Polyanionic polysaccharides reduce intra-abdominal adhesion and abscess formation in a rat peritonitis model.

BACKGROUND: Intra-abdominal infection is complicated by adhesion and abscess formation. We have assessed the adhesion- and abscess-reducing capacity of various solution volumes and concentrations of two polyanionic polysaccharides, hyaluronan (HA) and carboxymethylcellulose (CMC), in a rat peritonitis model. STUDY DESIGN: In 192 male Wistar rats a bacterial peritonitis was induced using cecal ligation and puncture. After 24 h the abdomen was reopened and the ligated cecum resected. Animals were randomized into three control groups, nine groups treated with various solution volumes (1 to 8 ml) containing different HA concentrations, and four groups treated with 1.7% CMC solution. Rats were killed at day 7, postoperatively, and adhesions were scored at five abdominal sites on a scale from 0 to 4. The presence and size of intra-abdominal abscesses were noted. RESULTS: Fifty-four rats (28%) prematurely died. There was no significant difference in mortality between treatment groups and controls. Treatment with CMC (P < 0.001) and low (0.2 and 0.4%) concentrations of HA (P < 0.005) significantly reduced intra-abdominal adhesion formation. High volumes of 0.2 and 0.4% HA were most effective (P = 0.01). The effect of CMC was volume independent. The incidence of abdominal abscesses was also significantly reduced by treatment with either CMC (P < 0.001) or low concentrations of HA (P < 0.001). With regard to abscess formation the effect was independent of the volume administered for HA, while low volumes of CMC were most effective (P < 0.005). CONCLUSION: Intraperitoneal treatment with either CMC or low-viscosity HA solution reduced intra-abdominal adhesion and abscess formation in a rat peritonitis model. The volume-induced reduction in adhesion formation suggests a hydroflotation effect of HA solution.

Removal of necrotic tissue with an ananain-based enzyme-debriding preparation.

An enzymatic debriding preparation was formulated with purified enzyme derived from a crude pineapple stem extract. The primary component of this preparation was the sulfhydryl protease ananain which represented >/=85% of the proteolytic activity. The remaining proteolytic activity in the preparation was contributed by a co-purifying homologous cysteine protease comosain. Taken together these two proteases provided a protein purity of greater than 95% as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This ananain-based enzyme preparation exhibited both gelatinolytic and fibrinolytic activity in vitro. Ananain-based enzyme preparation was formulated in a hydrophilic cream vehicle at concentrations ranging from 115 to 260 U/gm vehicle. Ananain-based enzyme preparation formulated in this fashion is referred to as Vianain debriding agent. Vianain was applied to partial-thickness cutaneous burn wounds produced in the skin of domestic pigs. A maximum of two 4-hour applications of Vianain provided complete debridement of eschar from the partial-thickness burn wounds as judged by light and electron microscopic analyses of biopsy specimens harvested before and after debridement. Wounds debrided with Vianain exhibited more rapid reepithelialization as compared with wounds that were not debrided. Wounds on pigs that were hyperimmunized to ananain-based enzyme preparation before burning and debridement with Vianain exhibited a similar enhancement in reepithelialization as compared with wounds treated with vehicle alone. The capacity of Vianain to debride necrotic tissue was also evaluated in a guinea pig ischemic ulcer model. Full-thickness ischemic lesions were created on the back of guinea pigs. Vianain was applied to the hardened necrotic tissue for 6 hours per day for up to a maximum of 5 days. Complete debridement of these wounds was accomplished within 4 to 5 days. Treatment of ischemic cutaneous ulcerations in this animal model with two commercially available enzyme-debriding agents provided little or no debridement of the necrotic tissue. In vitro, Vianain treatment of surgically debrided human tissue samples, obtained from patients with burn injury or cutaneous ulcers, showed that the protease preparation was effective in rapidly digesting these necrotic tissues.

Debridement of porcine burns with a highly purified, ananain-based cysteine protease preparation.

A novel enzymatic debriding agent was evaluated on experimental full-thickness porcine contact burns. This agent consists of a highly purified, ananain-based, cysteine protease preparation formulated in a hydrophilic cream vehicle. Debridement of full-thickness burns was found to be dependent on several factors including the concentration of enzyme in the vehicle, the duration of treatment, and the hydration status of the burn wound before treatment. With an optimized debridement regimen, burns were consistently debrided of all gelatinized tissue with two 5-hour treatments. Histologic evaluation of the debrided wounds revealed an acellular deeper dermis that was debrided of necrotic cellular debris; however, the collagen matrix of the deeper dermis remained intact. This observation was consistent with a demonstrated in vitro specificity of the ananain-based protease for gelatin over collagen. A direct comparison of debridement efficacy with sutilains ointment, showed the ananain-based, debriding enzyme preparation to provide more rapid debridement of gelatinized tissue. Enzymatically debrided wounds exhibited graft take only after surgical excision of approximately 1 mm of the remaining acellular, avascular dermis. This highly purified enzyme preparation offers the potential for rapid nonsurgical debridement of gelatinized burn tissue, but required additional surgical debridement for graft take in this porcine model.

Synthesis and characterization of a model extracellular matrix that induces partial regeneration of adult mammalian skin.

Regeneration of the dermis does not occur spontaneously in the adult mammal. The epidermis is regenerated spontaneously provided there is a dermal substrate over which it can migrate. Certain highly porous, crosslinked collagen-glycosaminoglycan copolymers have induced partial morphogenesis of skin when seeded with dermal and epidermal cells and then grafted on standard, full-thickness skin wounds in the adult guinea pig. A mature epidermis and a nearly physiological dermis, which lacked hair follicles but was demonstrably different from scar, were regenerated over areas as large as 16 cm2. These chemical analogs of extracellular matrices were morphogenetically active provided that the average pore diameter ranged between 20 and 125 microns, the resistance to degradation by collagenase exceeded a critical limit, and the density of autologous dermal and epidermal cells inoculated therein was greater than 5 x 10(4) cells per cm2 of wound area. Unseeded copolymers with physical structures that were within these limits delayed the onset of wound contraction by about 10 days but did not eventually prevent it. Seeded copolymers not only delayed contraction but eventually arrested and reversed it while new skin was being regenerated. The data identify a model extracellular matrix that acts as if it were an insoluble growth factor with narrowly specified physiochemical structure, functioning as a transient basal lamina during morphogenesis of skin.

Alteration of the electrophoretic mobility of human peripheral blood mononuclear cells following treatment with dimethyl sulfoxide.

Studies have been conducted to determine the effects of DMSO and freezing on the electrophoretic distribution of peripheral blood mononuclear cells. Sodium [51Cr]chromate was used to label the cells, and the distributions of cell number and cell-associated radioactivity were determined. Cells treated with DMSO had a narrower distribution of electrophoretic mobilities when compared with those not treated. DMSO-treated cells also demonstrated a more homogeneous distribution of radioactivity relative to the cell distribution than did the nontreated cells. The freezing of DMSO-treated cells did not result in any additional alteration of electrophoretic pattern compared to DMSO treatment alone. Analysis by linear categorization techniques indicated that the DMSO-treated and nontreated cells were completely distinguished by their electrophoretic behavior.

Wound tissue can utilize a polymeric template to synthesize a functional extension of skin.

Prompt and long-term closure of full-thickness skin wounds is guinea pigs and humans is achieved by applying a bilayer polymeric membrane. The membrane comprises a top layer of a silicone elastomer and a bottom layer of a porous cross-linked network of collagen and glycosaminoglycan. The bottom layer can be seeded with a small number of autologous basal cells before grafting. No immunosuppression is used and infection, exudation, and rejection are absent. Host tissue utilizes the sterile membrane as a culture medium to synthesize neoepidermal and neodermal tissue. A functional extension of skin over the entire wound area is formed in about 4 weeks.

51Cr and DF32P labeling of human blood cells in leukocyte-rich plasma.

Cell-rich plasma from human peripheral blood was labeled with disodium chromate (51Cr) and diisopropylfluorophosphate (DF32P), and the uptake and distribution of the radionuclides by granulocytes, lymphocytes, monocytes, and erythrocytes were studied. Velocity sedimentation through a 1-2% albumin gradient and electrophoresis in a Ficoll-sucrose gradient demonstrated that granulocytes migrated the fastest, followed by monocytes, and then by the mixture of lymphocyte-red cell populations. Monocytes accumulated four to five times as much 51Cr as the other leukocytes or the red blood cells. Erythrocytes showed a greater uptake of 51Cr than did granulocytes or lymphocytes. Granulocytes were preferentially labeled in vitro by DF32P, and the uptake of DF32P by lymphocytes was about one-third that by granulocytes. No measurable DF32P labeling of monocytes and only slight labeling of erythrocytes was observed by liquid scintillation counting of the labeled cells. These data have implications in the interpretation of in vivo survival studies of radiolabeled leukocytes.

Studies on the in vivo elution of 51Cr from baboon red blood cells.

Four anticoagulant solutions were added to baboon red blood cells prior to labeling with 51Cr to determine how each would influence the distribution of 51Cr within the red blood cells, the loss of 51Cr from the red blood cells after transfusion, and the calculated red cell survival value. The 51Cr label was detected in the hemoglobin and in the low molecular weight compounds within the red blood cells. The elution of 51Cr from labeled baboon red blood cells following transfusion could not be explained by the distribution of 51Cr between hemoglobin and low molecular weight compounds within the red blood cells.

In vitro interactions of 51Cr in human red blood cells and hemolysates.

The intracellular distribution of radioactivity was studied in normal and sickle erythrocytes labeled with sodium 51Cr chromate. Both types of cells had a higher fraction of 51Cr bound to hemoglobin when labeled in the presence of ACD at a pH of 7.09 than when labeled in the presence of CPD at a pH of 5.96. Citrate at a pH of 5.96 or less entered the red blood cells and decreased the 51Cr binding hemoglobin. Binding of 51Cr to hemoglobin within the red cells was also reduced when the ATP and DPG levels in the red blood cells were elevated. Studies with hemoglobin solution showed that 51Cr binding to hemoglobin was influenced by 2,3-DPG, ATP and citrate.

Gravity sedimentation analysis of human blood leukocytes.

A new method of sedimentation analysis of human blood leukocytes is described. Platelets, lymphocytes, monocytes, and polymorphonuclear cells isolated from normal human peripheral blood have been analyzed alone and in mixture by gravity sedimentation, employing a computerized scanning instrument. All four classes could be clearly resolved from each other exhibiting sedimentation velocities of 0.06 +/- 0.00, 1.04 +/- 0.11, 1.27 +/- 0.15 and 1.89 +/- 0.21 x 10(-4) cm/s, respectively, at 37 degrees C in a 2.5--6.25% Ficoll gradient in Medium 199. Less than 10(6) cells can be used for analysis. Possible applications of the method are discussed.

Cytotaxins after the sedimentation behavior of human granulocytes.

Human granulocytes from the peripheral blood of healthy donors were subjected to transient gravity sedimentation analysis in Ficoll density gradient columns (37 degrees C) containing different concentrations of Escherichia coli endotoxin-activated serum and medium 199. A dramatic serum concentration-dependent dispersion of the cells based on changes in sedimentation velocity was observed as a function of time, using a new optical scanning instrument. The phenomenon was virtually abolished in the presence of cytochalasin B, a known inhibitor of cellular chemotaxis. The width (second statistical moment) of the sedimenting cell distribution increased in a sigmoid fashion as a function of time regardless of cytotaxin concentration. This indicates that a slow and nonlinear response of the granulocytes to the cytotaxins occurs. This new kinetic method should be useful in examining an alternate manifestation of the chemoresponsiveness of phagocytic cells and of cell interactions in general.

In vitro function of granulocytes isolated from blood of normal volunteers using continuous-flow centrifugation in the IBM-aminco celltrifuge and adhesion-filtration leukapheresis using nylon fiber.

Granulocytes were harvested from each of five healthy male volunteers once by continuous flow centrifugation with the IBM-Aminco Celltrifuge, and once by adhesion filtration leukapheresis with nylon fiber. Granulocyte recovery and purity were significantly better with the filtration leukapheresis system than with continuous flow centrifugation. Measurements of trypan blue dye exclusion and muramidase activity were similar to those in control granulocytes regardless of the method of isolation. Granulocyte-stimulated oxygen consumption was diminished in granulocytes prepared by the adhesion filtration method, but normal in those prepared by continuous flow centrifugation with the IBM-Aminco Celltrifuge.

Oxygen consumption, platelet aggregation and release reactions in platelets freeze-preserved with dimethylsulfoxide.

Platelets were frozen with 4% or 5% DMSO at an overall rate of 2 to 3 C per minute and were stored at -80 C for as long as 10 months. They were washed with DMSO-plasma and acid-citrate-dextrose (ACD) solutions and were stored in 30 ml of autologous plasma at room temperature for about three hours before transfusion. Measurements were made of oxygen consumption, platelet aggregation and release reaction, platelet factor-3 and-4 activities, and platelet response to hypotonic stress. Platelet basal and latex stimulated oxygen consumption were found to be significantly impaired; platelet aggregation response to ADP, epinephrine, and collagen were decreased; platelet ATP and ADP content and release reactions were decreased; platelet antiheparin activity (platelet factor-4 level) was decreased; and the platelet response to hypotonic stress was impaired. What the results of these in vitro tests mean in relation to in vivo survival and hemostatic function of preserved platelets was not established.

The effect of cryogenic storage on human erythrocyte membrane proteins as determined by polyacrylamide-gel electrophoresis.

A study was conducted to determine the effects of freezing on the major membrane proteins of isolated human erythrocyte membranes. Membranes in low or normal ionic strength medium were frozen at slow or fast freezing rates. The membrane protein composition and elution of proteins from the membranes were studied utilizing polyacrylamide-gel electrophoresis in a sodium dodecyl sulfate or an acetic acid-urea-phenol solvent system. Neither a change in the composition of the membrane proteins nor any elution of membrane protein during freezing and thawing was observed. The data indicate that any human erythrocyte membrane damage during freezing and thawing was not related to a change in major membrane protein composition. Human red cell membranes were stable at --80 or --196degreesC in the absence of a cryoprotective agent.

The determination of leukocyte phagocytic oxidase activity by measurement of the initial rate of stimulated oxygen consumption.

A method for the estimation of leukocyte phagocytic oxidase activity is described, which is based upon the polarographic measurement of the initial rate of oxygen consumption of peripheral blood leukocyte suspensions during maximally active phagocytosis. Mean values are presented for 22 normal donors, and the effects of centrifugal force, serial washing, temperature of storage, and the presence of platelets in the incubation mixture are described and discussed.