A systematic approach to risk stratification and intervention within a managed care environment improves diabetes outcomes and patient satisfaction.

OBJECTIVE: To determine whether a comprehensive diabetes management program that included risk stratification and social marketing would improve clinical outcomes and patient satisfaction within a managed care organization (MCO). RESEARCH DESIGN AND METHODS: The 12-month prospective trial was conducted at primary care clinics within a MCO and involved 370 adults with diabetes. Measurements included 1) the frequency of dilated eye and foot examinations, microalbuminuria assessment, blood pressure measurement, lipid profile, and HbA(1c) measurement; 2) changes in blood pressure, lipid levels, and HbA(1c) levels; and 3) changes in patient satisfaction. RESULTS: Complete data are reported for the 193 patients who had been enrolled for 12 months; life table analysis is reported for all patients who remained enrolled at the study's end as well as for a comparative control group of 623 patients. For the 193 patients for whom 12-month data were available, the number of patients in the low-risk category (HbA(1c) <7%) increased by 51.1%. A total of 97.4% of patients with an HbA(1c) >8% at baseline had a change in treatment regimen. Patients at the highest risk for coronary heart disease (LDL >130 mg/dl) decreased from 25.4% at baseline to 20.2%. Patients with a blood pressure <130/85 mmHg increased from 23.8 to 44.6%. Of these patients, 63.0% had changes in medication. Patients and providers expressed significant increases in satisfaction with the program. CONCLUSIONS: The program was successful in initiating the recommended changes in the diabetic therapeutic regimen, resulting in improved glycemic control, increased monitoring/management of diabetic complications, and greater patient and provider satisfaction. These results should have great significance in the design of future programs in MCOs aimed at improving the care of people with diabetes and other chronic diseases.

Induction of TGF-beta1 by the matricellular protein SPARC in a rat model of glomerulonephritis.

UNLABELLED: Induction of TGF-beta1 by the matricellular protein SPARC in a rat model of glomerulonephritis. BACKGROUND: SPARC has been implicated as a counteradhesive and antiproliferative protein associated with deposits of extracellular matrix in renal disease. METHOD: We have examined the effect of recombinant SPARC containing a C-terminal His tag (rSPARC) in an acute model of mesangial cell injury that is induced in the rat by an antibody against the Thy1 antigen on the mesangial cell membrane. The recombinant protein was administered 24 hours after the induction of nephritis and was infused through day 4. RESULTS: rSPARC was localized to the renal glomeruli of rats treated with anti-Thy1 antibody. Type I collagen and fibronectin, as well as transforming growth factor-beta1 (TGF-beta1), were increased at day 5 in rats treated with rSPARC (N = 4, P < 0.05 vs. delivery buffer), but only minimal effects were seen on mesangial cell and endothelial cell proliferation. In primary cultures of rat mesangial cells, infusion of rSPARC was associated with increases in TGF-beta1 mRNA and in total, secreted TGF-beta1 protein. CONCLUSIONS: rSPARC stimulates expression of TGF-beta1 both in vitro and in vivo. Given the closely regulated expression of SPARC, TGF-beta1, and type I collagen in several animal models of glomerulonephritis, we propose that SPARC could be one of the major mediators of the induction of TGF-beta1 in renal disease.

Transfected CD59 protects mesangial cells from injury induced by antibody and complement.

CD59 is a complement regulatory protein on the glomerular cells that inhibits C5b-9 assembly and insertion. We employed an overexpression strategy to determine the functional significance of CD59 in mesangial cells. We made a CD59 expression vector tagged with FLAG utilizing site-directed mutagenesis and PCR, which allows transfected CD59 to be distinguished from the constitutively expressed protein. In stable clones, overexpressed CD59 was clearly detected immunocytochemically both by anti-FLAG and anti-CD59 antibody in a granular pattern. The overexpression of CD59 was also confirmed by Western blotting. To determine if overexpression of CD59 by mesangial cells protected these cells from C5b-9 attack, we performed complement-mediated cell lysis assays. CD59-transfected mesangial cells demonstrated marked resistance to complement-mediated cell lysis which was reversed in the presence of antibody to CD59. We also investigated the role of CD59 in protecting cells from the effects of membrane insertion of sublytic quantities of C5b-9. Overexpressed CD59 suppressed production of superoxide, one of the inflammatory mediators induced by sublytic C5b-9 attack. These results demonstrate directly that transfected CD59 functions as a potent protector of mesangial cells against both lytic and sublytic attack by C5b-9. CD59 may be an important regulator of complement-mediated disease in the glomerular mesangium.

Human serum amyloid A genes are expressed in monocyte/macrophage cell lines.

Serum amyloid A (apoSAA) is a family of proteins found, mainly associated with high density lipoproteins, in the blood plasma of mammals and at least one avian species, the Pekin duck. These proteins are present in small amounts under normal circumstances, but their concentration is capable of rising 100- to 1,000-fold in situations involving tissue injury or infection. Like classic acute phase proteins they are produced in the liver; however, expression of one of the apoSAA genes is known to occur in activated macrophages of mice. We examined three human macrophage precursor cell lines (THP-1, U-937, and HL-60), before and after differentiation with phorbol 12-myristate 13-acetate or 1 alpha,25-dihydroxy-vitamin D3, for apoSAA messenger (m)-RNA expression and found that: 1) induction of steady-state apoSAA mRNA by lipopolysaccharide, interleukin-1, or interleukin-6 required the presence of the synthetic glucocorticoid dexamethasone; 2) the three known active genes, apoSAA1, apoSAA2, and apoSAA4, were induced in THP-1 cells, whereas the pseudogene apoSAA3 was not; 3) differentiated and undifferentiated THP-1 cells expressed apoSAA mRNA, but U-937 cells expressed apoSAA mRNA (low levels) only after phorbol 12-myristate 13-acetate differentiation and HL-60 cells did not express apoSAA mRNA whether differentiated or not; 4) apoSAA protein was detectable immunologically at a low level in lyophilized medium from induced THP-1 cells. Our findings are compatible with the hypotheses that 1) apoSAA gene expression in human monocytes/macrophages in vivo is differentiation dependent; 2) activated macrophages provide a local source of apoSAA at sites of tissue injury or inflammation; 3) apoSAA is induced in tissue macrophages by local stimuli, under conditions that may not evoke the systemic acute phase response.

Expression of apolipoprotein serum amyloid A mRNA in human atherosclerotic lesions and cultured vascular cells: implications for serum amyloid A function.

Altered lipoprotein metabolism and vascular injury are considered to be major parts of the pathogenesis of atherosclerotic lesions. Serum amyloid A (SAA) is a family of acute-phase reactants found residing mainly on high density lipoproteins (HDL) in the circulation. Several functions for the SAAs have been proposed that could be important in atherosclerosis. These include involvement in cholesterol metabolism, participation in detoxification, depression of immune responses, and interference with platelet functions. Like other acute-phase reactants, the liver is a major site of SAA synthesis. However, studies in the mouse have revealed that several cell types including macrophages express SAA. Furthermore, we recently found that SAA mRNA expression can be induced in the human monocyte/macrophage cell line, THP-1. In the present study, human atherosclerotic lesions of coronary and carotid arteries were examined for expression of SAA mRNA by in situ hybridization. Surprisingly, SAA mRNA was found in most endothelial cells and some smooth muscle cells as well as macrophage-derived "foam cells," adventitial macrophages, and adipocytes. In addition, cultured smooth muscle cells expressed SAA1, SAA2, and SAA4 mRNAs when treated with interleukin 1 or 6 (IL-1 or IL-6) in the presence of dexamethasone. These findings give further credence to the notion that the SAAs are involved in lipid metabolism or transport at sites of injury and in atherosclerosis or may play a role in defending against viruses or other injurious agents such as oxidized lipids. Furthermore, expression of SAAs by endothelial cells is compatible with the evidence that SAA modulates platelet aggregation and function and possibly adhesion at the endothelial cell surface.

Preservation of RNA for in situ hybridization: Carnoy's versus formaldehyde fixation.

Tissues fixed with organic solvent fixatives such as Carnoy's solution are known to give poor and erratic results with in situ hybridization, whereas those fixed with paraformaldehyde produce more consistent results. To understand this difference and to improve the utility of Carnoy's-fixed tissue for in situ hybridization, we explored several parameters of RNA integrity and preservation. Carnoy's-fixed, paraffin-embedded livers and paraformaldehyde-fixed, paraffin-embedded livers of mice were compared for RNA extractability, degradation, and hybridizability. In addition, retention of RNA in tissue sections after sequential in situ hybridization treatments was compared. RNA was found to be easily extractable from Carnoy's-fixed liver and was well preserved, with only slight degradation of high molecular weight RNA. Conversely, only a small percentage of the RNA was extractable from paraformaldehyde-fixed liver unless the tissue was digested with protease. The extracted RNA was well preserved, without detectable degradation. Sections of tissue fixed in Carnoy's solution subjected to in situ hybridization retained only about 10% of their original RNA content and gave correspondingly weak in situ hybridization signals. Formaldehyde-fixed tissues retained much more of the RNA (about 45%) and produced strong in situ hybridization signals. Treatment of Carnoy's-fixed tissue sections with vaporous formaldehyde increased retention of RNA and provided in situ hybridization signals comparable with those of paraformaldehyde-fixed tissues.

Murine serum amyloid A3 is a high density apolipoprotein and is secreted by macrophages.

The serum amyloid A (SAA) proteins make up a multigene family of apolipoproteins associated with high density lipoproteins. They are of ancient origin; the finding of a highly homologous protein in mammals and ducks indicates that SAAs have been in existence for at least 300 million years. The interspecies similarity among the SAAs makes the mouse, in which they have been most thoroughly studied, a reasonable model to use for defining the function(s) of this family of proteins in humans. Originally it was observed that the SAA proteins were made in the liver and represented a set of proteins belonging to acute-phase reactants. SAA3 is a unique member of the SAA multigene family in mice in that its mRNA is also expressed in extrahepatic tissues by a variety of cell types, mainly macrophages and adipocytes. To date, nothing has been reported regarding the fate or function of the SAA3 translation product. To identify the SAA3 protein, we developed SAA3-specific antibodies by immunizing rabbits against a portion of SAA3 protein synthesized in a bacterial fusion protein expression system. Electroimmunoblot analysis of serum and lipoprotein fractions of it showed SAA3 to be associated with high density lipoproteins of mice treated with lipopolysaccharide. Furthermore, a continuous mouse macrophage cell line (J-774.1), when exposed to lipopolysaccharide, expressed SAA3 mRNA in a dose-dependent manner and secreted SAA3 protein. The expression and secretion of SAA3 by macrophages stimulated with lipopolysaccharide suggest a role for this SAA in local responses to injury and inflammation.

Serum amyloid A in the mouse. Sites of uptake and mRNA expression.

Murine serum amyloid A1 (SAA1) and serum amyloid A2 (SAA2) are circulating, acute phase, high density apolipoproteins of unknown function. To pursue issues relating to their possible function their uptake and formation were studied. Kinetics of SAA protein distribution and gene expression after acute phase stimulation by casein or lipopolysaccharide were examined using immunocytochemistry for protein and RNA blot and in situ hybridization with probes for SAA1 and SAA2 mRNA. After casein injection, interstitial cells of testes, cells of adrenal cortex, kidney proximal convoluted tubule epithelia, and some parafollicular cells of spleen took up SAA in a time pattern related to plasma SAA levels. Extrahepatic SAA1 and SAA2 mRNA were induced by lipopolysaccharide in kidney proximal and distal convoluted tubule epithelia, and SAA1 mRNA was induced in epithelial lining the mucosa of the ileum and large intestine, indicating that there may be more than one function for the apoSAA gene family related to site of and stimulus for expression.

Expression of the third member of the serum amyloid A gene family in mouse adipocytes.

Three homologous genes that code for three related proteins comprise the serum amyloid A (SAA) family in the mouse. Endotoxin induces equally vigorous expression of mRNAs for the three SAA genes in liver. In extrahepatic tissues SAA1 and/or SAA2 mRNAs have been found only in kidney and intestine, however, SAA3 is expressed in all extrahepatic tissues thus far examined. This observation raised the question: is SAA3 mRNA expressed by a single cell system dispersed throughout all tissues, or by differentiated cells of each tissue? This question was explored in various tissues by in situ hybridization with a single-stranded cRNA probe specific for SAA3 mRNA. We found expression in the liver of SAA3 mRNA by other cells as well as by hepatocytes. A common feature among extrahepatic tissues was SAA3 mRNA expression in adipocytes. SAA3 mRNA was also found in two nonadipose cells, Leydig cells of the testis, and some of the cells located in parafollicular zones of the spleen.

Rat tissues express serum amyloid A protein-related mRNAs.

Serum amyloid A (SAA) is a small (12 kDa) acute-phase apoprotein of high density lipoprotein found in mammals. It is also the precursor to amyloid protein A, the main protein constituent of fibrils found in amyloidosis secondary to chronic or recurrent inflammation--e.g., rheumatoid arthritis. However, rats do not develop amyloidosis and SAA is not an apoprotein of rat high density lipoprotein; thus rats appear to be an exception in regard to expression of SAA genes. We report here that rats do have representatives of the SAA gene family and express two distinct SAA mRNAs. Moreover, the pattern of genes expressed among tissues, and their induction by inflammatory agents, is similar to that of related mouse genes. RNA from various tissues of normal and injured rats was examined by RNA blot hybridization with SAA cDNA and complementary RNA probes for the three murine SAA genes. A SAA mRNA of approximately 400 nucleotides related to mouse SAA1 and SAA2 mRNAs reached a high level in liver 24 hr after injection of bacterial lipopolysaccharide. No extra-hepatic tissues were found to express the SAA1/SAA2-related mRNA. Turpentine induced two hepatic SAA1/SAA2-related mRNAs of approximately 400 and approximately 500 nucleotides in length. Liver SAA1/SAA2-related mRNA hybrid selected and translated in a wheat germ protein-synthesizing system, from lipopolysaccharide- and turpentine-injected rats, produced a single protein with an estimated molecular mass of 8 kDa. This rat liver SAA-related mRNA appears to lack a highly conserved coding region for portions of two amphipathic helical domains and the joining sequence. An mRNA related to mouse SAA3 was found expressed at a high level in lung after lipopolysaccharide but not following turpentine injection. This mRNA was also expressed at high levels in ileum and large intestine of control rats and was not found in the liver of control or challenged rats. These observations show that the SAA gene family is present and expressed in rats and that its expression is found under situations similar to those found in mice. This lends support for the importance of the SAA gene family in the response to injury by vertebrates.

Differential expression of the amyloid SAA 3 gene in liver and peritoneal macrophages of mice undergoing dissimilar inflammatory episodes.

The three active serum amyloid A (SAA) genes of mice, SAA 1, SAA 2, and SAA 3, are coordinately expressed in liver during acute and chronic inflammatory stimulation and experimental amyloidosis. The genes, primarily SAA 3, are also expressed extrahepatically. The apoprotein SAA 2 is the precursor of the amyloid A (AA) fibril protein that is deposited as insoluble fibrils extracellularly in spleen and other organs when amyloidosis occurs secondarily to inflammation. The exact cause of AA fibril formation is unknown. Amyloid enhancing factor is a high m.w. glycoprotein extracted from amyloidotic organs. Administration of amyloid enhancing factor alters experimental inflammation to bring about accelerated deposition of amyloid A fibrils first in spleen and later in other organs. In this study, hepatic and extrahepatic expression of the SAA genes were compared during accelerated amyloidosis relative to inflammation uncomplicated by amyloidosis. Differences in kinetics and pattern of SAA gene expression by resident peritoneal macrophages and liver were detected during four dissimilar inflammatory episodes. Macrophages expressed the SAA 3 gene solely, and to a greater extent in chronic than in acute inflammation. In accelerated amyloid induction, macrophage SAA 3 expression increased as SAA 1 and SAA 2 expression in liver decreased. However, alpha-1-acid glycoprotein expression remained elevated throughout the course of amyloid induction. The greatly increased expression of the SAA 3 gene by macrophages and decreased expression of the SAA 1 and SAA 2 genes in liver during amyloidosis, suggests that altered SAA gene expression may play a pathogenetic role in experimental amyloid deposition.

Amyloid A gene family expression in different mouse tissues.

Serum amyloid A (SAA) is a major acute-phase reactant and apoprotein of high density lipoprotein (HDL). SAA is encoded by a family of three active genes. We examined hepatic expression and searched for extrahepatic expression of the three SAA mRNAs after injection with casein or LPS. Studies using an SAA cDNA, which detects all three SAA mRNAs, revealed that after casein injection liver SAA mRNA was elevated approximately 1,000-fold. Adrenal gland expressed SAA mRNA at a low level (0.5% of hepatic level), and was the only extrahepatic tissue with elevated SAA mRNA after casein injection. The small intestine, primarily the ileum, and the large intestine of unstimulated control animals contained 5- and 15-fold higher SAA mRNA levels than control liver. LPS also elevated liver SAA mRNA approximately 1,000-fold. However, in contrast to casein injection, every extrahepatic tissue examined expressed SAA mRNA. Lung and kidney contained 2-5% and large intestine contained nearly 10% of SAA mRNA levels found in liver RNA. SAA mRNA levels were lower in the remaining tissues and ranged from 0.1% in the brain and pancreas to 1.0% in the small intestine, with the ileum containing 50-fold more than the duodenum. Analysis of liver with SAA1, SAA2, and SAA3 mRNA-specific oligonucleotide probes revealed that SAA1 and SAA2 mRNA were elevated approximately 50-fold higher than SAA3 mRNA after casein administration. LPS, however, induced all three SAA mRNAs equally. In extrahepatic tissues, SAA1, SAA2, and SAA3 mRNAs were expressed differentially and can be grouped into three general classes: tissues expressing all three genes, tissues expressing SAA1 and SAA3, and tissues expressing predominantly or only SAA3.

Amyloidogenesis. One serum amyloid A isotype is selectively removed from the circulation.

The deposits of fibrils found in amyloidosis of the A type are derived from only one of the three serum amyloid A (SAA) gene products, namely SAA2. In order to explore the mechanism of SAA isotype-specific amyloid protein AA deposition, the molecular kinetics of the serum amyloid proteins were examined in CBA mice during casein induction of amyloidosis. The presence of SAA mRNA in spleen was searched for; hepatic SAA1 and SAA2 mRNA levels, rates of specific protein synthesis and secretion by hepatocytes, and serum levels were measured during a 20-d period of amyloid induction. We observed the following: small amounts of amyloid substance appeared in the spleen by day 5 and increased steadily over the ensuing 15 d to occupy nearly 30% of splenic volume by day 20. No SAA mRNA was detected in spleen at any time during induction of amyloid formation. Total serum SAA levels peaked 1 d after we began casein treatment, and thereafter declined. This decline was accounted for entirely by a dramatic fall in SAA2, while SAA1 levels remained nearly constant throughout. The ratios of hepatic SAA2:SAA1 mRNA, as determined by in vitro translation, remained constant during the 20-d period, as did amounts of SAA1 and SAA2 synthesized and secreted by freshly isolated hepatocytes. These data indicate that the deposition of amyloid A protein derived from SAA2 is not due to local SAA production in spleen, nor excessive SAA2 production compared with SAA1, but involves the selective and accelerated removal of SAA2 from the circulating pool of both SAA1 and SAA2.

High-molecular-weight precursor of epidermal filaggrin and hypothesis for its tandem repeating structure.

Filaggrin is a histidine-rich protein that is intimately involved in mammalian epidermal keratinization. Using a combination of immunologic and in vivo pulse-chase studies with radiolabeled histidine and phosphate, we show that the phosphorylated precursor of both rat and mouse filaggrin has an apparent molecular weight much higher than previously realized (6 X 10(5) and 3.9 X 10(5), respectively). These high-molecular-weight filaggrin precursors can be rapidly labeled with histidine and extracted from the epidermis under denaturing conditions. More than half of the label incorporated in the precursor at 2 h is found in filaggrin at 24 h after injection, even though filaggrin is less than 10% of the size of the precursor. Limited proteolytic digestion of the precursor in vitro results in the formation of an oligomeric series of peptides based on a phosphorylated fragment slightly larger than filaggrin itself. More extensive digestion of this fragment shows that it is composed of filaggrin with few or no additional unrelated peptides, suggesting that the major part of the high-molecular-weight filaggrin precursor must be composed of repeated domains of filaggrin. Because the primary translation product of filaggrin mRNA is large, we propose that these domains are repeated in tandem. In addition, from molecular weight computations and peptide map analyses, we suggest that the filaggrins are themselves composed of multiple repeating units of an unidentified peptide of approximately Mr 8600. This value is derived from the molecular weights of filaggrin from several mammalian species that differ by integral multiples of 8600. A model for the structure of the high-molecular-weight precursor of filaggrin is presented.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidermal filaggrin is synthesized on a large messenger ribonucleic acid as a high-molecular-weight precursor.

Filaggrin is a keratin filament associated protein found in the differentiated cells of the epidermis. The mouse protein has a molecular weight of 26.5 X 10(3), while the molecular weight of rat filaggrin is 49 X 10(3), when analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. In order to clarify our understanding of the precursor form of filaggrin, RNA was isolated from mouse and rat epidermis and the poly(A+) fraction was translated in a cell-free protein synthesis system. Immunoprecipitation of translated proteins with specific antibody against rat filaggrin revealed a diffuse radiolabeled band with a molecular weight of approximately 250 X 10(3) on SDS-polyacrylamide gels. The size of filaggrin mRNA was estimated by sedimenting poly(A+) RNA through isokinetic sucrose gradients. Mouse filaggrin mRNA activity was located in the 30S region of the gradient, while rat filaggrin mRNA was located in the 34S region. The molecular weight of rat filaggrin mRNA was estimated to be 5 X 10(6) by electrophoresis in denaturing agarose gels containing methylmercury(II) hydroxide. A messenger RNA of this size could code for a polypeptide as large as approximately 600 kilodaltons. We conclude that filaggrin is synthesized as a large molecular weight precursor that must undergo substantial processing prior to formation of the mature form of the protein.

The signal sequence of ovalbumin is located near the NH2 terminus.

Ovalbumin, unlike other secretory proteins, is synthesized and secreted without cleavage of a hydrophobic signal peptide. Kinetic experiments were performed in a cell-free translation system to measure the minimum size of ovalbumin nascent chains required for binding of both the nascent chain and the corresponding mRNA to microsomal membranes derived from dog pancreas. Results of these experiments revealed that 50 to 60 amino acid residues are sufficient to bind ovalbumin-synthesizing polysomes to membranes in vitro. When microsomes with associated polysomes were isolated from chick oviduct, nascent ovalbumin chains longer than 50 residues were protected from proteolysis as long as the membranes remained intact, suggesting that the polypeptides were sequestered by the endoplasmic reticulum. We conclude that the functional signal for membrane translocation of ovalbumin becomes accessible when the nascent chain is 50 to 60 residues long. We speculate that the hydrophobic sequence which lies between residues 25 and 45 folds back on the preceding residues to form an amphipathic hairpin structure which is the signal element recognized by the membrane.