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.

Accumulation of glucosaminyl(acyl)phosphatidylinositol in an S3 HeLa subline expressing normal dolicholphosphomannose synthase activity.

Glucosaminyl(acyl)phosphatidylinositol [GlcN(acyl)PI], the third intermediate in the mammalian glycosylphosphatidylinositol (GPI) anchor pathway, is undetectable in most cells. This intermediate was previously shown to accumulate, however, in murine lymphoma mutant E and in yeast mutant dpm1, both of which lack dolicholphosphomannose synthase activity. Here we report that a mammalian HeLa S3 subline, denoted D, produces large amounts of GlcN(acyl)PI. The level of GlcN(acyl)PI in this subline is twice that in the murine lymphoma mutant E and 4 times that in the parental S3 line. This HeLa D subline differs from the previously reported mutants that accumulate GlcN(acyl)PI because no defects in the synthesis or utilization of dolicholphosphomannose were found. Kinetic analysis indicated that in this HeLa subline there is an increased rate of synthesis of GlcN(acyl)PI, whereas the rate of metabolism for this GPI is comparable to that in wild-type cells. Furthermore, HeLa D cells accumulate GlcN(acyl)PI without a block in the synthesis of the downstream mannosylated GPI anchor precursors and GPI-anchored proteins. These findings might be relevant for understanding the regulation of the GPI pathway.

In vitro evaluation of a relationship between human serum- or plasma-material interaction and polymer bulk hydroxyl and surface oxygen content.

To evaluate serum- or plasma-material interactions and a relationship between such interactions and membrane properties such as bulk hydroxyl percentage and surface oxygen percent, medical polymeric membranes with extremely different hydroxyl percentage and surface oxygen percentage were evaluated with normal human serums or plasmas. Six types of mini-membrane modules (hydroxyl & surface oxygen %) were studied including cellulose triacetate (CA; 0 & 34.7%), Cuprophan (CP; 31.5 and 37.4%), ethylene vinyl alcohol (EVAL; 30.4 and 25.3%), polyacrylonitrile (PAN; 1.5 and 10.2%), polysulfone (PS-F; 0 and 14.2%), and a polymer alloy of polysulfone (PS-K; 0 and 16.2%). Post-perfusion values of biochemical solutes and complement components for PAN and both PS membranes were smaller than those for sham, CA, CP, and EVAL membrane module perfusions. C3a and C4a concentrations showed no significant differences among all membranes except PS-K. Mononuclear cell transformation functions to all mitogens for serums in contact with CA and CP membranes were suppressed versus sham and PAN and both PS membranes. Fibrinogen concentration changes for plasma in contact with EVAL, PAN, and PS-F membranes were significantly smaller than sham, and a significant prolongation of APTT was found for only EVAL versus sham, CA, and CP. Higher surface oxygen percentages (CA, CP > EVAL > PAN, both PS) but not hydroxyl content were associated with lower protein adsorption and higher suppressive transformation function results. These results suggest that surface oxygen percentage may be an important indicator of biocompatibility.

Evaluation of materials for use in extracorporeal circulation. In vitro serum-material interaction.

Polymeric membranes used in extracorporeal circulation procedures of varying hydroxyl (-OH) percent were evaluated with normal human serum to detect differences between chemical composition and serum-material interactions. The materials were evaluated as hollow fibers built into modules, including polypropylene (PP; 0%), polyvinyl alcohol (PVA; 23.7%), ethylene vinyl alcohol (EVAL; 29.7%), Cuprophan (CP; 31.5%), and Hemophan (HP; 30.9%). Data from serum perfusions expressed as percent changes to sham (circuit minus module) showed that solute % decreases were from 0% to 10% in all materials except for PVA (10-22%). Complement activation product % increases were higher with PVA (606-4309%) and CP (48-567%), and mononuclear cell transformation functions (MNCTFs) were more suppressed with PVA (100 to 98%) and CP (10-18%). Despite EVAL and HP having an -OH %, complement activation products were relatively low with EVAL (less than 212%), HP (less than 13%), and PP (less than 131%). MNCTFs were stimulatory with the co-polymer of vinyl alcohol and ethylene (EVAL), and very suppressed by serums from PVA contact. Ethylene vinyl alcohol and modified cellulose (HP) had reduced complement activation despite higher or comparable bulk -OH. Bulk -OH content alone cannot explain the differences observed.

Effects of early intervention and stimulation on the preterm infant.

To test the hypothesis that early intervention can enhance the development of high-risk preterm infants, a prescribed multimodal sensory enrichment program, within a regional neonatal intensive care unit, was designed and implemented. Twenty-eight appropriate-for-gestational age infants with birth weights between 1,200 and 1,800 gm were selected for study. To prevent control group contamination by the enrichment procedure the first 14 infants were designated as the control group, and the next 14 as the treatment group. Treated infants had significantly higher developmental status than control infants, as measured by the Bayley Scales of Infant Development, at six months past the maternal expected date of confinement (F = 14.98, P < .001, and F = 16.46, P < .001 for the mental and motor scales, respectively). Mean infant weight gain per day and mean total weight gain during the hospitalization were not significantly different for the two groups although the treatment group received significantly less calories per kilogram per day than the control group (F = 9.02 P < .006). Our data suggest that a prescribed intervention program for high-risk preterm infants appears to enhance the quality of development as measured at six months past the expected date of confinement. Further studies are necessary to determine the long-term value of early intervention and the apparent ability of infants receiving an enrichment program to utilize calories more efficiently than control infants.