Endotoxin promotes the translocation of bacteria from the gut

Experiments were performed in mice to determine whether endotoxin could cause bacteria normally colonizing the gut to spread systemically, a process termed bacterial translocation. Endotoxin given intraperitoneally promoted bacterial translocation in a dose-dependent fashion from the gut to the mesenteric lymph node (MLN). The incidence of bacterial translocation to the MLN was similar whether the endotoxin was administered intramuscularly or intraperitoneally, although the number of bacteria colonizing the MLN was greater with intraperitoneal endotoxin. The incidence and magnitude of endotoxin-induced bacterial translocation were similar between CD-1 and C3H/HeJ (endotoxin-resistant) mice, indicating that bacterial translocation is not prevented by genetic resistance to endotoxin. Thus, it appears that the gut may serve as a reservoir for bacteria causing systemic infections during endotoxemia.     

Impairment of pulmonary macrophage function with total parenteral nutrition.

OBJECTIVE: The effects of total parenteral nutrition (TPN) administration on pulmonary macrophage function and host response to gram-negative pulmonary infection were evaluated. SUMMARY BACKGROUND DATA: Administration of TPN resulted in increased infectious complications in traumatized and perioperative patients, but underlying mechanisms are unclear. METHODS: Twenty-six male Wistar rats underwent central vein cannulation and were randomized to isocaloric feeding of a regular chow diet (RD) plus saline infusion or TPN without chow diet for 7 days. Pulmonary alveolar macrophage (PAM phi) superoxide production, Candida albicans phagocytosis and killing, and tumor necrosis factor (TNF) production in response to endotoxin (LPS) were assessed. Mesenteric lymph nodes (MLN) were cultured. A second group of rats (n = 6/group) were inoculated intratracheally with a sublethal dose of 9 x 10(9) live Escherichia coli per animal, and the lungs were cultured quantitatively 72 hours later to assess bacterial clearance. Finally, 11 RD-fed rats and 13 TPN-fed rats received intratracheal inoculation of 1.4 x 10(10) live E. coli and were included in follow-up. RESULTS: Administration of TPN was associated with a significant increase in bacteria positive MLN compared with those in the RD group (p < 0.01). Pulmonary alveolar macrophage superoxide production, Candida albicans phagocytosis and killing, TNF production, and pulmonary clearance of bacteria were decreased significantly in TPN-fed rats compared with those fed a regular chow diet (p < 0.05). These pulmonary macrophage function changes were associated with a significantly higher mortality in TPN-fed rats compared with RD-fed rats after higher dose pulmonary E. coli inoculation. CONCLUSIONS: Defective host pulmonary antimicrobial immune responses during TPN are associated with intestinal bacterial translocation, and may explain increased infectious complications.     

Strain differences in the attenuation of bone accrual in a young growing mouse model of insulin resistance

Skeletal fractures are considered a chronic complication of type 2 diabetes mellitus (T2DM), but the etiology of compromised bone quality that develops over time remains uncertain. This study investigated the concurrent alterations in metabolic and skeletal changes in two mouse strains, a responsive (C57BL/6) and a relatively resistant (C3H/HeJ) strain, to high-fat diet-induced glucose intolerance. Four-week-old male C57BL/6 and C3H/HeJ mice were randomized to a control (Con = 10 % kcal fat) or high-fat (HF = 60 % kcal fat) diet for 2, 8, or 16 weeks. Metabolic changes, including blood glucose, plasma insulin and leptin, and glucose tolerance were monitored over time in conjunction with alterations in bone structure and turn over. Elevated fasting glucose occurred in both the C57BL/6 and C3H/HeJ strains on the HF diet at 2 and 8 weeks, but only in the C57BL/6 strain at 16 weeks. Both strains on the HF diet demonstrated impaired glucose tolerance at each time point. The C57BL/6 mice on the HF diet exhibited lower whole-body bone mineral density (BMD) by 8 and 16 weeks, but the C3H/HeJ strain had no evidence of bone loss until 16 weeks. Analyses of bone microarchitecture revealed that trabecular bone accrual in the distal femur metaphysis was attenuated in the C57BL/6 mice on the HF diet at 8 and 16 weeks. In contrast, the C3H/HeJ mice were protected from the deleterious effects of the HF diet on trabecular bone. Alterations in gene expression from the femur revealed that several toll-like receptor (TLR)-4 targets (Atf4, Socs3, and Tlr4) were regulated by the HF diet in the C57BL/6 strain, but not in the C3H/HeJ strain. Structural changes observed only in the C57BL/6 mice were accompanied with a decrease in osteoblastogenesis after 8 and 16 weeks on the HF diet, suggesting a TLR-4-mediated mechanism in the suppression of bone formation. Both the C57BL/6 and C3H/HeJ mice demonstrated an increase in osteoclastogenesis after 8 weeks on the HF diet; however, bone turnover was decreased in the C57BL/6 with prolonged hyperglycemia. Further investigation is needed to understand how hyperglycemia and hyperinsulinemia suppress bone turnover in the context of T2DM and the role of TLR-4 in this response.     

Outcome following femur fracture and subsequent cecal ligation and puncture in endotoxin-sensitive (C3H/HeN) and endotoxin-resistant (C3H/HeJ) mice

This study examined the effect of sepsis following trauma in a reproducible model of sepsis--cecal ligation and puncture (CLP)--in endotoxin-sensitive (C3H/HeN) and endotoxin-resistant (CeH/HeJ) mice. Studies used CLP with a 25-gauge needle at different time intervals following injury, as induced by femur fracture (FF), to determine the effects of sublethal sepsis on survival after trauma. There was a 3% mortality for FF alone in both groups. Mortality in C3H/HeJ mice was not significantly increased over FF alone except when CLP followed FF by 3 days (45%, P less than 0.02, Chi-square). In contrast, C3H/HeN mice had significantly increased mortality rates (75 to 90%, P less than 0.001) versus FF alone at all intervals between FF and CLP. Mortality for FF plus CLP was significantly greater for C3H/HeN compared to C3H/HeJ (P less than 0.001) for all time intervals between FF and CLP. In conclusion, animals exposed to a septic episode following FF had significantly greater mortality than FF animals without a septic challenge. Endotoxin-sensitive mice had significantly higher mortality after CLP and significantly increased mortality when CLP followed FF (regardless of timing) compared to endotoxin-resistant mice.     

Interferon-gamma attenuates hemorrhage-induced suppression of macrophage and splenocyte functions and decreases susceptibility to sepsis.

Although it is known that interferon-gamma synthesis and macrophage functions are depressed after hemorrhage, it remains to be determined whether systemic administration of interferon-gamma has any effect on hemorrhage-induced depression of macrophage and splenocyte functions. To study this, C3H/HEN mice were bled to a mean blood pressure of 35 mm Hg, maintained for 60 minutes, and followed by adequate fluid resuscitation. The mice then received either 1000 units interferon-gamma or saline solution (vehicle). Peritoneal (pM phi) and splenic (sM phi) macrophages and splenocytes were isolated 24 hours later. PM phi antigen presentation was measured by coculturing pM phi with the D10.G4.1 cell clone. Major histocompatibility complex class II (Ia) antigen expression was determined by direct immunofluorescence. Cytokine release by pM phi, sM phi, and splenocytes was assessed with specific bioassays. For survival studies, mice were subjected to sepsis 3 days after hemorrhage. Treatment with interferon-gamma restored (p less than or equal to 0.05) hemorrhage-induced suppression of pM phi antigen presentation capacity and Ia antigen expression and increased (p less than or equal to 0.05) interleukin-1 and tumor necrosis factor release by pM phi and sM phi, as well as splenocyte proliferation (p less than or equal to 0.05). Interferon-gamma also decreased (p less than or equal to 0.007) the susceptibility to sepsis after hemorrhage. Thus interferon-gamma represents a potent agent for treating hemorrhagic shock-induced immunosuppression and for increasing the ability of the host defense system to combat bacterial infections after hemorrhage.     

Macrophage antigen presentation and interleukin 1 production after cecal ligation and puncture in C3H/HeN and C3H/HeJ mice

Following cecal ligation and puncture with a 25-gauge needle, endotoxin-sensitive C3H/HeN mice have a 45% mortality compared with no mortality in endotoxin-resistant C2H/HeJ mice. Macrophage production of interleukin 1 and antigen presentation were studied in these two strains of mice following cecal ligation and puncture at 2, 4, 8, 16, and 24 hours and at 2, 4, 6, and 8 days. Splenic macrophages were cultured with a T-helper cell clone (D10.G4.1), and antigen presentation and interleukin 1 production were measured by D10.G4.1 proliferation. Macrophage antigen presentation by C2H/HeJ mice was markedly increased compared with that in C3H/HeN mice at all times after cecal ligation and puncture, most strikingly at 2 days (185m740 cpm for C3H/HeJ mice vs 30,300 for C2H/HeN mice). Macrophage interleukin 1 production was significantly increased in C3H/HeJ mice vs C3H/HeN mice at all times after cecal ligation and puncture (except at 2 days) and was maximal at 8 days (25,000 cpm for C3H/HeJ mice vs 5190 for C3H/HeN mice). These data suggest that the differences in mortality after cecal ligation and puncture between these two strains of mice may relate to a supranormal response of macrophages of C3H/HeJ mice or to an inadequate response of macrophages of C3H/HeN mice.     

Genetic analysis of glucose tolerance in inbred mouse strains. Evidence for polygenic control

To determine genetic factors involved in diabetes susceptibility in inbred strains of mice, we initially evaluated differences in fed plasma glucose and insulin concentrations among six strains (AKR/J, C3H/HeJ, C57BL/6J, C57L/J, DBA/2J, and SWR/J). There was considerable variation in fed plasma glucose concentration, with C3H/HeJ mice the most glucose tolerant (174 +/- 7 mg/dl) and C57BL/6J mice the least glucose tolerant (252 +/- 7 mg/dl, P less than .0001 vs. C3H/HeJ mice). Glycosylated hemoglobin of C57BL/6J mice (4.0 +/- 0.06%) was also higher than that of C3H/HeJ mice (3.52 +/- 0.06%, P less than .0001). The fed plasma insulin concentration did not differ between these two strains. Glucose tolerance was further evaluated in overnight-fasted C3H/HeJ and C57BL/6J mice by an intraperitoneal glucose tolerance test (IPGTT). Although fasting plasma glucose did not differ, the most remarkable difference in plasma glucose during IPGTT between C57BL/6J and C3H/HeJ mice was noted at 30 min (489 +/- 29 vs. 227 +/- 20 mg/dl, P less than .001). To determine the number of genes involved in the phenotypic difference in glucose tolerance, C57BL/6J males were crossed with C3H/HeJ females (F1, C3H/HeJ X C57BL/6J), and the F1 hybrid females were backcrossed with C57BL/6J males (backcrossed, F1 X C57BL/6J). Plasma glucose after 30 min on IPGTT was 219 +/- 8 (n = 21), 456 +/- 18 (n = 23), and 292 +/- 13 (n = 23) mg/dl for C3H/HeJ, C57BL/6J, and F1 mice, respectively (P less than .001 for all comparisons).(ABSTRACT TRUNCATED AT 250 WORDS)     

Endotoxin tolerance from lipopolysaccharide pretreatment induces nuclear factor-kappaB alterations not present in C3H/HeJ mice

BACKGROUND: Lipopolysaccharide (LPS) activation of macrophage (MO) cytokine secretion requires activation and translocation of nuclear factor-kappaB (NF-kappaB). Endotoxin tolerance induced in LPS-responsive C3H/HeN MOs by LPS pretreatment results in decreased tumor necrosis factor (TNF) secretion and altered NF-kappaB activation. C3H/HeJ MOs have a genetic defect that renders them tolerant to LPS activation. We hypothesized that the alterations of NF-kappaB activation seen with LPS tolerance in HeN MOs would be present in HeJ mice. METHODS: MOs from C3H/HeJ and C3H/HeN mice were cultured with +/- 10 ng/mL LPS pretreatment for 24 hours and then stimulated with 1 to 1,000 ng/mL LPS. Activation of NF-kappaB was assayed by gel shift using a 32P-labeled specific oligonucleotide 30 minutes after LPS activation. TNF secretion 6 hours after LPS stimulation was measured by bioassay. RESULTS: LPS stimulation activated NF-kappaB in both HeN and HeJ MOs. We observed decreased NF-kappaB activation and a characteristic mobility shift in endotoxin-tolerant MOs from HeN mice that were not present in HeJ MOs. In contrast with the results in HeN mice, LPS pretreatment did not induce any alterations in NF-kappaB activation in HeJ MOs. LPS-stimulated TNF secretion was decreased in HeN MOs after LPS pretreatment. There was no change in TNF secretion in HeJ MOs, but, overall, TNF secretion by these cells was much less than that seen in HeN cells. CONCLUSION: MOs from C3H/HeN mice rendered LPS-tolerant by low-dose LPS pretreatment have alterations in activation of NF-kappaB not present in LPS-hyporesponsive C3H/HeJ mice.     

Mechanism of immunosuppression following hemorrhage: defective antigen presentation by macrophages

The mechanism by which simple hemorrhage profoundly impairs the proliferative response of T lymphocytes to mitogen and alloantigen, produces a defect in interleukin-2 generation, and increases the susceptibility to sepsis remains unknown. Since antigen presentation (AP) by the macrophage (M phi) plays a critical role in the antigen-specific activation of T-helper cells and lymphokine production, we investigated whether the function of the M phi as an AP cell is altered following hemorrhage. C3H/HEJ mice were bled to a mean BP of 35 mm Hg, maintained at that level for 1 hr, and then resuscitated. There was no mortality with this model. Control mice were not bled but otherwise treated identically. Immediately after resuscitation the mice were sacrificed and peritoneal M phi (PM phi) as well as splenic adherent cells (SAC) were harvested. AP function was tested by coculturing different numbers of PM phi and SAC with D10.G4.1 cells (2 x 10(4) cells/well) in the presence of conalbumin (300 micrograms/ml). This T-helper cell clone proliferates upon recognition of conalbumin in the context of Iak (a M phi surface membrane glycoprotein), thus directly reflecting M phi AP capability. After 72 hr of incubation, the cultures were pulsed with [3H]thymidine and harvested. D10.G4.1 proliferations induced via AP by PM phi and SAC from hemorrhaged-resuscitated mice were 29 and 24% of control, respectively (P less than 0.05). Thus, we conclude that AP by M phi following hemorrhage is defective despite adequate resuscitation, a mechanism which could explain the state of immunosuppression and enhanced susceptibility to sepsis.     

Role of the macrophage in the translocation of intestinal bacteria

To clarify the role of the macrophage in the translocation of intestinal bacteria, groups (n = 10) of Swiss Webster mice (Immunocompetent) and C3H/HeJ mice (macrophage defective) were given bacitracin/streptomycin in their drinking water to eliminate the majority of the intestinal microflora. These mice were then "monoassociated" with a streptomycin-resistant strain of Escherichia coli. Forty-eight hours later, E coli was present in all animals at a concentration of 10(11)/g of cecum. In four separate experiments, E coli was recovered from 100% of the mesenteric lymph nodes (MLNs) of the immunocompetent Swiss Webster mice and from 10%, 40%, 30%, and 50% of the MLNs of macrophage-defective C3H/HeJ mice. Swiss Webster mice were then similarly monoassociated by antibiotic decontamination followed by administration of antibiotic-resistant, fluorescein-labeled E coli in their drinking water; cohort groups of mice were given fluorescein-labeled latex beads (1 micron in diameter) in their drinking water. Two, four, and 11 days later, the MLNs were removed and single cell suspensions were analyzed in the fluorescence-activated cell sorter. The fluorescein label was detected exclusively in the macrophage (esterase-positive) population. These results support the hypothesis that intestinal macrophages may play a key role in the transport of intestinal particles (including bacteria) into extraintestinal sites.     

Effect of T cell modulation on the translocation of bacteria from the gut and mesenteric lymph node.

Although the ability of the gut-associated lymphoid tissue (GALT) to respond to orally ingested foreign antigens has been studied extensively, its function in preventing or limiting escape of resident gut bacteria has not been assessed. The following studies were performed to examine what role cell-mediated immunity (CMI) plays in this process. The ability of suppression of CMI to induce escape of gut bacteria (translocation) to the mesenteric lymph node (MLN) in immunocompetent mice whose gut flora was unaltered was examined. Administration of cyclosporine or anti-L3T4 antibody failed to induce translocation of indigenous gut bacteria after 7 or 14 days of treatment. Antithymocyte globulin (ATG) also failed to induce translocation after 7 days of treatment, despite depletion of all Thy 1, Lyt 1, L3T4, and Lyt 2 positive cells from the spleen, MLN, and intestine as demonstrated by immunofluorescent microscopy. Finally, cultures of the MLN, spleen, liver, and peritoneum of T cell-deficient BALB/c nude mice and their heterozygous T cell-replete littermates were also sterile, demonstrating that congenital suppression of T CMI also does not lead to translocation of indigenous gut bacteria. The role of CMI in limiting systemic spread of bacteria that were already translocating to the MLN was also examined. Translocation of Escherichia coli C25 to the MLN was induced by gastrointestinal (GI) monoassociation, which leads to translocation of E. coli C25 to the MLN in 80-100% of mice. Treatment with ATG during monoassociation failed to induce spread of E. coli C25 to the spleen, liver, or peritoneum, despite the same degree of T cell depletion achieved with ATG in the previous experiment. Monoassociation of conventional T cell-deficient BALB/c nude and heterozygous mice and germ-free T cell-deficient BALB/c nude and heterozygous mice also did not lead to spread of E. coli C25 beyond the MLN. However, in ATG-treated, conventional nude, and germ-free nude mice, the average number of translocating E. coli C25 per MLN was consistently higher. In separate experiments the ability of stimulation of T cell function to inhibit translocation of E. coli C25 was examined. Recombinant interleukin-2, 25,000 units, was administered intraperitoneally every 8 hours during exposure to E. coli C25. This reduced the incidence of translocation of E. coli C25 from 85% to 51% (p = 0.02). Suppression of CMI, either systemically or within the GALT, has a minimal influence on the mechanisms by which the normal gut flora are translocated to the MLN.(ABSTRACT TRUNCATED AT 400 WORDS)     

Elemental diet-induced bacterial translocation can be hormonally modulated.

BACKGROUND: The authors have previously documented that feeding mice an elemental diet resulted in bacterial translocation (BT) that could be prevented by the provision of dietary fiber. To test whether the protective effect of fiber was related to the stimulation of trophic gut hormones, the effects of sandostatin and bombesin were tested. METHODS: Mice fed either chow or the elemental diet were stratified into several groups and the ability of bombesin (10 micrograms/kg, tid) or sandostatin (100 micrograms/kg bid) to modulate BT was examined. After 14 days, mice were sacrificed and BT, cecal bacterial population levels, mucosal protein, and small bowel weight was measured. Segments of the ileum and jejunum were examined histologically. RESULTS: Incidence of elemental diet-induced BT (75%) was reduced by fiber (9%) or the administration of bombesin (13%) (p < 0.01). Although sandostatin did not promote BT in chow-fed mice, it reversed the protective effect of fiber on BT (75%) (p < 0.01). CONCLUSION: Elemental diet-induced bacterial translocation can be modulated hormonally and the beneficial effects of fiber on diet-induced BT appears to be hormonally mediated.     

Atherogenic high-fat diet reduces bone mineralization in mice.

The epidemiological correlation between osteoporosis and cardiovascular disease is independent of age, but the basis for this correlation is unknown. We previously found that atherogenic oxidized lipids inhibit osteoblastic differentiation in vitro and ex vivo, suggesting that an atherogenic diet may contribute to both diseases. In this study, effects of an atherogenic high-fat diet versus control chow diet on bone were tested in two strains of mice with genetically different susceptibility to atherosclerosis and lipid oxidation. After 4 months and 7 months on the diets, mineral content and density were measured in excised femurs and lumbar vertebrae using peripheral quantitative computed tomographic (pQCT) scanning. In addition, expression of osteocalcin in marrow isolated from the mice after 4 months on the diets was examined. After 7 months, femoral mineral content in C57BL/6 atherosclerosis-susceptible mice on the high-fat diet was 43% lower (0.73 +/- 0.09 mg vs. 1.28 +/- 0.42 mg; p = 0.008), and mineral density was 15% lower compared with mice on the chow diet. Smaller deficits were observed after 4 months. Vertebral mineral content also was lower in the fat-fed C57BL/6 mice. These changes in the atherosclerosis-resistant, C3H/HeJ mice were smaller and mostly not significant. Osteocalcin expression was reduced in the marrow of high fat-fed C57BL/6 mice. These findings suggest that an atherogenic diet inhibits bone formation by blocking differentiation of osteoblast progenitor cells.     

Oral Administration of Taurolidine Ameliorates Chronic DSS Colitis in Mice

Taurolidine (TRD) has antimicrobial and anti-inflammatory properties. However, the anti-inflammatory effects of TRD in inflammatory bowel diseases (IBD) have not been investigated. Here, we have analyzed the toxicity of TRD after oral long-term application in mice and examined the impact of oral TRD in a dextran sulfate sodium (DSS) model of experimental colitis. Female C57/BL6 mice received TRD in various concentrations (0.1% to 0.4%) for 60 days. Toxicity was evaluated by use of a disease activity index (DAI) and histological examination of major metabolic organs. Furthermore, the impact of 0.2% TRD on a chronic DSS colitis was examined by daily DAI, histological crypt damage score (CDS), bacterial translocation into mesenteric lymph nodes (MLN), and colonic expression of tumor necrosis factor (TNF) α, transforming growth factor (TGF) β, interleukin (IL)-1β, IL-6, cytochrome oxidase (COX)-2, and monocyte chemotactic protein (MCP)-1 by real-time polymerase chain reaction (PCR). Oral TRD administration for 60 days was well tolerated by the animals and did not show any toxic effects in terms of DAI and histological changes. TRD treatment of DSS colitis led to increased survival of 100%, compared to 33% in the untreated colitis group (p ≤. 005). Clinical amelioration was mirrored by significantly reduced DAI and CDS in the TRD treated colitis. Colonic cytokine expression and bacterial translocation into MLN showed no differences between both groups.

We thus report for the first time that oral application of TRD results in amelioration of an experimental IBD model. We hypothesize direct intraluminal antimicrobial effects of TRD as well as anti-inflammatory effects during the acute phase of DSS colitis.     

Oral administration of taurolidine ameliorates chronic DSS colitis in mice.

Taurolidine (TRD) has antimicrobial and anti-inflammatory properties. However, the anti-inflammatory effects of TRD in inflammatory bowel diseases (IBD) have not been investigated. Here, we have analyzed the toxicity of TRD after oral long-term application in mice and examined the impact of oral TRD in a dextran sulfate sodium (DSS) model of experimental colitis. Female C57/BL6 mice received TRD in various concentrations (0.1% to 0.4%) for 60 days. Toxicity was evaluated by use of a disease activity index (DAI) and histological examination of major metabolic organs. Furthermore, the impact of 0.2% TRD on a chronic DSS colitis was examined by daily DAI, histological crypt damage score (CDS), bacterial translocation into mesenteric lymph nodes (MLN), and colonic expression of tumor necrosis factor (TNF) alpha, transforming growth factor (TGF) beta, interleukin (IL)-1beta, IL-6, cytochrome oxidase (COX)-2, and monocyte chemotactic protein (MCP)-1 by real-time polymerase chain reaction (PCR). Oral TRD administration for 60 days was well tolerated by the animals and did not show any toxic effects in terms of DAI and histological changes. TRD treatment of DSS colitis led to increased survival of 100%, compared to 33% in the untreated colitis group (p < or = .005). Clinical amelioration was mirrored by significantly reduced DAI and CDS in the TRD treated colitis. Colonic cytokine expression and bacterial translocation into MLN showed no differences between both groups. We thus report for the first time that oral application of TRD results in amelioration of an experimental IBD model. We hypothesize direct intraluminal antimicrobial effects of TRD as well as anti-inflammatory effects during the acute phase of DSS colitis.     

Bone Response to In Vivo Mechanical Loading in Two Breeds of Mice

We investigated the bone response to external loading in C57BL/6J and C3H/HeJ mice, both breeds with low and high bone density, respectively. An in vivo tibial four-point bending device previously used for application of measured external loads in rats was adapted for mice. It delivered a uniform medio-lateral bending moment to the region of the tibia located 1–5.5 mm proximal to the tibio-fibula junction. The right legs of six C57BL/6J [low bone density (LBD)] and C3H/HeJ [high bone density (HBD)] mice were externally loaded in the device for 36 cycles/day at 2 Hz, 6 days/week for 2 weeks at 9.3 ± 0.9 N force, inducing estimated lateral periosteal surface compressive strains of 5121 ± 1128 με in C3H/HeJ (HBD) mice (n = 6), significantly higher than the estimated 3988 ± 820 με in C57BL/6J mice (n = 6) (mean ± SD). In addition, C3H/HeJ HBD mice (n = 11) were externally sham (pad pressure or no bending) loaded in the device for 36 cycles/day at 2 Hz, 3 days/week for 3 weeks at 9.3 ± 0.9 N force. Calcein injections for bone labeling were given at the 10th and 3rd days before sacrifice. At the end of the experiment, all mice were killed and both tibiae were removed, fixed, embedded, and cross-sectioned through the loaded region. Both tibiae were measured for marrow area (Ma.Ar), cortical area (Ct.Ar), total area (Tt.Ar), cross-sectional moment of inertia (CSMI), and periosteal and endocortical woven bone surface (Wo.B/BS), single-labeled surface (sLS), double-labeled surface (dLS), and total formation surface (FS/BS). Differences in all variables due to breed and loading (both bending and sham-bending) were tested by two-way analysis of variance (ANOVA) (P < 0.05). Ma.Ar, Tt.Ar, and CSMI were greater in C57BL/6J (LBD) than in C3H/HeJ (HBD) mice. Periosteal and endocortical woven bone and formation surface were increased significantly more by loading (bending) in C57BL/6J than in C3H/HeJ mice. Periosteal woven bone response due to sham-bending or sham-loading was significantly lower than due to bending loads in the C3H/HeJ mice. We conclude that the bone response to external loading is greater in LBD mice than in HBD mice. The high bone density of C3H/HeJ (HBD) mice is related to breed-specific factors other than the response to loading. Received: 5 March 1997 / Accepted: 8 April 1998     

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: cold exposure (up to 16 hr at 4 degrees C), a nontissue injury stress model; femoral fracture-amputation, a trauma model; and 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 less than 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 less than 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.     

Sepsis induces an early increased spontaneous release of hepatocellular stimulatory factor (interleukin-6) by Kupffer cells in both endotoxin tolerant and intolerant mice.

Previous studies have shown that hepatocellular function is significantly depressed early during sepsis and that this is associated with a marked increase in the circulating levels of the hepatocellular stimulatory factor (IL-6). It remains unknown, however, whether or not Kupffer cells (KC) are activated during sepsis and whether these cells are the major contributors to the increased circulating levels of this cytokine. The objectives of this study were, therefore, to determine whether or not during sepsis: (1) KC are stimulated in vivo to release IL-6, as compared to other cytokines; (2) KC differ from splenic macrophages (SM phi) in their ability to release cytokines; and (3) there is a difference in macrophage (M phi) cytokine release between endotoxin (ET)-tolerant (C3H/HeJ) and ET-intolerant (C3H/HeN) mice. To assess this, KC and SM phi were harvested at 1 or 24 hr from mice which had been subjected to polymicrobial sepsis by cecal ligation and puncture (CLP) or sham-operation. Following depletion of the nonadherent cells, KC and SM phi cultures were incubated for 24 hr in the presence or absence of 10 micrograms of ET/ml, and the levels of interleukin (IL)-1, IL-6, and TNF release were determined by bioassays. Sepsis induced an early (at 1 hr) in vivo stimulation of KC but not SM phi IL-6 release, irrespective of ET-tolerance/intolerance. However, the release of IL-1 or TNF was not markedly different for either CLP or sham KC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Promotion by burn stress of the translocation of bacteria from the gastrointestinal tracts of mice

A mouse burn model was established to test the effect of nonlethal thermal injury on the translocation of indigenous bacteria from the gastrointestinal (GI) tract to other organs. Specific pathogen-free (SPF) mice were given 15% or 30% total body surface area burns, and the mesenteric lymph nodes (MLNs), spleens, livers, blood, and peritoneal cavities were cultured for translocated bacteria at various time intervals. No viable aerobic, facultatively anaerobic, or strictly anaerobic bacteria of the indigenous flora grew in cultures from the MLNs of these mice. Consequently, SPF mice were antibiotic decontaminated and then colonized with Escherichia coli to develop a model in which E coli maintains abnormally high cecal population levels and translocates continuously to the MLN. These mice received 15% or 30% thermal burns four days after colonization with E coli. The incidence of bacterial translocation and the numbers of E coli translocating to the MLN, spleen, liver, blood, and peritoneal cavity increased with increasing burn area compared with controls. Mice receiving 15% burns could not clear intravenously challenged E coli from their bloodstream, MLN, or liver. Thus, burn stress promotes the translocation of bacteria from the GI tract to other organs to cause bacteremia.     

Water-soluble ethylhydroxyethyl cellulose prevents bacterial translocation induced by major liver resection in the rat.

Enteric bacteria might act as pathogens, translocating across the intestinal barrier to extraintestinal sites after major liver resection. In the current study, water-soluble ethylhydroxyethyl cellulose (EHEC) was administered before hepatectomy to evaluate the influence on bacterial translocation induced by major liver resection, phagocytic capacity by visceral and circulating macrophages, enteric bacterial population, and bacterial adherence on the intestinal surface in rats subjected to sham operation or to 70% or 90% hepatectomy. Oral or intravenous (IV) administration of EHEC reduced the incidence of bacterial translocation to mesenteric lymph nodes (MLN) and blood after major liver resection. Oral EHEC appeared more effective than IV administration in protecting against bacterial translocation to MLN in animals with 90% hepatectomy. Ethylhydroxyethyl cellulose (oral and IV) significantly diminished intestinal macrophage uptake capacity of 125I-labeled, heat-killed Escherichia coli as compared with animals without EHEC administration. Overgrowth or colonization of enteric bacteria after major liver resection could be prevented by oral or IV EHEC. Adherence of 14C-labeled, alive E. coli on the intestinal mucosa decreased after EHEC treatment in animals subjected to major liver resection. Systemic arterial pressure and intestinal blood flow markedly decreased from 1 hour and on after 90% hepatectomy. Intravenous administration of EHEC did not improve these alterations. Bacterial hydrophobicity and surface negative charge were significantly reduced 1 hour after bacterial culture with EHEC. Thus, EHEC appears to be a potent agent preventing translocation of enteric bacteria from the gut after major liver resection, by altering the surface characters of enteric bacteria, balancing the enteric microflora, inhibiting bacterial attachment onto the intestinal surface, and blocking phagocytosis by intestinal macrophages.