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Biomedical subjects

E Cocuzzi

Publications and source records attributed to E Cocuzzi.

8 recordsLinked to original sources
PubMed2001

Tears contain the complement regulator CD59 as well as decay-accelerating factor (DAF).

Previous studies have shown that DAF (or CD55), a cell surface inhibitor of autologous C3 activation, is present in tears and that > 90% of the C3 convertase regulatory activity in tear fluid resides in this protein (Lass JH et al., Invest Ophth Vis Sci 1990; 31:1136-48). This study investigated whether (i) the membrane cofactor protein (MCP or CD46), an additional factor that regulates C3 activation, and (ii) the membrane inhibitor of reactive lysis (MIRL or CD59), a cell surface regulator that acts to prevent formation of the membrane attack complex, are also present in tears, and if so, are functional. Two-site immunoradiometric assays showed that MCP is present in tears at low levels (42 + 8 ng/ml, n = 8) while CD59 is present at levels (222 + 78 ng/ml, n = 14) comparable to those of DAF (325 + 289 ng/ml, n = 12). The concentrations of CD59 (i) were increased two-fold or more in closed eye tears, and (ii) were decreased in reflex tears. Western blotting showed that CD59 protein in tears migrates with an apparent mol. wt similar to membrane CD59 protein. Phenyl-Sepharose adsorption and Triton X-114 partitioning of tear CD59 as well as of tear DAF however, showed that both proteins are devoid of GPI anchors. Assays using cobra venom factor-activated human serum and guinea pig erythrocytes showed that CD59 is functionally active in inhibiting autologous C5b-9-mediated lysis and, under constitutive conditions, accounts for > 85% of the C9 inhibitory activity in tear fluid.

CD55 Antigens↗
PubMed2001

Blockage of complement regulators in the conjunctiva and within the eye leads to massive inflammation and iritis.

The open environment of the eye is continuously subject to an influx of foreign agents that can activate complement. Decay-accelerating factor (DAF), membrane cofactor protein (MCP) and CD59 are regulators that protect self-cells from autologous complement activation on their surfaces. They are expressed in the eye at unusually high levels but their physiological importance in this site is unstudied. In the rat, a structural analogue termed 5I2 antigen (5I2 Ag) has actions overlapping DAF and MCP. In this investigation, we injected F(ab')2 fragments of 5I2 mAb into the conjunctiva and aqueous humor, in the latter case with and without concomitant blockage of CD59. Massive neutrophilic infiltration of the stroma and iris resulted upon blocking 5I2 Ag activity. Frank necrosis of the iris occurred upon concomitant intraocular blockage of CD59. C3b was identified immunohistochemically, and minimal effects were seen in complement-depleted animals and in those treated with non-relevant antibody. The finding that blockage of 5I2 Ag function in periocular tissues and within the eye causes intense conjunctival inflammation and iritis demonstrates the importance of intrinsic complement regulators in protecting ocular tissues from spontaneous or bystander attack by autologous complement.

Animals↗
PubMed2000

Release of complement regulatory proteins from ocular surface cells in infections.

PURPOSE: The decay accelerating factor (DAF or CD55) and the membrane inhibitor of reactive lysis (MIRL or CD59), two complement regulatory proteins that protect self cells from autologous complement-mediated injury, are attached to corneal and cqonjunctival epithelial cells by glycosylphos-phatidylinositol (GPI) anchors. We sought to 1) determine the frequency with which bacteria recovered from patients with infections of the eye elaborate factors that can remove these surface proteins from ocular cells, 2) determine the spectrum of bacteria from other sites that have similar effects, and 3) establish the time interval required for reconstitution of the two regulators. METHODS: Culture supernatants of 18 ocular isolates [P. aeruginosa (n = 3), S. marcescens (n = 1), S. epidermidis (n = 9), and S. aureus (n = 5)], and > 100 other clinical specimens isolated in the hospital's microbiology laboratory [P. mirabilis (n = 1), S. aureus (n = 65), S. epidermidis (n = 24), B. cereus (n = 12), H. influenzae (n = 15), and Enterobacter sp. (n = 21)] were incubated at 37 degrees C for various times with conjunctival epithelial cells, conjunctival fibroblasts or HeLa cells and the release of DAF and CD59 proteins from the surfaces of the cells analyzed by 2-site immunoradiometric assays and by Western blotting. The kinetics of recovery of DAF and CD59 expression on the cells was measured by flow cytometry. RESULTS: DAF and/or CD59 release from the cell monolayers varied from < 5% to > 99% at as much as a 1:81 dilution of the supernatant from some bacteria. On conjunctival epithelial cells, more than 8 hr was required for 44% recovery of DAF expression and for 50% recovery of CD59 expression. CONCLUSIONS: Bacteria produce phospholipases and/or other enzymes which can efficiently remove DAF and CD59 from ocular cell surfaces. This phenomenon may correlate with their in vivo pathogenicity.

Bacteria↗
PubMed2000

Decay-accelerating factor in tears of contact lens wearers and patients with contact lens-associated complications.

PURPOSE: Complement activation fragments have been detected in the anterior segment during 1) eye closure, 2) contact lens wear, and 3) in some contact lens-associated pathologies. The decay-accelerating factor (DAF), a membrane-associated complement regulatory protein that inhibits the central C3 amplification convertases of the cascade, is present on both the ocular surface and in tears. In this study, we measured levels of tear DAF in asymptomatic contact lens patients and in patients who presented with contact lens-associated complications. METHODS: Tears were collected from 55 patients using capillary pipettes. Subjects included normal non-contact lens wearing controls (N = 14), asymptomatic soft (N = 13) and rigid gas permeable (N = 5) wearers, and individuals with contact lens-induced acute red eye (CLARE) (N = 4), ulcerative keratitis (N = 3), giant papillary conjunctivitis (GPC) (N = 8), contact lens peripheral ulcers (N = 3), and infiltrative keratitis (N = 5). Levels of DAF were assessed using a two-site immunoradiometric assay using anti-DAF monoclonal antibodies. RESULTS: The mean concentration of DAF in normal controls was found to be 149+/-78 ng/ml, 117+/-59 ng/ml, and 111+/-86 ng/ml for noncontact lens patients, and asymptomatic soft and rigid gas permeable lens wearers, respectively. In the conditions of CLARE, infiltrative keratitis, and GPC, DAF concentrations were significantly reduced compared with normal noncontact lens controls. Compared with asymptomatic soft lens patients, the condition of infiltrative keratitis showed a significant reduction in tear DAF. CONCLUSIONS: This study documents a trend toward decreased levels of tear DAF in patients with the contact lens associated inflammatory conditions CLARE, GPC, and infiltrative keratitis. Tears of patients with infiltrates show the most significant reduction of tear DAF. The reductions may be associated with enhanced complement activation contributing to the pathogeneses of infiltrative keratitis and associated ocular surface diseases.

CD55 Antigens↗
PubMed1990

Post-translational modification of apolipoprotein B by transglutaminases.

The major form of cross-link found in apolipoprotein B was identified as N1N12-bis-(gamma-glutamyl)spermine, a product known to be formed through the catalytic action of transglutaminases (EC 2.3.2.13). N1-(gamma-Glutamyl)spermine was present in a trace amount but epsilon-(gamma-glutamyl)lysine cross-links, which are formed during fibrin formation in plasma, were not detected. In the presence of catalytic amounts of plasma Factor XIIIa (a thrombin-dependent extracellular transglutaminase) or cellular transglutaminase (a cytosolic enzyme), apolipoprotein B and other plasma apolipoproteins (A-I, A-II and C) underwent covalently bridged polymerization and served as amine acceptor substrates. These results suggests that transglutaminases may participate in the covalent modification of apolipoproteins, either in the physiological state or during pathogenesis.

Animals↗
PubMed1989

Transglutaminase expression in rat parotid gland after isoproterenol stimulation.

Transglutaminases (E.C. 2.3.2.13) are calcium-dependent enzymes that catalyze the covalent cross-linking of proteins, and occur in multiple molecular forms in a variety of tissues. Distribution of each form of transglutaminase varies with different tissues. Studies were undertaken to characterize the form of transglutaminase expressed in rat parotid gland, and to examine a possible physiological role for the enzyme. It was found that chronic treatment of rats with the beta-adrenergic agonist isoproterenol (IPR) resulted in the induction of parotid transglutaminase activity. The properties of this transglutaminase appeared to be distinct from those of the well-characterized guinea pig liver cytosol transglutaminase (TGase C). The findings that protein polymerization (observed on SDS-PAGE) and incorporation of radioactive putrescine, a polyamine, into protein occur in the presence of exogenous transglutaminase and calcium indicated that certain rat parotid salivary proteins are or could be substrates for this enzyme. Analysis of proteolytic digests of rat parotid salivary proteins on an amino acid analyzer and by high-performance liquid chromatography also indicated that these salivary proteins contain gamma-glutamyl derivatives of primary amines (e.g., polyamines or lysine), post-translational products of transglutaminase catalysis. The possible physiological function of this enzyme in the oral cavity might be stabilization of proteinaceous structures during normal oral homeostasis and/or woundhealing.

Animals↗
PubMed1986

The biochemistry of epsilon-amino groups of lysine residues from apolipoprotein B of human low density lipoprotein.

The availability of epsilon-lysine residues of apolipoprotein B in LDL for chemical or enzymic modification was investigated. Amino acid analyses of detergent-solubilized apolipoprotein B, following cyanoethylation with acrylonitrile, revealed that 10% of the lysine in apolipoprotein B were unreactive. The unreactive residues were associated with the most hydrophobic subfraction of apolipoprotein B. Since apolipoprotein B has a high molecular weight a study was undertaken to determine whether lysine residues were crosslinked to glutamic acid via epsilon-(gamma-glutamyl)lysine as demonstrated for fibrin. Apolipoprotein B was digested exhaustively with proteases. The content of epsilon-(gamma-glutamyl) lysine was determined by chromatography and isotope dilution. In contrast to earlier reports for serum LDL the data showed that less than 0.01 moles of lysine/mole of LDL apolipoprotein B were present as epsilon-(gamma-glutamyl)lysine in plasma LDL. It was determined also that the crosslinks were not found in apolipoprotein B during clotting since LDL was not a substrate for clotting factor XIII which forms the bond in fibrin. Furthermore, the lipoprotein contained no inherent transglutaminase activity. It is concluded that the lysine residues in LDL, which are unreactive to cyanoethylation, can not be detected in the digests as epsilon-(gamma-glutamyl)lysine.

Acrylonitrile↗