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S S Twining

Publications and source records attributed to S S Twining.

At least 55 records · Page 3Linked to original sources
PubMed1987

Vitreous macrophage elicitation: generation of stimulants for pigment epithelium in vitro.

Vitreous and macrophage samples were tested for the ability to stimulate proliferation and cell migration in cultured rabbit retinal pigment epithelium (RPE). A macrophage invasion was elicited by the intravitreal injection of latex particles in rabbits and after 3 days the vitreal macrophages were collected. The macrophages themselves, macrophage-conditioned culture medium, and macrophage-incubated vitreous had modest effects on RPE proliferation, but significantly stimulated RPE migration. A portion of the migration activity may be due to macrophage-derived proteases acting on normal vitreous. Mitogenic and additional migration-stimulating activity may also arise from adjacent tissues or from a breakdown of the blood-vitreous barrier that accompanies a macrophage invasion. A macrophage ingress into the vitreous may provide part of the stimulation for the migration and proliferation of RPE in conditions such as proliferative vitreoretinopathy.

Animals↗
PubMed1986

Relationship between proteases and descemetocele formation in experimental Pseudomonas keratitis.

Proteases are involved in the pathogenesis of Pseudomonas aeruginosa infections of the cornea. Although there are many potential roles for these enzymes, involvement in corneal stroma destruction with subsequent descemetocele formation and/or corneal perforation is an important example. This study examined the relationship of elastase and alkaline protease to corneal destruction as indicated by descemetocele formation. The protease content of the overnight Mueller-Hinton broth cultures of various strains of P. aeruginosa correlated with the production of descemetoceles both in the rabbit trauma model of Pseudomonas keratitis and upon intrastromal injection of filtrates of the overnight culture media. The levels of alkaline protease correlated better with descemetocele formation than those of elastase. The influx of inflammatory cells was not correlated with descemetocele formation within 24 to 48 hrs.

Animals↗
PubMed1986

The immune system in experimental Pseudomonas keratitis. Model and early effects.

A model to study the immune system in Pseudomonas keratitis was developed using defined flora rats (WAG/RijMCW) that have not been exposed to Pseudomonas aeruginosa. One group of rats was made immunocompetent towards P. aeruginosa by intraperitoneal injection of phenol-killed P. aeruginosa while a second group remained naive to this organism. Corneas of both groups were scratched centrally with a 21-g needle, before inoculation with 2 X 10(8) P. aeruginosa organisms. Corneas of control animals were either only scratched or only inoculated with the bacterium. At 18 hr, the naive animals were killed. In naive rat corneas, light and electron microscopy showed bacteria throughout the cornea, polymorphonuclear leukocytes (PMNs) distributed from the limbus towards the center, and little stromal degradation. In contrast, massive corneal degradation was observed in the immunocompetent rats; PMNs were present, but no bacteria were observed free in the stroma. The total acid protease content was higher in the immunocompetent than in the naive rat corneas, a possible reason for the observed difference in corneal degradation. This difference was not due to increased numbers of PMNs since nearly equal numbers of PMNs were counted after enzymatic disaggregation of both types of corneas. Glycogen-induced peritoneal PMNs from both types of rats migrated equally well towards P. aeruginosa culture media and media of corneas incubated with this bacterium. The authors conclude that immune recognition is (1) involved in the corneal host response to P. aeruginosa and (2) required for efficient phagocytosis by PMNs but not their recruitment.

Animals↗
PubMed1986

Role of leukocytes in ocular inflammation of tyrosinemia II.

In the animal model of tyrosinemia II only corneas from tyrosine(tyr)-fed rats produce chemoattractants in organ culture. To study the role of neutrophils (PMNs) in production of these chemoattractants, leukocytes (WBCs) were depleted using i.p. cyclophosphamide (CP). Saline (SAL)-treated rats maintained 18,375 +/- 894 WBC/mm3 (mean +/- SEM) with 4168 +/- 424 PMNs. Rats receiving CP (150 mg/kg day 0, 75 mg/kg day 4) has 1565 +/- 170 WBC (565 +/- 129 PMN) on day 3, and 398 +/- 68 WBC (19 +/- 5 PMN) on day 8. Rats ate a low-protein +/- 5% tyr diet on days 4-8. Only SAL-treated tyr-fed rats developed plaque-like gray epithelial lesions; histopathology showed corneal epithelial necrosis, stromal edema, and epithelial and stromal PMN infiltration. Control and CP-treated tyr-fed rat corneas showed no inflammation. On day 8 corneas were cultured in RPMI 1640 + 5% heat-inactivated fetal bovine serum. After 3 days, supernatants were assayed for chemotactic activity (leading front method); data were expressed as the percentage of peritoneal PMN migration relative to 5% zymosan-activated rat serum. The mean total migration toward 75% supernatant from SAL-treated, tyr-fed rat corneas was 79%, whereas migration toward corneal supernatants from controls and CP-treated tyr-fed rats ranged from 42-48%. Corneal extracts were assayed for proteolytic activity. WBC depletion prevented the increase in cathepsin B- and D-like activities present in tyr-fed corneas, suggesting that PMNs were a major source of these enzymes. The data suggest that WBC depletion reduces both corneal inflammation in vivo and the production of chemotactic activity by tyr-fed corneas in culture.

Animals↗
PubMed1985

Acid proteases and histologic correlations in experimental ulceration in vitamin A deficient rabbit corneas.

Although xerophthalmia due to severe vitamin A deficiency is the leading cause of childhood blindness in the underdeveloped countries, little is known about the proteases (other than collagenase) that are involved in the degradative mechanism. The degree of cellular autolysis and stromal degradation observed histologically in early stages of xerophthalmia and in ulcerating corneas in vitamin A deficient rabbits in this study were, in general, proportional to the levels of the proteases studied. The only major histologic and ultrastructural alteration observed in early xerophthalmic corneas was autolysis of superficial epithelial and stromal cells. In contrast, in the ulcerating corneas the stroma was infiltrated heavily with inflammatory cells and extensive stromal degradation was observed in the central necrotic region of the lesions. Maximal proteolytic activity toward hemoglobin was observed at pH 3.3 for corneal extracts from normal (N) and pair-fed control (C) rabbits and rabbits with early xerophthalmia (X) and ulcerating xerophthalmia (U) corneas. This activity was a cathepsin D-like enzyme per cornea that had a ratio of 1:1:3:16 in the N, C, X, and U corneas. The ratio of cathepsin B-like activity per cornea for N, C, X, and U corneas was 1:2:2:10.

Animals↗
PubMed1984

Fluorescein isothiocyanate-labeled casein assay for proteolytic enzymes.

A simple inexpensive sensitive protease assay was developed using soluble fluorescein isothiocyanate (FITC)-labeled casein. Casein was reacted with FITC to form the fluorescein thiocarbamoyl derivative. This substrate is cleaved by trypsin, chymotrypsin, elastase, subtilisin, and thermolysin in a linear time-dependent manner. These enzymes can be measured in the nanogram and subnanogram range using this assay. The assay is reproducible, has a low blank, and uses casein, which resembles natural substrates of most proteases.

Animals↗
PubMed1983

A pepsinogen from dog stomach.

1. A pepsinogen has been purified from dog stomach in 20% yield. 2. At pH values below 4 it spontaneously converts to a pepsin, the first order rate constant at pH 2, 22 degrees C, being 11 min-1. 3. The pepsinogen is stable to denaturation at pH values up to 9, in contrast to the pepsin which is irreversibly denatured above pH 7. 4. Its proteolytic activity against denatured hemoglobin is equal to that of pig pepsin, but in the milk-clotting assay at pH 5.5 its has only 14% the activity.

Amino Acids↗
PubMed1983

A pepsinogen from rainbow trout.

1. A pepsinogen, Ia on the basis of its electrophoretic mobility, from rainbow trout stomach, has an optimum pH near 2 for activation. 2. The cognate pepsin is denatured at pH values above 7, in contrast to the zymogen, which is slightly more alkali-stable. It has an optimum pH of 3 for proteolysis of denatured hemoglobin. 3. The intrinsic reactivity of the zymogen and pepsin (rates of activation and of proteolysis, respectively) are quite high, but as they operate at the environmental temperature of the fish, are remarkably similar to rates of activation and proteolysis by mammalian pepsinogens and pepsins.

Animals↗
PubMed1981

Preparation and activation of a spin-labeled pepsinogen.

gen derivative has been prepared, using 3-[[2,5-dioxo-1-pyrrolidinyl)oxy]carbonyl]-2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrolyl-1-oxy with 3 spin-labels/protein molecule. Two are located in the peptide that is released first (1-16) and the other is in the secondarily removed sequence (17-44). Activation of the labeled zymogen is a faithful model of native pepsinogen activation because the two processes are closely related in rate, pH dependence of rate, and sites of peptide bond cleavage. The ESR signal associated with the bound label changes during activation due to release of the activation peptide. The rate of release (0.5 min(-1) at pH 2, 22 degrees C) is an order of magnitude slower than is the rate of activation, i.e., cleavage of the peptide bond that holds the peptide to the enzyme. Activation in the presence of pepstatin, however, results in peptide release (2 min(-1) at pH 2, 22 degrees C) nearly as fast as activation occurs. At pH values between 2.5 and 3, there is a lag in the change of the ESR signal following acidification. This indicates accumulation of an intermediate with pH 8.5 labile activity that still has its activation peptide attached. The strong temperature dependence of the rate of activation (26 kcal/mol) is reflected neither in reported characteristics of pepsin catalysis nor in the measured rate of release of the activation peptide from the enzyme (13 kcal/mol).

Animals↗
PubMed1981

Genetic control of the immune response to myoglobin. V. Analysis of the cross-reactivity of 12 myoglobins with sperm-whale myoglobin antisera of inbred mouse strains in terms of substitutions in the antigenic sites and in the environmental residues of the sites.

The determination of the entire antigenic structure of myoglobin has made it possible to focus attention on the molecular factors controlling and influencing immune recognition. Recently, using antisera raised against sperm-whale myoglobin (Mb) in six different host species and investigating their reactions with Mb from 15 species, we showed that the binding capacity of an antigenic site is influenced by substitutions within site residues as well as within the residues close (within 6.0 å) to the sites. Based on these effects it was possible to correlate, at least in qualitative terms, the expected effects of the substitutions in each Mb and its observed cross-reaction with antisera to sperm-whale Mb. In the present work, these correlations were tested using sperm-whale Mb antibodies raised in four inbred mouse strains and in which the amount of antibodies directed to each antigenic site was determined. The amounts of 125I-labelled antibodies that could be bound maximally be each of 12 Mb variants were determined. Because of genetic control, the response to some antigenic sites was not expressed and therefore permitted us to evaluate with a good degree of confidence the effects of amino acid replacements in various myoglobins upon the reactivity of the sites. The values of cross-reaction expected for each Mb variant from these considerations agreed well with the values found experimentally. The results confirm unambiguously that the major factors affecting the cross-reactions of proteins can be attributed to substitutions within the sites and within environmental residues of the sites.

Animals↗
PubMed1980

The antibody response to myoglobin is independent of the immunized species. Analysis in terms of replacements in the antigenic sites and in environmental residues of the cross-reactions of fifteen myoglobins with sperm-whale myoglobin antisera raised in different species.

The recent determination of the entire antigenic structure of sperm-whale myoglobin with rabbit and goat antisera has permitted the examination of whether the antigenic structure recognized by antibodies depends on the species in which the antisera are raised. Also, by knowledge of the antigenic structure, the molecular factors that determine and influence antigenicity can be better understood in terms of the effects of amino acid substitutions occurring in the antigenic sites and in the environmental residues of the sites. In the present work, the myoglobins from finback whale, killer whale, horse, chimpanzee, sheep, goat, bovine, echidna, viscacha, rabbit, dog, cape fox, mouse and chicken were examined for their ability to cross-react with antisera to sperm-whale myoglobin. By immunoadsorbent titration studies with radioiodinated antibodies, each of these myoglobins was able to bind antibodies to sperm-whale myoglobin raised in goat, rabbit, chicken, cat, pig and outbred mouse. It was found that the extent of cross-reaction of a given myoglobin was not dependent on the species in which the antisera were raised. This indicated that the antibody response to sperm-whale myoglobin (i.e. its antigenic structure) is independent of the species in which the antisera are raised and is not directed to regions of sequence differences between the injected myoglobin and the myoglobin of the immunized host. Indeed, in each antiserum from a given species examined, that antiserum reacted with the myoglobin of that species. The extent of this auto-reactivity for a given myoglobin was comparable with the general extent of cross-reactivity shown by that myoglobin with antisera raised in other species. The cross-reactivities and auto-reactivities (both of which are of similar extents for a given myoglobin) can be reasonably rationalized in terms of the effects of amino acid substitutions within the antigenic sites and within the residues close to these sites. These findings confirm that the antigenicity of the sites is inherent in their three-dimensional locations.

Amino Acid Sequence↗
PubMed1979

Use of immunoadsorbents for the study of antibody binding to sperm whale myoglobin and its synthetic antigenic sites.

Conditions for preparing immunoadsorbents of sperm-whale myoglobin and its five synthetic antigenic sites and for desorption of radiolabeled antibodies from the immunoadsorbents were studied. In immunoadsorbent titration studies, the sum of the amounts of antibodies bound in the plateau (maximum binding) by the adsorbents of the five sites accounted quantitatively for the entire (100%) antibody response to sperm-whale myoglobin.

Animals↗
PubMed1979

Genetic control of immune response to sperm whale myoglobin in mice. II. T lymphocyte proliferative response to the synthetic antigenic sites.

The genetic control of T lymphocyte proliferative response to the five synthetic antigenic sites of myoglobin, two synthetic nonantigenic control peptides, and one "nonsense" peptide was determined in independent and recombinant strains of mice. In all the strains examined, the nonantigenic control peptides and the "nonsense" peptide did not invoke a response in myoglobin-primed mice. Further, when mice were not primed with whole myoglobin, no response was obtained with any of the antigenic sites. Haplotypes H-2d, H-2f, and H-2s are higher responders to sites 1 and 2, whereas haplotypes H-2d and H-2s are high responders to site 5. Response to site 3 may be controlled by a non-H-2-linked gene. Site 4 can stimulate H-2b and H-2k haplotypes that are nonresponders to the whole myoglobin. Studies with the recombinant strains suggested that Ir genes to sites 1 and 2 map in the I-A subregion and I-C subregion and were designated Ir-Mb-1,2(A) and Ir-Mb-1,2(C). Ir genes to sites 4 and 5 mapped only in the I-A subregion and were designated Ir-Mb-4(A) and Ir-Mb-5(A). These studies suggest that individual antigenic sites in a molecule are controlled by unique Ir genes.

Animals↗