretic analysis was consistent with agarose gel profiles that revealed increased apoB-rich VLDL, IDL and LDL and decreased apoAl-rich HDL2 fractions. Cholesterol transported by LDL of AgNO3 treated hamsters was double that of controls while the pool of HDL-cholesterol was only two-thirds that of controls. Fasting triglyceride and cholesterol secretion rates were depressed sharply at 24 h. After E. coli lipopolysaccharide (LPS) injection, apoSAA-HDL particles bound avidly to cultured peritoneal macrophages (mos) but in vitro exposure of tissue mos to LPS did not alter the binding characteristics of either control-or apoSAA...

Dietary n-3 fatty acids and arthritis

Abstract. We have evidence that dietary fish oil (FO) decreases severity of collagen‐induced arthritis (CIA), changes the fatty acid composition of macrophage (Mϕ) membrane phospholipids, decreases Mϕ synthesis of prostaglandins (PGs), changes chemotactic ability of Mϕs, and affects metabolism of acute phase proteins. Gender also has pronounced effects on susceptibility to CIA and Mϕ prostaglandin profiles. The mechanisms by which dietary n‐3 fatty acids may act to alleviate symptoms of CIA, as well as interactions of dietary n‐3 and n‐6 fatty acids and gender are discussed. We suggest that the ability of FO diets to influence favourably the course of chronic inflammatory diseases is mediated via alterations in n‐6 fatty acid metabolism and that intrinsic differences in n‐6 fatty acid metabolism may account not only for our reported gender differences in incidence and severity of CIA, but also the well‐documented sexual dimorphism in immune/inflammatory responses in general.

Use of soluble, collagenous peptides from medium of chick calvaria cultures as substrate for prolyl hydroxylase.

Abstract Underhydroxylated collagenous proteins accumulate in the media of embryonic chick calvaria cultured in the presence of α,α′-dipyridyl for 24 h. These soluble collagenous proteins, when labeled with radioactive proline, were shown to be a specific, stable, and highly efficient substrate for in vitro measurement of prolyl hydroxylase. The ability of the media proteins to serve as a substrate for prolyl hydroxylase was abolished by digestion with purified bacterial collagenase. This method of substrate preparation provides a soluble, efficient, economical substrate for routine prolyl hydroxylase assays, and permits the accumulation of sufficient quantities of substrate of one specific activity to serve for extended periods of time.

Temporal alterations of intracellular Na, K, Ca, Mg, and PO4 in muscle beneath the burn wound

Three days after a single hindlimb scald in the rat, soleus muscle from the burned limb, but not from the contralateral unburned limb, shows an elevation in glucose uptake and utilization, an increase in protein turnover, and a loss of responsiveness to insulin. To assess the relationship of extra- and intracellular electrolyte concentrations to the changes, rats were scalded on one hindlimb and compared to controls at 4 hr, 1 day, and 3 days postburn. There were no pronounced changes in serum sodium, potassium, or calcium levels of burned rats. Serum concentrations of magnesium and phosphate, however, were elevated 4 hr postburn but were significantly lower than controls 3 days later. Intracellular electrolytes in soleus muscles from the unburned limb of burned rats were comparable to those in control muscles. In contrast, thermal injury resulted in profound intracellular electrolyte alterations in soleus muscles from the burned limb. Four hours postburn, the intracellular sodium concentration was elevated 210% (P < 0.001) whereas the concentrations of potassium, magnesium, and phosphate were reduced 44, 24, and 41% (P < 0.001), respectively. In the ensuing 3 days, all four ions returned to, or closely approached, the intracellular concentrations in control muscles. Unlike these transient alterations, thermal injury led in burned limb muscles to a progressive accumulation of cellular calcium reaching values 18-fold higher (P < 0.001) than control values 3 days postburn. The data suggest that this increase in cellular calcium could be linked to the chronic increase in glucose metabolism and protein turnover which develop in thermally injured muscle.

Macrophage function in azocasein-induced amyloidosis in CBA/J and A/J mice

It is known that a single gene governs resistance to amyloidosis induced by azocasein in A/J compared to CBA/ Jmice. We wished to determine whether this trait might be linked to differences in macrophage activation as measured by Feγ-receptor (Fcγ-R) and C3h receptor (C3b-R) phagocytosis of sheep erythrocytes and/or TNF secretion by adherent resident peritoneal cells. Macrophages from preamyloidotic and amyloidotic CBA/J mice showed marked enhancement of Feγ-R and C3h-R binding activity. Macrophages from A/Jmice receiving multiple azocasein injections showed a significant increase in Fcγ-R but not in C3b-Rphagocytic indices. Adherent peritoneal cells from control and azocasein-treated A/J Write produced higher TNF levels than control and azocasein-treated CBA/J mice. In both strains, peritoneal macrophage TNF secretion was depressed after 10 azocasein injections. Our findings differ from previous studies in which the amyloid deposition phase was associated with normal or depressed phagocytosis. On the oth...

Dietary modulation of apolipoprotein serum amyloid A (apoSAA) metabolism and prevention of amyloidosis in aging C57BL/6J and SJL/J mice

Abstract The specific effect of acute inflammation on apoSAA and lipoprotein metabolism was determined in two strains of mice that are prone to develop spontaneous age-associated systemic amyloidosis. C57BL 6J and SJL J mice were maintained on purified diets, relatively rich in fat and differing only with respect to protein constituents, i.e., casein (20%) versus soy protein (20%). After 18 months, aging mice of either strain and on either diet were injected subcutaneously with AgNO3 thus inducing a powerful acute phase response as evidenced by increases in plasma LDL and apoSAA-rich HDL. None of the mice in each of the dietary groups developed amyloidosis. Cholesterol levels were elevated during the acute phase response in both mouse strains, although the hypercholesterolemia was less pronounced in C57BL 6J mice and in SJL J mice fed soy protein compared with casein. Control plasma triglyceride levels were lower in C57BL 6J mice in both dietary groups compared to the SJL J strain. By contrast, C57BL 6J mice fed either protein increased the triglyceride concentration, whereas in SJL J mice, triglyceride levels were not altered by casein but were decreased significantly by soy protein diets. Oir findings show for the first time that dietary protein intake modulates the acute phase response in rodents. Furthermore, the data support the concept that alterations in lipid metabolism during the acute phase response may represent a protective mechanism whereby HDL-cholesterol and phospholipids are directed to sites of inflammation for connective tissue repair.

Increased Binding of Acute Phase Lipoproteins by Peritoneal Macrophages (MØs) in Mice

Serum amyloid A protein (SAA), synthesized in response to an acute inflammatory stimulus, is transported in serum in association high density lipoprotein. We have compared peritoneal macrophage binding of lipoprotein fractions isolated from normal mice with those isolated from mice 18 hrs after intraperitoneal injection of 100 ug purified endotoxin. Lipoproteins were isolated by ultracentrifugation at d > 1.21 g/ml and the apol ipoproteins labelled with I by the iodine monochloride method. Labelled lipoproteins were incubated with adherence purified peritoneal macrophages and cell associated radioactivity measured. Cellular uptake of acute phase lipoproteins was six to seven-fold greater that uptake of lipoproteins from normal animals. Treatment of the cells with glutaraldehyde prior to exposure to the lipoproteins abolished the difference in uptake.

A micromethod for the purification of lysyl oxidase

Abstract A simple, efficient, and rapid procedure for the purification of lysyl oxidase is described. This method utilizes an affinity scheme involving powdered elastin-hydroxyethylmethacrylate hydrogels and high-performance liquid chromatography and permits the study of enzyme from sources which contain limited amounts of enzyme, such as aortic smooth muscle cells in culture.