Effects of recombinant bactericidal, permeability-increasing protein on bacterial translocation and pulmonary neutrophil sequestration in burned mice.

Burn injury induces bacterial translocation (BT) from the gut in multiple animal models. Etiologic factors contributing to BT may be an ischemia-reperfusion injury to the gut, the release of inflammatory cytokines, oxygen metabolites and other mediators, and cytotoxic effects mediated by endotoxin (lipopolysaccharide). Bactericidal, permeability-increasing protein is a neutrophil granule protein with potent bactericidal and lipopolysaccharide-neutralizing activities. The use of this protein has not been previously reported in a burn-injury model. The purpose of this study was to determine whether recombinant bactericidal, permeability-increasing protein (rBPI23) affects the incidence of BT and myeloperoxidase content in lung tissue (a measure of leukocyte sequestration) in a burn-injury model. Mice received a 32% total body surface area, full-thickness, scald burn, and 10 mg/kg body weight rBPI23 in saline solution was given by intraperitoneal injection at 0, 3, and 6 hours after the burn. Control animals received intraperitoneal saline solution only. All animals received a total of 1 ml saline solution intraperitoneally immediately after burn injury for fluid resuscitation. At 24 hours after burn injury, mesenteric lymph nodes (MLN) were harvested, homogenized, and plated. Lung tissue was harvested and assayed for myeloperoxidase. Burned mice treated with rBPI23 had significantly (p = 0.005, Fisher's Exact Test, two-tailed) decreased incidence of BT, compared to burned mouse controls. Leukosequestration into lung tissues was not affected by rBPI23. Postburn administration of rBPI23 reduces but does not abolish the incidence of BT after burn injury in mice, perhaps by reducing intestinal injury during burn shock and the ischemia-reperfusion period by inhibiting the effects of lipopolysaccharide. An alternate explanation may be that rBPI23 could increase clearance and killing of bacteria by host defenses.

Burn injury results in decreased gastric acid production in the acute shock period.

Gastrointestinal complications including acute stress ulcerations occur after burn injury. The causes of acute gastric derangements are multiple, and tissue ischemia in the acute period of burn shock may promote breakdown of the gastric mucosal protective barrier. We compared gastric pH in mice after 25% total body surface area, full-thickness murine burn injury with that in unburned control animals. Animals were anesthetized with methoxyflurane and were resuscitated with 1 ml normal saline solution immediately after burn. Animals were killed at intervals up to 24 hours after burn injury, stomachs were removed and opened, and gastric mucosal pH was measured by use of a surface pH probe. In other animals mixed venous blood was obtained via direct inferior vena cava puncture, and blood gas analysis was performed at intervals up to 24 hours after burn injury. Unexpectedly, gastric mucosal pH increased in burned mice compared with that in controls. The peak difference, greater than one log pH unit, occurred at 3 hours after burn injury (pH 4.45 burn vs pH 3.34 control, p < 0.00001), and this difference was maintained through 12 hours (pH 4.88 burn vs pH 3.20 control, p < 0.005). In this model, shock was observed to begin as early as 1 hour after burn injury and reached its maximal period (base deficit, -27.8 mEq/L) at 12 hours after burn injury. In view of the higher gastric pH in burned mice with concomitant profound shock, gastric acid production appears to be impaired during this time, which suggests acute postburn gastrointestinal ulcerations may be primarily due to ischemia. Prevention of organ ischemia may play a key role in the prevention of acute gastric ulcerations after burn injury.

Effects of high-dose vitamin C administration on bacterial translocation and lung neutrophil sequestration in burned mice.

Severe burn injury produces shock and induces acute gastrointestinal derangements that may disrupt mucosal integrity and facilitate bacterial translocation (BT) to mesenteric lymph nodes, accompanied by endotoxemia. Antioxidant treatments may be beneficial after shock by acting as scavenger agents for highly reactive oxygen intermediates. We studied the effects of high dosages of vitamin C, a water-soluble antioxidant, on the incidence of BT and on levels of lung myeloperoxidase in burned mice. Myeloperoxidase is primarily found in neutrophils, and levels of myeloperoxidase in tissues reflect neutrophil sequestration. The doses of vitamin C used were equivalent on a weight basis to 1 gm/hr administered to humans over a 24-hour period, doses that have shown efficacy in improvement of resuscitation in other experimental burn models and currently are being used in clinical trials in patients with burns. Mice were anesthetized and received 32% total body surface area, full-thickness burn injury to the dorsum, followed by injection of 1 ml of Ringer's lactate (RL) for resuscitation. Mice were divided into three groups: (1) unburned, received anesthesia and RL injections; (2) burned, received vitamin C (14 mg/kg/hr) in 1 ml RL by intraperitoneal injection immediately after burn and via subcutaneous injection (0.5 ml) at 6 and 12 hours after burn; (3) burned, received identical injections of RL alone (control animals). Mesenteric lymph nodes were removed by use of sterile technique at 24 hours after burn and cultured; any growth was considered evidence of BT. The incidence of BT in burned mice was not altered by administration of vitamin C (normal, 10% BT; burn, 41.37% BT; burn+vitamin C, 45.83% BT). Similarly, burned animals that received vitamin C or RL alone did not differ in the levels of myeloperoxidase in the lungs (normal, 0.015 +/- 0.003 U/gm; burn, 0.2231 +/- 0.029 U/gm; burn+vitamin C, 0.281 +/- 0.041 U/gm).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of small-volume bolus treatment with intravenous normal saline and 7.5 per cent saline in combination with 6 per cent dextran-40 on metabolic acidosis and survival in burned mice.

Standard murine burn models include the administration of intraperitoneal (i.p.) saline solutions which are intended to resuscitate the animals during subsequent burn shock. Prehospital administration of small volumes of concentrated salt solutions has been recommended for the early treatment of haemorrhagic shock, and have also been utilized for burn shock. We studied the effects of bolus intravenous (i.v.) hypertonic saline (HS) or hypertonic saline/dextran-40 (HS + DEX) on animal survival and acid-base balance following 25 per cent total body surface area, full-thickness burn injury in mice. I.v. injections were administered via a tail vein immediately prior to burn injury. Some mice received 1 ml i.p. normal saline (NS) immediately after burn injury. Acid-base balance of vena caval blood was measured during the period of maximal metabolic acidosis following burn injury (12 h postburn). Preburn i.v. administration of 5 ml/kg of HS or HS+DEX, followed by 1 ml i.p. NS, only slightly decreased the degree of metabolic acidosis compared to animals receiving i.p. fluid alone, the standard resuscitation regimen for burned mice. Preburn i.v. administration of 0.2 ml volumes of HS or HS + DEX, without i.p. fluid administration, resulted in extremely high mortality. Immediate preburn i.v. administration of HS or HS + DEX did not eliminate metabolic acidosis in this murine burn model, and markedly increased the mortality when subsequent i.p. fluids were not administered. The degree of metabolic acidosis in the murine experimental burn model has not previously been clearly described. Furthermore, adequate fluid resuscitation of these animals may be difficult to achieve without indwelling vascular catheters which could deliver continuous i.v. fluids following burn injury.

Supplementation of an elemental enteral diet with alanyl-glutamine decreases bacterial translocation in burned mice.

Although there are many reports of the importance of early enteral feeding in maintaining gastrointestinal integrity and preventing bacterial translocation (BT) following burn injury, no diet has been shown clinically to protect the GI tract postburn. Several studies suggest that glutamine (GLN) may benefit gut integrity following injury, shock and other stress. Unfortunately, the free amino acid GLN is unstable in solution. Alanyl-glutamine (ALA-GLN), a soluble form of GLN, maintains long-term stability in solution and could be supplemented to conventional liquid enteral diets. We studied the effects of ALA-GLN supplementation of the elemental diet Vivonex TEN on effecting BT in mice following 32 per cent TBSA full skin thickness burns. Groups A-D were burned. Group A (30 mice) was fed standard rodent chow, which contains extremely high (clinically non-useable) levels of protein. Group B (51 mice) was fasted 24 h, then fed chow 24 h. Group C (64 mice) was fed Vivonex TEN, and Group D (65 mice) received Vivonex TEN plus ALA-GLN (GLN equivalent, 14 g/l). A control group (Group E) consisted of 22 normal mice (no burn injury, chow diet). Mice were assessed for BT by sterile harvesting and plating of mesenteric lymph node tissue, 48 h postburn. Plates were considered positive if any bacterial growth was noted. Non-burned mice exhibited no BT, while burn-fasted mice showed a 64.3 per cent incidence of BT (P = NS).(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial translocation in burned mice after administration of various diets including fiber- and glutamine-enriched enteral formulas.

OBJECTIVE: Severe burn injury can produce acute gastrointestinal derangements which may facilitate bacterial translocation to mesenteric lymph nodes. We studied the effects of feeding different dietary formulations on bacterial translocation in burned mice. DESIGN: Prospective, blinded, nonrandomized laboratory study. SETTING: Research laboratory. SUBJECTS: One hundred sixty-nine female, outbred, CF-1 mice, 8 to 12 wks of age. INTERVENTIONS: Anesthetized mice received a 32% total body surface area, full-thickness burn injury. Mice were then fed with: a) mouse chow; b) a low-residue enteral formula; c) a high-protein, high-fat enteral formula; d) an enteral formula with high concentrations of supplemental glutamine; or e) an enteral formula that contains soy fiber. MEASUREMENTS AND MAIN RESULTS: Burned mice that were fed the low-residue enteral formula demonstrated increased mortality rate (21.2%, p = .05) compared with chow-fed mice in the 2-day postburn period (0 mortality); other burn-diet groups had intermediate mortality rates. In surviving mice, bacterial translocation was found to be: a) lowest in the group fed chow (31.0%) and the high glutamine formula (30.8%); b) intermediate in the group fed formula and soy fiber (44.8%, NS compared with burn-chow group); and c) highest in the group receiving the low-residue enteral formula (73.1%, p < .005) and high-protein, high-fat enteral formula (59.3%, p < .05). CONCLUSIONS: Dietary composition markedly affects bacterial translocation in this animal burn model. Commercial enteral diets containing fiber and high concentrations of glutamine provide protection for the gut after burn injury and reduce the occurrence of bacterial translocation in this animal model.

Reduction of bacterial translocation and intestinal structural alterations by heparin in a murine burn injury model.

Burn injury produces acute gastrointestinal (GI) derangements that may predispose the burn victim to bacterial translocation (BT). We studied the effects of heparin on gastrointestinal (GI) anatomic alterations and BT after 25% and 32% total body surface area (TBSA), full-thickness murine burn injuries. Heparin (100 U/kg) was administered with 1 mL of normal saline (NS) resuscitation solution immediately postburn and 4 hours and 18 hours postburn in volumes of 0.5 mL NS. Mice with 25% TBSA burns treated with heparin maintained small intestine weight, measured 24 hours postburn, and ileal mucosal height was preserved, whereas burned, untreated mice lost organ weight and mucosal height. Bacterial translocation was decreased in mice with 25% TBSA burn injuries treated with heparin (35.0% vs. 10.7%, p < 0.025). After 32% TBSA burn injuries, BT was also decreased in heparin-treated animals (64.3% vs. 31.6%; p < 0.025). Analysis of mixed venous blood gases showed that heparin did not affect the severe metabolic acidosis that follows burn injury in this animal model, indicating that general tissue perfusion was not improved. Heparin administered in the acute postburn period ameliorates GI structural and functional damage in this murine burn model and decreases BT.

Direct comparison of a cultured composite skin substitute containing human keratinocytes and fibroblasts to an epidermal sheet graft containing human keratinocytes on athymic mice.

This study compares two techniques for making cultured skin substitutes: a composite graft made of human fibroblasts and keratinocytes on a collagen-glycosaminoglycan membrane (CG) and a cultured epidermal cell sheet graft (CEG), without a dermal component. The "take" and quality of these cultured skin substitutes were evaluated by placing them on full-thickness, excised wounds of athymic mice. These cultured skin substitutes were placed onto 2-X-2-cm wounds created on athymic mice. Mice were sacrificed at days 10, 20, and 42 with histologic sections obtained for light, electron, immunofluorescent, and immunohistochemical microscopy. "Take" was determined separately by a direct immunofluorescent stain for human leukocyte ABC antigens. There were ten mice of each graft type with at least two animals sacrificed at each time point. Results showed positive "take" for all animals. Grossly, there was little difference between the two graft types, with the CEG having occasional blister formation. By light microscopy, the CEG had a dissociation of dermis from epidermis until day 42, which was never apparent with the CG. By day 42, the CG had increased dermoepidermal interdigitations similar to rete ridges, with a mature epithelium. Neither of these findings were seen with the CEG. Immunofluorescent and immunohistochemical microscopy for type IV collagen and laminin, as well as electron microscopy, showed similar retardation of basement membrane formation with the CEG. Using this model, the composite graft had significant advantages over the epidermal sheet graft in the closure of full-thickness wounds.

Pharmacokinetics of DepoFoam gentamicin delivery system and effect on soft tissue infection.

Infections of burn and soft tissue wounds are often difficult to treat with systemic antibiotics since drug delivery to the wound may be suboptimal and high doses may result in toxicity. DepoFoam particles, a novel lipid-based drug delivery system, are composed of phospholipid membranes, enclosing multiple aqueous chambers into which pharmacologic agents can be encapsulated for local drug delivery. We encapsulated gentamicin (GENT) in DepoFoam particles with an average yield of 81% +/- 8 SD for 10 preparations. Encapsulated GENT was incubated in human plasma with t1/2 of 21 days, demonstrating stability in vitro. In vivo pharmacokinetics were determined by injecting CF-1 mice subcutaneously (sc) with a single dose of 0.5 mg of free (nonencapsulated drug) or DepoFoam GENT. At intervals postinjection the sc tissue was excised and blood was obtained by inferior cava puncture and both were assayed for GENT levels. At 0.5, 2, 6, and 24 hr following drug administration there was a significant difference between GENT levels in the tissue achieved with the encapsulated drug and free drug with n = 3-4 at each time point for each group (P < 0.01). By 24 hr following administration of free drug there was minimal detectable GENT in the tissues, while therapeutic levels of GENT remained in tissue at 24 hr following DepoFoam GENT injection. Serum GENT peaked at 30 min for both the DepoFoam (5 micrograms/ml) and free drug (10 micrograms/ml) and was undetectable by 2 hr (n = 3 each group).(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitation of cytokine levels in skin graft donor site wound fluid.

We quantified endogenous levels of multiple cytokines in skin graft donor site wounds in patients with small to moderate-sized burn injuries. Thirteen patients aged 11 months to 61 years with mean TBSA burn of 4 +/- 1 per cent underwent placement of occlusive wound dressings on partial skin thickness donor site wounds. Fluid was aspirated from beneath the dressing on postoperative day 1 and every subsequent 24 h until no further fluid could be obtained. Interleukin-1 alpha (IL-1) and tumour necrosis factor-alpha (TNF-alpha) levels were determined by enzyme-linked immunosorbent assay (ELISA). Epidermal growth factor (EGF), basic-fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF) were measured by an enzyme immunoassay (EIA). We found substantial levels of EGF and TNF-alpha in the donor site wound fluid in all 13 patients; detectable levels of bFGF in five patients; and elevated levels of IL-1 in three patients. There were no detectable levels of these cytokines in normal human serum. In contrast, there were no measurable levels of PDGF in any patient's wound fluid; the mean level in serum was 1.5 ng/ml +/- 0.2 s.e.m. Studies of cytokines in the normal wound healing environment may help in the design of future therapies to augment wound healing.

Effects of fluid resuscitation, burn eschar excision, and blockade of afferent pain responses on bacterial translocation and acid-base balance after murine burn injury.

We tested effects of fluid resuscitation, early burn excision/grafting, and blockade of afferent stimuli from the burn wound on bacterial translocation and acid-base balance after murine burn injury. Burn excisions were performed with patients either 15 minutes or 2 hours after burn injury under anesthesia, and excised wounds were immediately closed with murine allograft skin. Twenty-four hours after 25% total body surface area (TBSA) burn injury and 48 hours after 32% TBSA injury, mesenteric lymph nodes were cultured. Incidences of bacterial translocation in 25% and 32% TBSA burns were 31.6% and 68.4% of animals, respectively. Burned animals were in severe shock, and metabolic acidosis reached a nadir 12 hours after burn injury, with base deficit -27.8 +/- 0.6 mEq/L; 5% to 10% of animals died acutely after burn injury. After excision/grafting of burned mice at 2 hours after burn injury, the incidence of bacterial translocation was unchanged (35.7% with 25% TBSA burn, 73.3% with 32% TBSA burn), and mortality did not change. When 32% TBSA excisions were performed exactly 10 minutes after burn injury, four of the 13 mice died within several hours, and five (55.5%) of the nine survivors translocated. Rates of bacterial translocation in mice receiving anesthesia or excision/grafting without burn injury were 15.0% and 20%, respectively (p = NS compared with normal mice). Subcutaneous implantation of normal or burned skin into normal animals neither elicited shock nor increased the incidence of bacterial translocation. Increasing amounts of fluid resuscitation in the 25% TBSA burn model provided only delayed improvement of acid-base balance; increased amounts of fluid did not decrease bacterial translocation.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of corticotropin releasing factor on acid-base alterations and bacterial translocation in a murine model of thermal injury.

Corticotropin-releasing factor (CRF) is a 41 amino acid polypeptide produced by the hypothalamus which has been shown to decrease inflammation and tissue oedema when administered following burns, cold and acid injuries in some animal models, and to increase mesenteric blood flow. We determined whether systemic administration of CRF to burned mice would decrease metabolic acidosis and protect the gastrointestinal (GI) tract from ischaemic injury leading to bacterial translocation (BT). Synthetic CRF was administered by intraperitoneal injection in doses of 20 and 200 micrograms/kg to mice immediately following 25 and 32 per cent TBSA burn injuries; the doses were repeated at 8 and 16 h postburn. Severe metabolic acidosis, measured 12 h after burn injury, was not improved in mice which received CRF treatment. Bacterial translocation, measured by quantifying bacteria in mesenteric lymph nodes harvested from animals 48 h postburn, was also not decreased with CRF treatment. CRF does not improve general tissue perfusion nor decrease GI derangements leading to bacterial translocation in this animal model of burn injury.

Epidermal growth factor limits structural alterations in gastrointestinal tissues and decreases bacterial translocation in burned mice.

BACKGROUND: Burn injury produces acute gastrointestinal derangements that may predispose to bacterial translocation (BT). We studied effects of recombinant human epidermal growth factor (r-HuEGF), a gastrointestinal trophic hormone, on gastrointestinal alterations and BT after murine burn injury. METHODS: r-HuEGF was administered 1 and 12 hours after burn injury in a dose of 4 micrograms per animal subcutaneously after 25% and 32% total body surface area (TBSA) scald burn. Small bowel and gastric weight and histologic factors were studied, and BT was measured by culturing mesenteric lymph nodes. RESULTS: Mice treated with r-HuEGF maintained gastric and small intestine weight measured 24 hours after burn injury, and ileal mucosal height was preserved, whereas burned-untreated mice lost organ weight and mucosal height. BT was decreased significantly in mice with 32% TBSA burn injury treated with r-HuEGF after injury (burn, 64.2% of animals had BT; burn-r-HuEGF, 34.6% had BT; p < 0.05). After 25% TBSA burn injury, BT was also decreased in r-HuEGF-treated animals (burn, 31.4% of animals had BT; burn-r-HuEGF, 14.3% had BT), but the difference was not statistically significant (p < 0.1). CONCLUSIONS: r-HuEGF improves intestinal and gastric structure in mice 24 hours after burn injury and decreases BT after 32% TBSA burn injury.

Cytotoxicity to human leukocytes by topical antimicrobial agents used for burn care.

We tested two topical antimicrobial agents (TAAs), silver sulfadiazine and mafenide acetate, to determine their cytotoxic effects when human lymphocytes and neutrophils were incubated with the agents in vitro for 30 minutes. Dilute concentrations of both TAAs markedly inhibited neutrophil respiratory burst activity and mitogen-stimulated lymphocyte proliferation (p < 0.05). The components of silver sulfadiazine (silver and sulfadiazine) were separately tested, and each component inhibited both neutrophil and lymphocyte functions. Mafenide acetate markedly decreased intracellular Ca+2 flux in lymphocytes. The effects of the TAAs were partially reversed when cells were washed and resuspended in medium after they were exposed in vitro to the TAAs. Commonly used TAAs may contribute to local immune dysfunction in the patient with burns. Because evidence suggests that T lymphocytes may participate in wound healing, prolonged treatment with TAAs may also effect certain aspects of wound healing.

Temporal analysis of human leucocyte surface antigen expression and neutrophil respiratory burst activity after thermal injury.

Sepsis, the major cause of morbidity and mortality after burn injury, is related to multiple immune derangements. Using monoclonal antibodies and two-colour flow cytometry to identify surface antigens, peripheral blood mononuclear cell (PBMC) populations were analysed and correlated with lymphocyte proliferation assays for 21 days postinjury. In addition, in vitro expression of activation antigens by mitogen-stimulated PBMCs was analysed during the time period. Twenty-nine burn patients were studied, with burn injuries ranging from 19 to 97 per cent TBSA; PBMCs from human volunteers were used for control cells. Patients received aggressive enteral nutritional support starting day 1 postburn and underwent early excision and grafting of wounds; no patients developed sepsis during the study period. The most consistent changes in PBMCs after thermal injury were decreased percentages of total T cells (CD3+), T helper/inducer cells (CD4+), and T suppressor/cytotoxic cells (CD8+); the percentages of natural killer (CD16+) cells were not altered. Expression of surface 'activation' antigens on CD4+ and CD8+ cells (HLA-DR, interleukin-2 receptor and transferrin receptor) after mitogen stimulation was significantly depressed as early as 1 day postburn. An early monocytosis was seen on day 1 postburn, but decreases were found on days 4 and 7. Monocyte expression of HLA-DR antigen was suppressed throughout the study. Lymphocyte proliferation after mitogen stimulation and the responses of lymphocytes in mixed lymphocyte culture were suppressed postburn. Neutrophil respiratory burst responses were supranormal on days 1 and 7 postburn, but the differences were not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of a device for measuring adherence of skin grafts to the wound surface.

Adherence of a biological graft to the wound surface is the most important factor influencing the ultimate success of graft viability. A machine has been developed to test the adherence of biological graft materials to a substrate such as a wound surface. The peeling mode, which yields reproducible quantitative measurements of adherence, is a standard method for testing adhesives. The device is designed to continuously measure the force required to peel the graft from the substrate at a constant rate. This force is a function of the energy of adhesion per unit area of adhered surface. This device has been used to measure the peeling force of (2 x 2 cm) skin grafts which are applied to full-thickness wounds on mice. Results of tests on adherence of autografts on mice show that the peeling force increases significantly with time over the first 9 days of healing. Thus, this device is useful in quantitative comparison of various skin grafting techniques and artificial grafts.

Effect of indomethacin and Impact liquid diet administration on inflammatory mediator production by murine splenocytes after burn injury.

Production of prostaglandin E2 (PGE2) and tumor necrosis factor-alpha (TNF-alpha) by various cells is increased after injury, and these mediators are implicated in the downregulation of immune responses. We attempted to modulate the production of immune suppressive mediators by inhibiting prostaglandin production in a murine model that had a burn covering 25% of the total body surface area. Two treatments were performed. One was the intraperitoneal administration of indomethacin after burn injury at 2 mg/kg day for 10 days. The control group received saline solution injections. The other treatment was the ad libitum administration of a commercial diet after burn injury for 10 days. This diet contained mixed fats, including omega-3 and omega-6 fatty acids. The control group received standard mouse food. On days 1, 5, and 10 after burn injury, spleens were removed aseptically, and splenocyte cultures were established and stimulated with phytohemagglutinin. TNF-alpha and PGE2 concentrations were determined in culture supernatants at 48 hours with the use of commercial enzyme-linked immunosorbent assay kits. Splenocytes from burned animals produced elevated levels of both mediators in culture supernatants, reaching significant levels of TNF-alpha on day 10 after burn injury and of PGE2 on days 5 and 10 after burn injury. Neither the administration of indomethacin for 10 days nor the administration of the commercial diet for 10 days decreased production of these mediators in culture. However, cells in culture may escape the in vivo regulating effects of biologic modifying agents.

Immunologic alterations in a murine model of hemorrhagic shock.

OBJECTIVE: To study multiple immune parameters in mice subjected to severe hemorrhage without fluid resuscitation. STUDY DESIGN: Controlled animal study. Anesthetized, female mice were hemorrhaged by tail bleeding. Immune parameters (spleen T-cell proliferation and activation, intracellular calcium flux, cytokine production, peritoneal neutrophil respiratory burst, and survival after intra-abdominal septic challenge) were measured at 24, 48, and 72 hrs after hemorrhage. MEASUREMENTS AND MAIN RESULTS: T-cell proliferation was decreased in animals after two 20% blood volume hemorrhages, 30 mins apart; single 30%, 40%, and 50% blood volume hemorrhages did not depress proliferation. "Helper/inducer" T cells from twice-hemorrhaged mice showed decreased expression of activation antigens (interleukin-2 receptor, Ia) after mitogen stimulation. In contrast, "suppressor/cytotoxic" T cells displayed increased activation, shown by augmented expression of interleukin-2 receptor and Ia antigens. Leukocyte production of prostaglandin E2, a mediator frequently implicated in immune down-regulation, was unaffected by hemorrhage. Secretion of tumor necrosis factor-alpha (TNF-alpha) in culture was increased when cells were harvested 48 hrs after injury. Intracellular calcium flux in stimulated lymphocytes was decreased 24 hrs after hemorrhage, suggesting deranged intracellular signal transduction. Respiratory burst activity of peritoneal neutrophils was unchanged following hemorrhage. When animals were subjected to septic challenge, the survival rate was markedly decreased after two hemorrhages (when sepsis was induced 24 hrs after hemorrhage). By 72 hrs posthemorrhage, most of the immunologic alterations, including resistance to septic challenge, had resolved. CONCLUSIONS: This uninstrumented hemorrhagic shock model allows quantification of multiple immune derangements. Immune suppression was identified after two smaller (20% blood volume) hemorrhages, but not after a single, larger hemorrhage. Immune derangements are maximal at 24 hrs posthemorrhage, and resolve in the subsequent 48 hrs.

[Bacterial translocation. Its role in the etiology of sepsis and multiple organ failure]. / Translocación bacteriana. Papel en la etiología de la sepsis y la falla de múltiples órganos.

Under certain conditions or diseases the microorganisms that normally inhabited in the gastrointestinal tract reach the mesenteric lymph nodes, the portal circulation, the intra and extraperitoneal organs and the systemic circulation creating the possibility of infection, sepsis and multiple organ failure. This phenomenon has been termed bacterial translocation and although was described few decades ago, today it has regain critical importance due to the association to the multiple organ failure syndrome in critical and severely injured patients. In this review a series of pathologies where the translocation of bacteria has been demonstrated are described as well as the possible therapeutics maneuvers.

[Bacterial translocation in a model of intestinal obstruction. II. Bacteriological study and role of cellular immunity]. / Translocación bacteriana en un modelo de obstrucción intestinal. II. Estudio bacteriológico y papel de la inmunidad celular.

Bacterial translocation (Bt) from the gastrointestinal (GI) tract to systemic organs creates the possibility of Infection and sepsis in a great number of pathologic entities. In a mouse model of Intestinal Obstruction (IO), we evaluated the type of micro-organisms and the organs that bacteria frequent translocated. At 24 hours post-10, positive cultures where obtained at the MLN, portal, systemic circulation and peritoneal cavity, establishing that the translocation is bi-directional. The more frequent bacteria isolated were the Streptococcus group D, Proteus mirabilis, Escherichia coli, Pseudomonas sp., an clostridium. BT occurs at 24 hour post-OI and was due to increased intestinal permeability, at 48 hrs BT increased and related to the physical disruption of the mucosal barrier in the intestinal mucosa. Cell mediated immunity (CMI) response in this model was not altered, although a progressive decrease was observed at 48 hrs it was not significant, suggesting that the CMI play no role in the pathogenesis of BT. In the Control-Laparotomy group, CMI response was increased significantly at 48 hours, suggesting that a simple laparotomy boost the immune defense response.