Michael D. Peck

The process of microbial translocation

The process of microbial translocation was studied using Candida albicans, Escherichia coli, or endotoxin instilled into Thiry-Vella loops of thermally injured guinea pigs and rats. Translocation of C. albicans occurred by direct penetration of enterocytes by a unique process different from classical phagocytosis. Translocation between enterocytes was not observed. Internalization was associated with a disturbance of the plasma membrane and brush border, but most internalized organisms were not surrounded by a plasma membrane. Passage of the candida into the lamina propria appeared to be associated with disruption of the basal membrane with extrusion of cytoplasm of the cell and candida. Organisms in the lamina propria were commonly phagocytized by macrophages but also were found free in lymphatics and blood vessels. Translocation of E. coli and endotoxin also occurred directly through enterocytes rather than between them, but translocated endotoxin diffused through the lamina propria and muscular wall of the bowel wall by passing between rather than through the myocytes. These descriptive phenomena provide new insight into the role of the enterocyte and intestinal immune cells in the translocation process.

Arginine Supplementation and Its Effect on Established Peritonitis in Guinea Pigs

To evaluate the efficacy of supplemental arginine with nutritional support in the presence of sepsis, eighty-eight gastrostomized female Hartley guinea pigs were implanted with osmotic pumps effusing an Eschericia coli/Staphylococcus aureus mixture. Animals were randomized and infused for two weeks with isocaloric and isovolumetric diets containing 0%, 2%, 4%, or 6% supplemental arginine as arginine hydrochloride. Survival was 12/22 (54%) in 0%, 9/22 (41%) in 2% and 4%, and 2/22 (9%) in 6%. Analysis by chi-square test of independence was significant (p = 0.0141) with 6% survival lower than the others. Median survival was 11 days in 0%, 8 days in 2% and 6%, and 9 days in 4%. Median survival was longer in 0% than in 2% or 6% (Kruskal-Wallis ANOVA: p = 0.02). Nitrogen balance was significantly lower in 6% compared to 0% on days 2 through 10, and lower than 2% and 4% on days 6 and 9. Nitrogen balance was higher in 0% than in 2% on days 4, 6, 10, and 13. Serum albumin and C3 were lower in all experimental groups than normal controls (ANOVA: p = 0.01). Comparison of liver, spleen, adrenals, gastrocnemius, and carcass weights, cell-mediated immunity as determined by contact sensitivity to DNFB, and transferrin showed no significant differences. There was a positive dose-response effect seen amongst the experimental groups for the amino acids arginine, ornithine, and citrulline in relation to the amount of supplemental arginine. This study suggests that dietary arginine supplementation does not enhance survival in a guinea pig model of established peritonitis.

A new model for studying nutrition in peritonitis. The adverse effect of overfeeding.

In guinea pigs fed ad libitum, controlled intraperitoneal infusion of bacteria by an implanted 7-day osmotic pump resulted in peritonitis or abscess formation with a 50% survival 14–18 days after pump implantation. Administration of 125 kcal/kg/day of a diet found to be optimal for burned guinea pigs by continuous pump controlled feedings via a previously placed gastrostomy was well-tolerated, with a 62.5% mortality by Day 17. Administration of only 100 kcal/kg/day caused weight loss of approximately 17% after 16 days, but fewer animals died (42.8%, p = NS). Feeding either 150 kcal/kg/day or 175 kcal/kg/day caused death in all 25 animals (p< 0.001) and their survival time was slightly shortened (p = NS) when compared with animals receiving 100 or 125 kcal/kg/day. This is the first animal model of peritonitis that permits incisive dissection of the relative influences of dietary composition on outcome, because survival can be extended to 2 weeks or more in the presence of continuing sepsis.

The Role of Protein and Calorie Restriction in Outcome From Salmonella Infection in Mice

We studied the separate effects of protein and calorie restriction in mice challenged with Salmonella typhimurium, an intracellular pathogen eliminated by cell-mediated immunity. Female A/J mice (n = 73) were placed on one of eight solid diets for 3 weeks. Animals were weighed at the beginning and the end of the feeding period. Diets were adjusted by two factors. The total amount of protein in the diet was 1%, 5%, 20%, or 40% by weight. The diets were fed to half the mice in quantities of 3 g and to the other half at 1.5 g per mouse per day. At the end of 3 weeks, mice were injected intraperitoneally with bacteria and mortality was observed for 2 weeks. Mortality was related to protein intake and was significantly higher in the 1% and 5% groups (chi 2: p = .0021). However, mortality was lower in the calorie-restricted groups (chi 2: p = .0242). Although caloric intake did not affect cell-mediated immunity, the response to 2,4-dinitrofluorobenzene was greater in the low protein groups. Lymphoproliferative responses in the mixed lymphocyte response were not affected by either caloric or protein intake. Lymphoproliferative responses to both lipopolysaccharide and phytohemagglutinin were affected by dietary protein but not by caloric intake; proliferative responses were higher in the low-protein groups. We conclude that protein restriction can increase mortality in this model. On the other hand, short-term calorie restriction can improve survival.

The effect of dietary fatty acids on response to Pseudomonas infection in burned mice

Since fatty acids influence prostaglandin synthesis, and since both fatty acids and prostaglandins modulate immune function, we investigated the hypothesis that manipulation of dietary fats would affect survival after infection in a murine burn model. Mice were fed for 2 to 3 weeks with diets containing different types and amounts of fat. They were then subjected to a 20% flame burn and infected with Pseudomonas aeruginosa. Survival in the group fed 40% of total calories as fish oil had significantly higher mortality than those fed safflower oil. This difference was not noted at lower fat levels. Similar groups of animals were sacrificed the day after injection. Splenic macrophage production of PGE2 was significantly lower in the fish-oil group, but production of LTB4 and TXB2 were not affected. In vitro tests of T- and B-cell function were not different amongst groups. We conclude that manipulation of dietary fats can alter outcome in this murine model of infection after thermal injury.

Low protein diets improve survival from peritonitis in guinea pigs.

Enteral diets with different protein content were tested to determine their effect on outcome in a model of protracted bacterial peritonitis. Hartley guinea pigs were provided with gastrostomies, and 1 week later, osmotic pumps were implanted into the peritoneal cavity to allow for continuous release of live bacteria over the course of 1 week. Three days after pump implantation, the animals began receiving isocaloric enteral diets that contained 5%, 10%, 15%, or 20% of total calories as protein. After 2 weeks of observation, the survivors were killed. All animals lost weight during the 2-weeks period, but there was no difference in weight lost. Nitrogen balance correlated with dietary protein. The mortality rate was significantly higher in the groups that received 15% and 20% of total calories compared with the group that received 5% (p less than 0.05). Although dietary protein in the 5% group was insufficient for meeting the nutritional needs of the animal, survival was best in this group. Possible explanations are that protein restriction in this model may either augment host defence or impair bacterial virulence.

Short-Term Dietary Lipid Manipulation Does Not Affect Survival in Two Models of Murine Sepsis

Dietary lipid manipulation has been shown to have various effects on the immune system, depending on the amount of fat, degree of saturation, and type of fat used. In this study we investigated the role of different sources of fat at different levels on the survival of mice in two models of peritonitis, one with Pseudomonas aeruginosa and the other with Salmonella typhimurium. CF1 mice were pair-fed diets with 5% or 40% of total calories as fat. The source of fat used was coconut oil, oleic acid, safflower oil, or fish oil. Three other diets were tested as well, one with no fat, one with only 0.5% of total calories linoleic acid as the only source of fat, and a control diet that had 12% of total calories as corn oil. At the end of 2 weeks of feeding the experimental diets, mice were challenged with Ps aeruginosa intraperitoneally and mortality was recorded over 1 week. After 3 weeks of feeding the experimental diets, mice were challenged with S typhimurium and mortality was recorded over 2 weeks. No significant differences were seen on survival among groups fed different levels of fat, as well as different sources of fat. We conclude that, overall, 2- and 3-week manipulation of dietary fat does not affect outcome from infection in these models.

Composition of fat in enteral diets can influence outcome in experimental peritonitis.

An animal model of protracted acute peritonitis was used to study the effects on survival of different amounts and types of dietary fat. Guinea pigs (n = 132) were provided with gastrostomies and allowed to recover. Intraperitoneal osmotic pumps were then placed, allowing for effusion of viable bacteria during the next week. Three days after pump implantation, the animals were randomized to receive one of nine diets, differing only in fat content (3.5%, 14%, or 56% of total calories) and fat composition (100% Microlipid [primarily safflower oil, rich in linoleic acid], 100% MaxEPA [fish oil containing 35% omega-three fatty acids], or a 50:50 mixture of both oils). At the end of 2 weeks, survival rates were compared. Among all types of fat used, the level of fat did not affect outcome, with survival rates of 13% (6 of 45) at the low level of fat used, 30% (13 of 44) at the medium level, and 26% (11 of 43) at the high level (P greater than 0.10). However fat composition significantly influenced survival across all levels of fat, with a 39% (17 of 44) survival rate in the groups given equal amounts of Microlipid and MaxEPA, compared either to 21% (9 of 44) for the 100% Microlipid groups, or to 9% (4 of 44) for the 100% MaxEPA groups (p less than 0.05). A second experiment was performed with 58 animals fed five different mixtures of fats, all at 14% of total calories. Diets were made with fat ratios of 100:0, 75:25, 50:50, 25:75, and 0:100 Microlipid to MaxEPA. The survival rate in the 50:50 group was 83% (10 of 12), which is significantly higher than the survival rates with any of the others (p less than 0.05). It is concluded that the amount of fat in the diet does not strongly influence outcome in this model. However an equal mixture of safflower and fish oils significantly improves survival rates compared to diets made with single-oil preparations.

Survival in Septic Guinea Pigs Is Influenced by Vitamin E, but Not by Vitamin C in Enteral Diets

Oxygen-free radicals are produced during sepsis, and may contribute to cell injury and dysfunction. We studied the effect of different levels of vitamins E and C in the diet fed enterally to septic guinea pigs. Sixty-four female guinea pigs were provided with gastrostomies and allowed to recover. Intraperitoneal osmotic pumps were then implanted that provided effusion of Escherichia coli and Staphylococcus aureus for the next 7 days. Three days after pump implantations, the animals were started on one of nine diets. The diets were isocaloric and isonitrogenous, and differed only in the amounts of vitamins E and C. Three levels of each vitamin were used, based on the Recommended Daily Allowance (RDA). The feedings were continued for 2 weeks, during which time mortality was observed. The amount of vitamin C had no effect on outcome, with mortality rates of 68% (15/22) in the 1 x RDA group, 73% (16/22) in the 5 x RDA group, and 65% (13/20) in the 25 x RDA group. However, vitamin E altered outcome significantly, with mortality rates of 86% (18/21) in the 1 x RDA group, 45% (10/22) in the 3 x RDA group, and 76% (16/21) in the 9 x RDA group. Mortality in the 3 x RDA group was significantly lower than that in the 1 x RDA group and in the 9 x RDA group.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of free radical scavengers on outcome after infection in burned mice.

Thermal injury generates free radicals from various cellular populations, and modulation of free radical activity with scavengers may improve outcome. Balb/c mice were infected with Ps. aeruginosa the day after burn injury and mortality rates observed. Tocopherol was given by gavage daily for 4 days starting 2 days before burn. Survival was improved in the groups receiving 25 and 250 IU/kg compared to control. In other groups, tocopherol was given after burn, but mortality rates were not changed. Treatment with the oxygen radical scavengers ascorbic acid, and a combination of superoxide dismutase and catalase had no effect on mortality. We conclude that ascorbic acid, superoxide dismutase, and catalase do not appear to benefit survival after thermal injury and infection in this murine model. Tocopherol given in high doses of at least 25 IU/kg by gavage preceding injury and continued thereafter for 2 days did improve survival, but this benefit was not seen if tocopherol was not started until after injury.