Aladdein Mattar

Effect of probiotics on enterocyte bacterial translocation in vitro

Abstract Enteral probiotics such as Lactobacillus casei GG (LGG) have been used in the treatment of a variety of intestinal disorders in infants and children, including diarrhea, malabsorption, and Clostridium difficile colitis. We have previously demonstrated that the probiotic bacterium LGG has an inhibitory effect on bacterial translocation (BT) in a neonatal rabbit model. However, this in-vivo model is limited for investigating the cellular and molecular mechanisms responsible for probiotic inhibition of BT. The purpose of this study was to determine the efficacy of LGG in reducing the rate of Escherichia coliC25 (E. coli C25) translocation using an in-vitro enterocyte cell-culture model. Human colonic carcinoma (Caco-2) enterocytes were seeded in porous filters in the apical chamber of a two-chamber cell- culture system and grown for 14 days to confluence. The monolayers were incubated at 37 °C with LGG for 180 min. Non-adherent LGG was washed away prior to a 120-min incubation period with 105 CFU E. coli C25. E. coli that had translocated across the enterocyte monolayer were quantified by growing basal-chamber media samples on gram-negative bacteria-specific MacConkeys agar. In order to determine monolayer integrity, transepithelial electrical resistance (TEER) was measured across Caco-2 cells treated with LGG and E. coli. Statistical analysis was by ANOVA with P < 0.05 considered significant. LGG inhibited E. coli translocation at all LGG concentrations tested. The TEER ratio was not significantly altered by addition of LGG or E. coli (0.9 ± 0.03 vs 0.8 ± 0.05). These results demonstrate that the probiotic bacterium LGG inhibits BT of E. coli C25 in a dose-dependent manner in an in-vitro cell-culture model. This model should be valuable in investigating the cellular and molecular mechanisms involved in the inhibition of pathological enteral bacteria by probiotic agents.

Attenuating Burn Wound Inflammatory Signaling Reduces Systemic Inflammation and Acute Lung Injury

The relationship between local inflammation and the subsequent systemic inflammatory response is poorly described. In a burn injury model, the dermal inflammatory response may act as an ongoing trigger for the systemic inflammatory response syndrome (SIRS) and subsequent systemic complications. We hypothesized that topical attenuation of burn wound inflammatory signaling will control the dermal inflammatory source, attenuate SIRS, and reduce acute lung injury. Mice received a 30% total body surface area burn. Subgroups were treated with specific p38 MAPK inhibitor or vehicle, which was topically applied to wounds. Topical p38 MAPK inhibition significantly reduced burn wound inflammatory signaling and subsequent systemic expression of proinflammatory cytokines and chemokines. In vitro macrophage functional assays demonstrated a significant attenuation in serum inflammatory mediators from animals receiving the topical inhibitor. Topical p38 MAPK inhibition resulted in significantly less pulmonary inflammatory response via reduction of pulmonary neutrophil sequestration, pulmonary cytokine expression, and a significant reduction in pulmonary microvascular injury and edema formation. Although dermal activating transcription factor-2, a downstream p38 MAPK target, was significantly reduced, there was no reduction in pulmonary activating transcription factor-2 expression, arguing against significant systemic absorption of the topical inhibitor. These experiments demonstrate a strong interaction between dermal inflammation and systemic inflammatory response. Attenuating local inflammatory signaling appears effective in reducing SIRS and subsequent systemic complications after burn injury.

Topical p38MAPK inhibition reduces dermal inflammation and epithelial apoptosis in burn wounds.

ABSTRACT Thermal injury induces dermal inflammatory and proapoptotic signaling. These phenomena extend burn wound size and trigger a systemic inflammatory response, factors known to adversely affect outcomes. p38MAPK is known to trigger inflammatory responses and induce epithelial proapoptotic genes. We hypothesize that topical p38MAPK inhibition will attenuate excessive inflammatory and apoptotic signaling and reduce dermal tissue loss. Rats were given a 30% total body surface area partial thickness burn or sham injury. Some of the animals were treated with a p38MAPK inhibitor or vehicle, which was applied directly to the wound. Dermal inflammation was investigated with enzyme-linked immunosorbent assay, reverse transcriptase polymerase chain reaction, myeloperoxidase assay, and Evans blue extravasation. Apoptotic changes were detected using terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and Caspase-3 in situ staining. Burn injury activated dermal p38MAPK and induced a significant rise in dermal IL-6, TNF-&agr;, and IL-1&bgr; expression. Neutrophil sequestration, microvascular damage, and hair follicle apoptosis were significantly elevated after injury. Topical p38MAPK inhibition significantly attenuated downstream dermal p38MAPK targets, proinflammatory cytokine expression, neutrophil sequestration, and microvascular injury. A significant reduction in hair follicle apoptosis was seen. This study demonstrates the attenuation of burn-induced cellular stress by topical application of p38MAPK inhibitors. Blunting early excessive inflammatory signaling may be an efficient strategy to improve patient outcomes after burn injury.

Topical nanoemulsion therapy reduces bacterial wound infection and inflammation after burn injury

BACKGROUND Nanoemulsions are broadly antimicrobial oil-in-water emulsions containing nanometer-sized droplets stabilized with surfactants. We hypothesize that topical application of a nanoemulsion compound (NB-201) can attenuate burn wound infection. In addition to reducing infection, nanoemulsion therapy may modulate dermal inflammatory signaling and thereby lessen inflammation following thermal injury. METHODS Male Sprague-Dawley rats underwent a 20% total body surface area scald burn to create a partial-thickness burn injury. Animals were resuscitated with Ringers lactate solution and the wound covered with an occlusive dressing. At 8 hours after injury, the burn wound was inoculated with 1 x 10(6) colony-forming units (CFUs) of Pseudomonas aeruginosa. NB-201, NB-201 placebo, 5% mafenide acetate solution, or 0.9% saline (control) was applied onto the wound at 16 and 24 hours after burn injury. Skin was harvested 32 hours postburn for quantitative wound culture and determination of inflammatory mediators in tissue homogenates. RESULTS NB-201 decreased mean bacterial growth in the burn wound by 1,000-fold, with only 13% (3/23) of animals having P. aeruginosa counts greater than 10(5) CFU/g tissue versus 91% (29/32) in the control group (P < .0001). Treatment with NB-201 attenuated neutrophil sequestration in the treatment group as measured by myeloperoxidase assay and by histology. It also significantly decreased levels of proinflammatory cytokines (interleukin [IL]-1beta and IL-6) and the degree of hair follicle cell apoptosis in skin compared to saline-treated controls. CONCLUSION Topical NB-201 substantially decreased bacterial growth in a partial-thickness burn model. This decrease in the level of wound infection was associated with an attenuation of the local dermal inflammatory response and diminished neutrophil sequestration. NB-201 represents a novel potent antimicrobial and anti-inflammatory treatment for use in burn wounds.

Attenuating burn wound inflammation improves pulmonary function and survival in a burn-pneumonia model.

Objective:We previously showed that topical inhibition of inflammatory signaling in burn wounds reduced systemic inflammatory response and burn-induced pulmonary inflammation. We hypothesized that this topical intervention would attenuate burn-induced lung injury, improve pulmonary function, protect lungs from bacterial invasion, and reduce mortality. Design:Controlled, in vivo, laboratory study. Setting:University laboratory. Subjects:Female mice, 8–10 wks old. Interventions:Animals received 30% total body surface area burn followed by topical application of a specific inhibitor of p38 mitogen-activated protein kinase, a key inflammatory signaling pathway, or vehicle to the wound. Twenty-four hours after injury, pulmonary collagen deposition and pulmonary function were assessed. One day postburn, some of the animals received intratracheal instillation of Klebsiella pneumoniae and were subsequently monitored for 7 days. Measurements and Main Results:Topical inhibition of p38 mitogen-activated protein kinase significantly decreased pulmonary collagen deposition and prevented a decline in pulmonary function at 1 day after burn injury. Compared with sham controls, animals with burn injury had a significantly higher mortality in response to intratracheal bacterial challenge. Application of p38 mitogen-activated protein kinase inhibitor to the burn wound attenuated pulmonary neutrophil infiltration and reduced the mortality rate to a level experienced by sham controls. Conclusions:Inflammatory source control in burn wounds with topical p38 mitogen-activated protein kinase inhibition attenuates acute lung injury, avoids pulmonary dysfunction, protects lungs from bacterial challenge, and improves survival.

Local wound p38 MAPK inhibition attenuates burn-induced cardiac dysfunction

BACKGROUND Topical inhibition of activated p38 MAPK within burn wounds attenuates the local and systemic inflammatory response. In this study, we investigated the effects of local activated p38 MAPK inhibition on burn-induced cardiac dysfunction. METHODS Using a standardized rat model of scald burn injury, rats were given a 30% total body surface area partial thickness burn or sham injury, and the wounds were treated with an activated p38 MAPK inhibitor (SB) or vehicle. Systemic blood pressure measurements were recorded in vivo followed by in vitro assessment of sarcomere contraction in single-cell suspensions of isolated cardiomyocytes. RESULTS Systolic blood pressure or maximum left ventricular pressures in vivo and peak cardiomyocyte sarcomere contractility in vitro were significantly reduced after burn injury. These functional deficits were abolished 24 h after burn injury following local p38 MAPK inhibition. In vitro incubation of normal cardiomyocytes with homogenate from burned skin or burn serum resulted in a similar pattern of impaired cardiomyocyte contractility. These effects were reversed in normal cardiomyocytes exposed to burn skin homogenates treated topically with a p38 MAPK inhibitor. A Western blot analysis showed that cardiac p38 MAPK activation was not affected by dermal blockade of activated p38 MAPK, arguing against systemic absorption of the inhibitor and indicating the involvement of systemic cytokine signaling. CONCLUSION Topical activated p38 MAPK inhibition within burned skin attenuates the release of proinflammatory mediators and prevents burn-induced cardiac dysfunction after thermal injury. These results support the inhibition of burn-wound inflammatory signaling as a new therapeutic approach to prevent potential postthermal injury multiorgan dysfunction syndrome.

Functional contribution of CXCR2 to lung injury after aspiration of acid and gastric particulates

The effects of individual ELR+ CXC chemokines have been documented in experimental models of acid aspiration. However, aspiration lung injury would be influenced by the combined effects of these chemokines and other factors related to their function. Therefore, the role of the chemokine receptor CXCR2 was examined in lung injury induced by aspiration of acid and acid with gastric particulates. Anesthetized mice were given intratracheal injections of saline, acid solution, or acid containing gastric particles. Within 6 h, bronchoalveolar lavage fluid neutrophils and albumin increased relative to the severity of the insult. Immunohistochemistry and RT-PCR demonstrated striking increases in pulmonary expression of CXCR2 after aspiration. In CXCR2-deficient mice, neutrophil recruitment to airways was significantly reduced after aspiration of either acid or acid with particles. However, lung injury scores were unaffected in Ccr2-/- mice in the acid + particles group. Esterase-stained lung tissue demonstrated that focal aggregates of inflammatory cells contained neutrophils in the Ccr2-/- mice. These studies suggest CXCR2 and its ligands are dominant mediators of neutrophil recruitment to airways after aspiration. However, CXCR2-independent mechanisms recruit neutrophils into areas of cellular aggregation after aspiration of acidified gastric particulates.