Magnitude Of Bacterial Translocation Research Articles

Effect of secretory immunoglobulin A on bacterial translocation in an enterocyte-lymphocyte co-culture model.

Intestinal secretory immunoglobulin A (sIgA) plays an important role in gut mucosal immunity in vivo; however, in-vitro enterocyte models for studying the mechanisms of these effects are lacking. This study utilizes a cell-culture model to investigate the effect of sIgA on bacterial translocation (BT) across human enterocytes co-cultured with human lymphoid cells (Raji cells). This model is intended to mimic in-vivo enterocyte/lymphocyte interactions found in intestinal follicle-associated epithelia. Human Caco-2 enterocytes were grown to confluence on porous filters in the apical chamber of a two-chamber cell-culture system. After differentiation, human B lymphoid cells (Raji cells) were added to the basolateral surface of Caco-2 monolayers for 3 days' co-culture, followed by washing away of unincorporated Raji cells. Transepithelial electrical resistance (TEER) was used to measure tight-junction permeability. Monolayers were treated with or without sIgA, IgG (negative control), or mannose (positive control). BT across the cell monolayer was determined 1.5 h after addition of Escherichia coli. Statistical analysis was by the Kruskal-Wallis test, P below 0.05 considered significant. In co-culture monolayers treated with sIgA, IgG, or mannose, there was no significant effect on TEER; however, the magnitude of BT across cells treated with sIgA (1.3 +/- 0.4 log10CFU/ml) and mannose (1.6 +/- 1.1 log10CFU/ml) was significantly decreased compared to PBS (3.9 +/- 0.4 log10CFU/ml) and IgG (2.9 +/- 0.6 log10CFU/ml) controls (P < 0.05). sIgA BT inhibition was dose-dependent. BT inhibition by sIgA and mannose was additive (0.5 +/- 1 log10CFU/ml). Inhibition of BT was negated when sIgA and mannose were removed by washing prior to E. Coli addition (3.6 +/- 0.5 log10CFU/ml), suggesting that both inhibitors act through bacterial binding.

The effect of phospholipids and fatty acids on tight-junction permeability and bacterial translocation.

The activity of phospholipase A2 (PLA2) is elevated in the intestinal epithelia of patients with inflammatory bowel disease (IBD). We recently reported that PLA2 mediates hydrolysis of phosphatidylcholine (PC) to lysophosphatidylcholine (L-PC) when both are applied to the apical surface of cultured EC monolayers, resulting in increased bacterial translocation (BT) and decreased transepithelial electrical resistance (TEER). Free fatty acids (FFA) are the other products of this reaction, however, their effect on Caco-2 cell permeability has not been reported. In addition to PC, other luminal phospholipids are present at the surface of the enterocyte. PLA2 may also mediate the hydrolysis of luminal phospholipids other than PC. The aim of this study was to examine the effects of phospholipids other than PC and common FFA on intestinal epithelial permeability and BT. Human Caco-2 enterocytes were grown to confluence on porous filters in the apical chamber of a two-chamber cell-culture system. Monolayer integrity and tight-junction permeability were measured as TEER. First, common FFA released by PC hydrolysis were determined using thin-layer chromatography (TLC). In separate experiments, monolayers were treated with phosphatidylethanolamine (PE), lysophosphatidylethanolamine (L-PE), or palmitoleic acid, oleic acids, linoleic acids, and arachidonic acid solubilized in solution with PC. The magnitude of BT was determined 2 h after treatment by adding Escherichia coli C25 to the apical chamber followed by quantitatively culturing basal-chamber samples. Statistical analysis was by the Kurosaki-Wallis test. TLC of PC samples incubated with PLA2 on the apical surface of Caco-2 monolayers demonstrated the production of palmitoleic acid, oleic acids, linoleic acids, and arachidonic acid. L-PE significantly decreased TEER compared to controls, but to a lesser degree than L-PC alone. L-PE had no effects on BT. Palmitoleic acid and oleic acid likewise significantly decreased TEER compared to controls, however, less than L-PC. All FFA tested had no effect on BT. Phospholipids applied to the apical surface of enterocytes, such as those found in vivo in mucus, can be hydrolyzed by the enzyme PLA2 resulting in lysophospholipid and FFA species that can alter enterocyte monolayer permeability. However, FFA and L-PL, other than L-PC, appear to have no effect to stimulate BT. This observation may have clinical implications in the pathogenesis and treatment strategies for IBD patients in whom enterocyte PLA2 activity has been shown to be elevated.

The effect of phospholipase A2 on bacterial translocation in a cell culture model.

The activity of phospholipase (PL)A2 is elevated in the intestinal epithelia of patients with inflammatory bowel disease (IBD). Recently, we reported that lysophosphatidylcholine (L-PC), the PLA2 hydrolysis product of phosphatidylcholine (PC), stimulates bacterial translocation (BT) in an enterocyte cell-culture model. These two observations stimulated us to examine the effects of extracellular PLA2 on intestinal epithelial permeability. Human Caco-2 enterocytes were grown to confluence on porous filters in the apical chamber of a two-chamber cell-culture system. Monolayer integrity and tight-junction permeability were measured by dextran blue (DB) permeability and transepithelial electric resistance (TEER). Monolayers were treated with PC, L-PC, or PLA2 with and without PC. The magnitude of BT was determined 2 h after treatment by adding Escherichia coli to the apical chamber followed by quantitatively culturing basal chamber samples. Thin-layer chromatography (TLC) was utilized to verify PLA2 hydrolysis of PC to L-PC. Statistical analysis was performed by one-way analysis of variance. The magnitude of BT across monolayers pretreated with PLA2 + PC significantly increased compared to either PC or PLA2 (6.83 +/- 0.069, 2.41 +/- 0.46, and 3.06 +/- 1.14 log10 colony forming units/ml, respectively, P < 0.05). Absence of DB-permeability in any group confirmed monolayer integrity. TLC of PL samples harvested from the apical monolayer surface confirmed PC hydrolysis. PLA2 mediates hydrolysis of PC to L-PC when both are applied to the apical surface of cultured enterocyte monolayers, resulting in increased BT and increased TEER with no damage to monolayer integrity. These observations may have implications in the pathogenesis and treatment strategies for IBD.

The effect of mucin on bacterial translocation in I-407 fetal and Caco-2 adult enterocyte cultured cell lines.

Although the intestinal mucosa forms a crucial barrier between the host and the environment, bacterial translocation (BT) occurs frequently in neonates and may be a source of sepsis. The intestinal mucous gel layer is thought to be a vital component of the gut barrier and is composed, in part, of a family of glycoproteins known as mucins. Our aim was to study the effects of mucin on BT in an enterocyte cell-culture model using a fetal (I-407) and an adult (Caco-2) intestinal cell line. I-407 and Caco-2 cells were grown to confluence on porous filters in a two-chamber Transwell system. The integrity of the monolayers was confirmed by transepithelial electrical resistance (TEER) and permeability using the macromolecule dextran blue. Cells were treated with mucin (40 mg/ml) prior to inoculation of 1 x 10(6) Escherichia coli C25. The magnitude of BT was determined quantitatively by culturing the samples from the basal chamber of the wells and was expressed as log 10 [Colony Forming Units (CFU)/ml]. Statistical analysis was performed by the Mann-Whitney U test with statistical significance at P < 0.05. Mucin inhibited BT across both fetal and adult cultured enterocyte monolayers; however, the inhibitory effect was less on the fetal cells compared to the adult cells. Dextran-blue studies showed that monolayers were intact throughout the experiments. Despite 98% inhibition of BT, mucin had a statistically significant effect on post-bacterial inoculation TEER in Caco-2 cells and no effect in I-407 cells. The ability of mucin, a mucous-barrier glycoprotein, to inhibit BT across immature intestinal enterocytes, as in the neonate, may be diminished compared to mature adult enterocytes.

THE GUT

The aim of this study was to test the hypothesis that the gut is capable of becoming a cytokine-generating organ following either a lethal or nonlethal inflammatory insult. Adult male rats were given an intraperitoneal challenge with saline, or with a nonlethal (.1 mg/g) or LD50 (.5 mg/g) dose of zymosan. Mesenteric lymph nodes, efferent mesenteric lymph, liver, spleen, and blood (portal and systemic) were obtained at 2, 4, 6, 8, or 10 h post challenge. Organs, lymph, and blood were tested for bacterial translocation (BT); blood and lymph were assayed for tumor necrosis factor (TNF) and IL-6. After .1 mg/g zymosan, BT was limited to the mesenteric lymph node complex only; .5 mg/g zymosan promoted BT to blood, mesenteric lymph, and organs (p < .05 vs. control or .1 mg/g zymosan). The magnitude of portal bacteremia was greater than systemic bacteremia (p < .003). Serum TNF peaked at 2 h (p < .05 vs. control), and serum IL-6 peaked at 4-6 h (p < .05 vs. control) post zymosan challenge. Portal and systemic bioactivity was similar for either cytokine, and serum bioactivity did not correlate with zymosan dose. TNF bioactivity was increased in the mesenteric lymph at 2 h post challenge with .5 mg/g zymosan only (p < .05 vs. control or .1 mg/g zymosan). IL-6 bioactivity was increased in the mesenteric lymph at 4 through 10 h post zymosan challenge (p < .05 vs. control), but was similar with either dosage of zymosan. In conclusion, the gut may be capable of producing cytokines in response to an inflammatory stimulus, even in the absence of portal or systemic spread of bacteria. The magnitude of the cytokine response does not correlate with the magnitude of bacterial translocation.

Relationship between extent of burn injury and magnitude of microbial translocation from the intestine.

The gut can be a source of sepsis after thermal injury. In the present study the relationship between the extent of burn injury and magnitude of bacterial translocation was investigated. Mice underwent 0%, 10%, 20%, 30%, or 50% total body surface area full-thickness burn and simultaneous gavage with 1 x 10(10) 14C-labeled Escherichia coli. mesenteric lymph nodes, liver, spleen, peritoneal fluid, and burn wound were excised 4 hours after burn injury. Residual radioactivity and bacterial colony counts were measured, and percentages of viable organisms were calculated. Results showed that the rate of translocation of 14C E. coli increased proportionally with the burn size, reaching a maximum at 30%. The cutaneous eschar collected a remarkable amount of labeled bacteria, suggesting enteric microflora as a possible source of contamination of the burn wound via endogenous routes. The percentage of viable organisms in the tissues demonstrated that the ability of mesenteric lymph nodes, liver, and eschar to clear translocated bacteria was directly affected by the severity of the burn injury.

Effect of stress and trauma on bacterial translocation from the Gut

Previously, we established that bacteria contained within the gut can cross the GI mucosal barrier and spread systemically, a process termed bacterial translocation. Three models were used to extend this work: (1) cold exposure (up to 16 hr at 4°C), a nontissue injury stress model; (2) femoral fracture-amputation, a trauma model; and (3) thermal injury (30% third-degree burn), a trauma model with retained necrotic tissue. CD-1 mice either with a normal GI microflora or who were monoassociated with Escherichia coli C-25 were subjected to sham or actual stress or trauma. The animals were sacrificed at various times postinsult and the ceca, mesenteric lymph nodes (MLN), spleens, and livers were quantitatively cultured. Neither the incidence nor the magnitude of bacterial translocation was increased in the cold-exposed animals compared to control mice. The incidence of bacterial translocation to the systemic organs was higher in the animals with a normal flora receiving femoral fracture amputation (11%) ( P < 0.02) than in animals receiving a thermal injury (1%) or sham-injured control mice (0%). In contrast, the incidence of translocation to the liver or spleen was higher in burned mice monoassociated with E. coli C-25 (60%) ( P < 0.01) than in E. coli monoassociated mice sustaining femoral fracture amputation (17%). Stress alone (cold exposure) does not promote bacterial translocation; however, trauma, especially in combination with retained necrotic tissue, promotes bacterial translocation. Thus bacteria colonizing the gut can invade systemic organs after trauma, especially when the normal ecology of the gut flora has been disrupted.