Lee Carrick

Purification and partial characterization of a collagenolytic enzyme from Pseudomonas aeruginosa

A proteinase from Pseudomonas aeruginosa exhibiting collagenolytic activity was purified 1575-fold with a recovery of 24% by use of chemical and chromatographic technics. The enzyme preparation appeared to be homogeneous when subjected to chromatographic, electrophoretic and ultracentrifugational analyses. A standard state sedimentation coefficient of 2.10 S was calculated and further analyses indicated that the enzyme had a molecular weight of 17 500 and dimerizes under certain conditions to yield an apparent molecular weight of 34 000. In addition to insoluble collagen, the enzyme catalyzed the hydrolysis of congocoll, azocoll, soluble collagen and casein, but did not attack orcein-elastin, azoalbumin, p-toluene eulfonyl-L-arginine methyl ester, benzoyl-L-tyrosine ethyl ester, and the hexapeptide N-benzyloxycarbonyl-glycyl-L-prolyglycylglycyl-L-prolyl-L-alanine. Enzymatic activity against congocoll was 6-fold greater at pH 7.5 in Tris with HCl than in phosphate buffer at the same ionic strength. Cobalt, and to a lesser extent, Zn2+ appeared to activate the enzyme, especially in phosphate buffer. NcCN and p-chloromercuribenzoate did not appreciably inhibit enzyme activity, while (NH4)2 SO4, EDTA and cysteine displayed a significant inhibitory effect under certain conditions.

The artificial granuloma 1: In vitro lymphokine production by pulmonary artificial hypersensitivity granulomas☆

Abstract Artificial pulmonary hypersensitivity granulomas were induced around methylated bovine serum albumin (MBSA)-coated beads in sensitized mice. Seven-day-old lesions were isolated and explanted to tissue culture where they were incubated in the presence of MBSA (without serum) for 24 hr. Supernatants from foreign-body granulomas induced around plain beads cultured identically contained no chemotactic and macrophage migration inhibitory materials. The supernatants from the isolated hypersensitivity granuloma cultures contained chemotactic and macrophage migration inhibitory materials which were partially purified and separated by ion-exchange chromatography and gel filtration. The apparent molecular weight, thermostability, and the sensitivity to enzymatic digestion of these fractions was consistent with those reported for other murine lymphokines.

Experimental Studies on Mice Challenged Subcutaneously with Pseudomonas aeruginosa

Summary By use of the subcutaneous route, chronic Pseudomonas aeruginosa infections were established in normal mice undebilitated by burn wounds or leukopenic agents. Using a 21-day holding period, an LD50 value of 4.6 × 108 colony forming units was obtained. After subcutaneous infection, the dermis was completely necrosed with the lesions reaching deep into the subcutaneous tissue and musculature within 3-4 days. Ecthyma gangrenosum-like skin lesions at the site of infection appeared during this time period. By 7-15 days all mice exhibited a systemic infection. Both the livers and lungs showed a great deal of hemorrhage and frequently contained large necrotic foci, while the kidneys showed petechial hemorrhage and occasional renal abscesses. The susceptibility to infection was markedly increased by use of various antineoplastic agents and suprarenal hormones. However, the type of tissue damage or severity was not significantly altered as compared to infected mice which had not received any of the chemical agents. This investigation was supported by the Office of Naval Research Grant N-00014-69-0235-0002, the National Institutes of Health general research support Grant 5 S01 RR 05384, and the National Science Foundation Graduate Traineeship Grant GZ 2033.

The artificial granuloma: III. Collagen synthesis during the cell-mediated granulomatous response as determined in explanted granulomas

Abstract Pulmonary granulomatous inflammations form in response to persistent live or inanimate irritants. The chronic inflammatory reactions cause tissue damage and terminate in fibroses which aggravate the pathology of granulomatous diseases. Fibroblast activity within pulmonary granulomas has not yet been examined. Presently, foreign body, artificial hypersensitivity, and “infectious” granulomas were generated around beads or schistosome eggs in the murine lung. At weekly intervals granulomas were removed from homogenized lungs, the explants were pulsed in vitro with [14C]proline, and the incorporation of label into collagen and noncollagenous protein was measured. Collagen synthesis was verified by collagenase digestion and amino acid analysis of labeled proline and hydroxyproline in hydrolyzates. Collagen synthesis was strongest in the hypersensitivity, weakest in the foreign body explants (schistosome > hypersensitivity bead > foreign body lesions). Peak synthesis occurred in the 1-week-old lesions when 20–40% of the label was incorporated into collagen. Systemic antigen injections given to hypersensitive granuloma-bearing mice significantly elevated collagen synthesis in the explants. Lymphokine-active supernatants from cultured hypersensitivity granulomas given ip to mice bearing nonimmune lesions caused a similar enhancement. Significantly less enhancement was obtained after administration of supernatants from foreign body granulomas. It is concluded that the state of delayed hypersensitivity which maintains the granulomatous inflammation also plays a role in fibroblast activation.

Experimental studies on hematogenously induced renal damage in the rabbit due to Pseudomonas aeruginosa.

Summary Chronic systemic infections of rabbits were established by intravenous inoculation of 4 × 108 P. aeruginosa cells in order to study the sequence of events leading to severe kidney damage. Renal lesions were detected by the fifth- to seventh-day postinfection, as were lesions in the liver and lungs. Progressive azotemia led to death by the 12th-16th day. Lesions in the kidneys, lungs and liver were characterized terminally by intense mononuclear cell infiltrates, hemorrhage, and microabscess formation. Mononuclear cells also appeared to be the predominant responsive cell early in infection. There appeared to be no difference in the susceptibility to infection or severity of renal lesions between rabbits with surgically induced unilateral ureteral obstruction and nonobstructed rabbits. This project was supported in part by the Office of Naval Research, Grant No. N-00014-69-A-0235-0002 and by the Renal Fund of Childrens Hospital of Michigan.

Experimental Studies on Mice Subcutaneously Challenged with Heat-Killed Cells of Pseudomonas aeruginosa

Summary Mice were found to be generally refractory to sc challenge with heat-killed cells of Pseudomonas aeruginosa and did not die unless unusually high concentrations were employed. Approximately 38.6% of the animals receiving a single, sublethal dose of 1 × 1010 dead cells developed black, crusty, necrotic skin lesions within 3 to 5 days. No major gross and histopathological changes were detected in internal organs. If the animals were sc administered sublethal doses of either live or dead cells of P aeruginosa 16 days prior to sc challenge, then the incidence of black lesions rose to 78.6 and 50% of the animals, respectively. Of several antineoplastic agents tested, only methotrexate significantly affected the 72-hr LD50 resulting in a drop to 1.8 × 109 from 3.4 × 1010 cells. However, both methotrexate and actinomy-cin D decreased the incidence of the black lesions. This investigation was supported by the Office of Naval Research Grant No. N-00014-69-0235, the National Science Foundation graduate traineeship Grant No. GZ 2033, and the National Institutes of Health general research support Grant No. 5 S01 RR05384.