Winnicott goes to the movies: the false self in Ordinary People.

Winnicott's theories of development, while appearing metaphorical and impressionistic, actually offer a remarkably consistent explanation for pathological character formation as an outcome of environmental failure. He suggested that faulty mothering can lead to a chain of disturbing internal psychic events that necessitate a reorganization in the child. A major pathological resolution is the formation of a false self and false self bonds. Winnicott's recommendations for treating the crippling effects of the early traumata proceed logically from his concepts of developmental pathology. The film, Ordinary People, offers a way of understanding the tragedy and then the hope stemming from the application of Winnicott's concepts.

Features of autoimmunity in the abdominal aortic aneurysm.

PURPOSE: Previous work has revealed that nonspecific abdominal aortic aneurysms (AAAs) have a prominent infiltration of inflammatory cells and that soluble extracts of AAA tissue are rich in immunoglobulins. These observations raise the question whether autoimmune mechanisms play a role in the pathogenesis or progression of AAA disease. The hypothesis of this investigation was that IgG purified from aneurysmal specimens would be immunoreactive with normal components of the aortic wall (by means of immunohistochemistry) and with soluble proteins extracted from normal aortic tissue (by Western immunoblotting methods). METHODS: Immunoglobulin G extracted from AAA homogenates was used to detect immunohistochemical reactivity to connective tissue components in fixed sections of normal aorta obtained from an organ donor. Immunoblotting techniques were used to compare the reactivity of IgG (detected with secondary goat antihuman antibody) from 14 patients with AAA with soluble proteins extracted from normal and aneurysmal aortas. Immunoglobulins G purified from extracts obtained from nine patients with no AAA were used for control experiments. RESULTS: A unique band at approximately 80 kd was visualized when the filters were probed with IgG from 11 (79%) of 14 patients with AAA compared with only one (11%) of nine control subjects (P = .002 by Fisher's exact test). Immunoglobulins G from patients with AAA codistributed with matrix fibers in normal aortic sections, particularly in the adventitia (suggestive of a microfibrillar component). CONCLUSION: Our findings suggest that there are autoimmune features of AAA disease that might not only be informative in terms of AAA origin but also lead to more precise forms of pharmacologic down-regulation of disease progression.

Cellular localization of matrix metalloproteinases in the abdominal aortic aneurysm wall.

PURPOSE: This study explores the source(s) of the matrix-degrading proteinases, matrix metalloproteinase 1 (MMP-1; interstitial collagenase), matrix metalloproteinase 3 (MMP-3; stromelysin 1), and matrix metalloproteinase 9 (MMP-9; gelatinase B), previously implicated in abdominal aortic aneurysm (AAA) development. The possible involvement of the plasmin cascade in the activation of these proteinases was also explored by examining the presence of the urokinase-type plasminogen activator (uPA) in aneurysm wall. METHODS: Immunohistochemical techniques were used to detect the presence of MMP-1, MMP-3 and MMP-9 proteins and uPA in fixed, paraffin-embedded tissue sections from AAA (n = 10) and control (n = 2) aortas. RESULTS: The MMP-9 protein was localized to mononuclear cells in the AAA wall. Dual-labeling techniques confirmed the identity of these cells as macrophages. The MMP-3 protein and uPA were also detected primarily in the macrophage-like mononuclear cells infiltrating the aneurysmal aorta. Immunoreactive material to MMP-1 was demonstrated in mesenchymal cells of the AAA wall suggesting alternative expression and delivery of this enzyme in AAA. CONCLUSIONS: This work establishes the role of macrophages in the delivery, expression, and possible activation of matrix destructive proteinases during AAA pathogenesis and suggests a role for the activation of MMPs in the progression of the disease.

Cytokines that activate proteolysis are increased in abdominal aortic aneurysms.

BACKGROUND: Abdominal aortic aneurysm (AAA) disease is characterized by an increase in proteolysis and loss of matrix components. The cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta), products of activated macrophages and T cells, are known to increase the production of matrix-degrading enzymes in some pathological states. METHODS AND RESULTS: Seven AAA and five control aortic tissue extracts were assayed for TNF-alpha and IL-1 beta with ELISA. TNF-alpha was elevated significantly in AAA extracts compared with controls (86 +/- 34 pg/mg of total protein versus 1 +/- 1 pg/mg of total protein; P < .001). IL-1 beta concentration also was significantly increased in the AAA specimens (48 +/- 14 pg/mg of total protein versus 12 +/- 5 pg/mg of total protein; P < .05). Immunoblotting demonstrated secreted forms of TNF-alpha in the AAA extracts, and possible membrane-bound forms were observed when the tissues were detergent-extracted. Known forms of IL-1 beta also were observed on immunoblots of AAA tissue extracts. CONCLUSIONS: The presence of TNF-alpha and IL-1 beta in AAA tissue underscores the importance of the infiltrating inflammatory cells present in the media and adventitia of aneurysmal aortic wall and further implicates an inflammatory process in the pathogenesis of AAA.

Possible key role for plasmin in the pathogenesis of abdominal aortic aneurysms.

BACKGROUND: Activation of proteolysis is characteristic of abdominal aortic aneurysm (AAA) disease, and by substrate gel enzymography with casein the most conspicuous proteinase of AAA wall has a molecular weight of approximately 80 kd. This activity has been resolved into separate metalloproteinase and serine proteinase (SP) components, by binding out the metalloproteinase by affinity to tissue inhibitor of metalloproteinases. Because plasmin plays a key role in activating members of the metalloproteinase family, the following experiments were done to test the hypothesis that the unknown SP is plasmin. METHODS: Immunoblots, immunoprecipitations, and immunohistochemistry were performed by conventional methods with a specific polyclonal antibody to plasminogen. RESULTS: Immunoblot analysis of extracts from AAA specimens (n = 7) revealed dense bands corresponding to known molecular forms of plasmin. Only trace amounts were detected in control aortic extracts (n = 4). When samples were equalized for total protein, the mean amount of immunoreactive material in the 80 kd band (in densitometric units) was 216 +/- 44 (SEM) for AAAs versus 27 +/- 15 (SEM) for controls (p < 0.01). The residual 80 kd SP activity on casein substrate gel enzymography was quenched by immunoprecipitation with the specific antibody. Immunohistochemistry revealed strong reactivity of the AAA wall. CONCLUSIONS: Because plasmin plays a key role in the cascade for activation of the matrix metalloproteinase (including collagenase and the metalloelastases), the present results suggest that plasmin may be important in the sequence of events leading to the destruction of aortic matrix in AAA.

Identification of matrix metalloproteinases 3 (stromelysin-1) and 9 (gelatinase B) in abdominal aortic aneurysm.

A prominent metalloproteinase activity with an apparent molecular mass of 80 kD and additional activities at 67 through 70, 50, and 32 kD have been observed on casein, gelatin, and elastin gel zymography in extracts from abdominal aortic aneurysms (AAAs). The forms at 80, 50, and 32 kD were isolated by affinity to recombinant tissue inhibitor of metalloproteinases, and the 80-kD and 50-kD components were shown to be derived from matrix metalloproteinase-9 (MMP-9). The relative electrophoretic mobility of these forms under reducing and nonreducing conditions corresponds to those of MMP-9 generated by MMP-3 (stromelysin-1) cleavage, and the active forms of MMP-3 at 45 and 35 kD were detected in aneurysmal extracts under reducing conditions by using specific antibody. Confirmation that the major proteolytic activity observed at 80 kD is MMP-9 was also demonstrated by immunoprecipitation of the activity with specific antibody. Comparative immunoblots of tissue extracts from 10 typical AAA patients, using specific antibody against MMP-9, revealed bands at 92, 82, 67, 51 through 53, 27, 23, and 20 kD under reducing conditions; six aortic control specimens displayed negligible immunoreactivity. This report is the first to show that known activated forms of MMP-3 and MMP-9 are present in the aneurysmal aortic wall and that they may play a role in the destruction of aortic matrix in AAA disease.

Analysis of elastin cross-linking and the connective tissue matrix of abdominal aortic aneurysms.

BACKGROUND: Studies of the connective tissue matrix of abdominal aortic aneurysms (AAAs) have yielded conflicting results, and the glycosaminoglycan content has not been previously reported. The present work was done to evaluate the matrix components of AAAs, including the cross-link content of the residual elastin. METHODS: Aortic specimens from AAAs and controls were sequentially extracted with salt, Brij, and urea; and the residual pellets were the subject of further studies. Elastin was purified by hot alkali treatment; other matrix components were determined by conventional methods. RESULTS: Elastin content of the purified material was reduced in AAA. The cross-link content, desmosine+isodesmosine, was also reduced in AAA as a ratio to insoluble matrix dry weight. However, the cross-link content as a ratio to valine in the purified elastin was normal. The amino acid profiles of representative AAA and controls elastin preparations were similar to that of reference elastin. The amino acid content of the insoluble matrix of AAA revealed a significant reduction of protein (controls = 820 +/- 40 micrograms/mg versus AAA = 700 +/- 20 micrograms/mg, p < 0.05); the collagen content was unaltered. The content of glycosaminoglycan in AAA was noted to be significantly reduced (controls = 33.5 +/- 3.4 micrograms/mg versus AAA = 17.1 +/- 2.0 micrograms/mg, p < 0.05). CONCLUSIONS: The data do not support the hypothesis of a primary cross-link deficiency in elastin of AAA; but the reduced contents of protein and glycosaminoglycans in AAA suggests basic biochemical alterations in the diseased aorta that warrant further investigation.

Matrix metalloproteinases in abdominal aortic aneurysm: characterization, purification, and their possible sources.

One of the most consistent observations in abdominal aortic aneurysm (AAA) disease is the disorganization and disruption of elastin and other matrix components of the aortic wall. The enzymatic basis for the biochemical features of AAA has been investigated beginning with the demonstration on substrate gel enzymography of a typical "profile" of proteinase activities in AAA tissue extracts which degrade gelatin, casein and elastin. A recombinant TIMP-1 affinity column was developed and three of the elastolytic/caseinolytic activities with approximate molecular weights of approximately 80 kDa, approximately 50 kDa and approximately 32 kDa were partially purified from these extracts. Affinity for rTIMP-1 suggests that these enzymes are members of the matrix metalloproteinase (MMP) family. High molecular weight forms of two MMPs, collagenase (MMP-1) and stromelysin-1 (MMP-3), were also isolated from the AAA tissue on this column; active forms of MMP-1 could be demonstrated by immunoblotting techniques in this preparation under reducing conditions. Infiltrating inflammatory cells are known sources of these proteolytic activities; analysis of these cell populations in the aneurysmal aortic wall using fluorescence-activated cell counting revealed a fifty-fold increase in macrophages (a well-known source of matrix-degrading enzymes) as well as a significant increase in lymphocytes.

Analysis of human acid beta-glucosidase by site-directed mutagenesis and heterologous expression.

Structure/function relationships of acid beta-glucosidase, the enzyme deficient in Gaucher disease, were evaluated by characterizing the proteins expressed from cDNAs encoding normal and mutant enzymes. Twenty-two Gaucher disease mutations or created mutations were expressed in Spodoptera frugiperda (Sf9) cells and analyzed for catalytic properties, stability, inhibitor binding, and modifier interactions. Many Gaucher disease mutations encoded highly disruptive amino acid substitutions (e.g. P289L and D409V) and produced severely compromised proteins with very reduced activity (kcat < 1% of normal) and/or stability. Six mutant enzymes had sufficient catalytic activity (kcat approximately 5-30% of normal) for extensive studies. The highly conservative substitutions, i.e. F216Y or S364T and V394L, led to severe, but selective, abnormalities of enzyme stability or large decreases in catalytic activity, respectively. The T323I, N370S, and V394L enzymes interacted abnormally with active site-directed inhibitors and localized these residues to the glycon binding region. Selected mutant enzymes were poorly activated by phosphatidylserine (V394L, L444P, and R463C) or by saposin C (L444P and T323I), indicating that the enzyme sites for interaction with these activators were within the carboxyl one-third of the enzyme. Substitutions of Ser, Glu, and/or Gly at residues Asp-443 and/or Asp-445 demonstrated important steric roles for these residues in the active site, but neither is the catalytic nucleophile. Together with previous studies, the present analyses provide an insight into the pathogenesis of Gaucher disease and the functional organization of acid beta-glucosidase.

Demonstration of interstitial collagenase in abdominal aortic aneurysm disease.

This study was performed to evaluate the presence of interstitial collagenase, now known as matrix metalloproteinase-1 (MMP-1), in specimens of abdominal aortic aneurysms (AAA). Eight AAA and four control infrarenal aortas were evaluated. After homogenization and extraction of soluble proteins, immunoblots of the extracts equalized for protein content were performed with a specific antibody to MMP-1. Under native conditions, immunoreactive material was distributed between M(r) 27 kDa to > 106 kDa. When the extracts were reduced and denatured, immunoreactive bands were detected in AAA at the expected M(r)'s of the secreted isoforms (57 and 52 kDa), whereas control aortic extracts had low levels of detectable immunoreactive material. Only extracts from AAA demonstrated significant immunoreactivity to the lower M(r) isoforms (22, 25, and 27 kDa), which correspond to reported cleavage products of MMP-1. Preliminary immunofluorescent studies of AAA localized MMP-1 to cells present in the adventitia of AAA. These findings will help to resolve disagreement in the recent literature regarding the presence of collagenolytic activity in AAA disease.

Reaching children of the uninsured and underinsured in two rural Wisconsin counties: findings from a pilot project.

Debates about the accessibility, costs, and coverages of health care for the population at large have recently accelerated. This paper addresses some of the demographic, health, and fiscal ramifications of creating a preventive health care bridge to children in uninsured and underinsured families in two rural Wisconsin counties. The study findings revealed that the initial health status of children making a preventive health visit under a minimal copayment plan was noticeably worse than the status of those who had the free Early and Periodic Screening, Diagnosis, and Treatment (EPSDT) program available to them on a more or less continual basis. Upon their first visit, the children who did not have access to a free EPSDT program had a greater number of medical and dental health problems and fewer preventive dental care visits than their EPSDT contemporaries. Beyond a greater number of problems, however, we found no noticeable differences between the two groups in the types of health problems present (i.e. the clinical distribution of the problems was similar across the two groups). This paper also contrasts referral completion rates and rates of diagnostic confirmation of identified problems between the two groups. Finally, we provide estimates of the cost of coverage for each unprotected child.

Processing, secretion, and biological properties of a novel growth factor of the fibroblast growth factor family with oncogenic potential.

We recently reported that the protein encoded in a novel human oncogene isolated from Kaposi sarcoma DNA was a growth factor with significant homology to basic and acidic fibroblast growth factors (FGFs). To study the properties of this growth factor (referred to as K-FGF) and the mechanism by which the K-fgf oncogene transforms cells, we have studied the production and processing of K-FGF in COS-1 cells transfected with a plasmid encoding the K-fgf cDNA. The results show that, unlike basic and acidic FGFs, the K-FGF protein is cleaved after a signal peptide, glycosylated, and efficiently secreted as a mature protein of 176 or 175 amino acids. Inhibition of glycosylation impaired secretion, and the stability of the secreted K-FGF was greatly enhanced by the presence of heparin in the cultured medium. We have used the conditioned medium from transfected COS-1 cells to test K-FGF biological activity. Similar to basic FGF, the K-FGF protein was mitogenic for fibroblasts and endothelial cells and induced the growth of NIH 3T3 mouse cells in serum-free medium. Accordingly, K-fgf-transformed NIH 3T3 cells grew in serum-free medium, consistent with an autocrine mechanism of growth. We have also expressed the protein encoded in the K-fgf protooncogene in COS-1 cells, and it was indistinguishable in its molecular weight, glycosylation, secretion, and biological activity from K-FGF. Taken together, these results suggest that the mechanism of activation of this oncogene is due to overexpression rather than to mutations in the coding sequences.

Human acid beta-glucosidase: affinity purification of the normal placental and Gaucher disease splenic enzymes on N-alkyl-deoxynojirimycin-sepharose.

Two sepharose-bound 1-deoxynojirimycin N-alkyl derivatives, N-(9-carboxynonyl)- and N-(11-carboxyundecyl)-deoxynojirimycin, were used for the affinity purification of acid beta-glucosidase (beta-Glc) from normal and type-1 Ashkenazi Jewish Gaucher disease (AJGD) sources. The capacities of these nondegradable inhibitor supports were 0.5 and 0.75 mg of normal beta-Glc/ml of settled gel, respectively. The purified normal enzyme (14-18% yield) had a specific activity of 1.6 X 10(6) nmol/h/mg protein and was homogeneous as evidenced by a single protein species of Mr = 67,000 on sodium dodecylsulfate-polyacrylamide gel electrophoresis and reverse phase high-performance liquid chromatography (HPLC). Microsequencing demonstrated a single N terminus, and the sequence of the first 22 N-terminal amino acids was colinear with that predicted from the beta-Glc cDNA. Amino acid composition analyses of beta-Glc revealed a high content (35%) of hydrophobic amino acids. The N-decyl-deoxynojirimycin support facilitated the purification of the residual enzyme from type-1 AJGD spleen to about 7,500-fold in four steps with a yield of about 11%. These new affinity supports provided improved stability, capacity and/or specificity compared to other affinity or HPLC methods for purifying this lysosomal glycosidase.