Bacterial translocation induces proinflammatory responses and is associated with early death in experimental severe injury

Objective

An experimental model of severe injury with great lethality was studied to define the impact of bacterial translocation on survival and on inflammatory response.

Methods

Forty-one rabbits were divided into two groups: A, femur myotomy; and B, myotomy and fracture of the femoral bone. Vital signs and survival were recorded. Serum circulating endotoxins (lipopolysaccharides; LPS) were determined and tissue cultures were performed at necropsy. A subgroup of animals was sacrificed at 48 h post injury; LPS was determined in abdominal aorta and portal vein, apoptosis of spleen cells was assessed by flow cytometry, and ex vivo production of tumor necrosis factor alpha by splenocytes was measured.

Results

Tissue bacterial burden was increased in animals that died early (i.e., within 48 h after injury) versus rabbits that died later. Portal vein LPS at 48 h was increased in group B compared with group A, whereas circulating LPS did not differ. No difference in apoptosis of either lymphocytes or macrophages of the spleen was found in group B compared with group A. Following stimulation with LPS or phytohemagglutinin, tumor necrosis factor α production by splenocytes of group B was greater than that of group A.

Conclusions

Bacterial translocation primes enhanced proinflammatory responses and it is associated with early death in severe trauma.     

Anomalous origin of the right coronary artery arising from the circumflex artery

A case of anomalous origin of the right coronary artery discovered among 3100 selective coronary arteriograms is described. This artery was arising from the circumflex artery. The position, distribution, and configuration of this coronary artery was as a normal right coronary artery, except that its origin was in the peripheral segment of the circumflex artery. This anomalous origin is very rare and seems not to give rise to any clinical significance.     

Role of soluble triggering receptor expressed on myeloid cells in inflammatory bowel disease

AIM: To investigate the probable role of soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) in the pathogenesis of inflammatory bowel disease (IBD). METHODS: Fifty-eight patients were enrolled; nineteen healthy volunteers served as controls; 8 patients were diagnosed with Crohn's disease, and 31 with ulcerative colitis. Clinical and endoscopic activity indexes of patients with Crohn's disease and ulcerative colitis respectively were estimated. Upon admission blood was sampled; sTREM-1 and TNFαwere measured by an immunoassay and malondialdehyde (MDA) by the thiobarbitourate assay, after passage through an HPLC system. RESULTS: Median±SE of TNFαof controls, patients with Crohn's disease and patients with ulcerative colitis were 6.02±3.94, 7.98±5.08 (P = NS vs controls), and 8.45±4.15 ng/L (P = 0.018 vs controls) respectively. Respective values of sTREM-1 were 53.31±32.93, 735.10±197.17 (P = 0.008 vs controls) and 435.82±279.71 ng/L (P = 0.049 vs controls). sTREM-1 was positively correlated with Crohn's disease activity index and clinical and endoscopic activity indexes of ulcerative colitis (P = 0.002, 0.001 and 0.009, respectively). sTREM-1 of patients with ulcerative colitis was positively correlated with TNFa (P = 0.001). CONCLUSION: sTREM-1 seems to behave as a novel mediator in IBD in correlation with the degree of the inflammatory reaction of the intestinal mucosa.     

Early apoptosis of monocytes contributes to the pathogenesis of systemic inflammatory response and of bacterial translocation in an experimental model of multiple trauma

The objective of this study was to investigate the occurrence of apoptosis of monocytes in an experimental model of multiple trauma and its probable correlation to bacterial translocation. Thirty-two rabbits were applied in three groups: A, controls; B, myotomy of the right femur; and C, myotomy and fracture of the right femur. Blood was sampled for the estimation of endotoxins [lipopolysaccharide (LPS)], tumour necrosis factor (TNF)-alpha, malondialdehyde (MDA) and isolation of peripheral blood mononuclear cells (PBMCs). PBMCs, derived after centrifugation over Ficoll, were incubated in flasks and apoptosis of non-adherent lymphocytes and adherent monocytes was estimated after staining for Annexin-V and flow cytometry. TNF-alpha of supernatants of cultured monocytes was also determined. Tissue segments were cultured after death. Median survival of groups A, B and C was > 14, > 14 and 9.00 days, respectively. Apoptosis of lymphocytes in group C was higher than group A at 2, 4 and 48 h and of monocytes in group C higher than group A at 2 and 4 hours. LPS in group C was higher than group A at 2, 4 and 48 h. Apoptosis of lymphocytes and monocytes was correlated positively with serum TNF-alpha and negatively with TNF-alpha of monocyte supernatants. Cultures of organ segments of group A were sterile. Pseudomonas aeruginosa was isolated from liver, lung and spleen in five animals in group B (45.45%) and in six in group C (54.54%). Early apoptosis of blood monocytes supervened after multiple trauma; the phenomenon was accompanied by apoptosis of blood lymphocytes and subsequent bacterial translocation.     

Change of annexin binding of monocytes as an expression of cellular response to <Emphasis Type="Italic">Candida albicans</Emphasis>: down-regulation in severe sepsis

To study the differences of monocyte activation by albicans and non-albicans species of Candida and its change in sepsis, peripheral blood mononuclear cells were isolated from 17 healthy volunteers and 26 patients with severe sepsis/shock, and incubated in the absence/presence of heat-killed (HK) isolates of four different Candida species and purified β-D-glucan from C.albicans. Experiments were repeated in the presence and absence of inhibitors of intracellular activation pathways. Expression of annexin V on cells membranes of monocytes and lymphocytes, cytoplasmic activity of caspase-3, and DNA fragmentation of monocytes were studied. Membrane expression of annexin V on viable monocytes of healthy volunteers decreased significantly after incubation with C.albicans but not with non-albicans species. The decrease was dose-dependent from the Candida inoculum and by the concentration of β-D-glucan. A relationship with inhibition of apoptosis was found as the activity of caspase-3 activity, and the level of DNA fragmentation were also decreased. Incubation in the absence/presence of inhibitors showed that the decrease by annexin V expression resulted by activation of the dectin-1 pathway and Raf-1 by β-D glucan. The decrease of annexin V(+)/PI(?) expression was not shown on monocytes of patients with severe sepsis/shock, where no effect of inhibitors was found. Decrease of annexin V binding on monocytes can be viewed as a selective response to C.albicans partly effected through activation of dectin-1. This response is down-regulated after a septic insult.     

In Vitro Elution of Daptomycin by a Synthetic Crystallic Semihydrate Form of Calcium Sulfate,Stimulan

A synthetic crystallic semihydrate form of calcium sulfate, Stimulan, was evaluated as a biodegradable carrier for the daily in vitro elution of daptomycin. Daptomycin and Stimulan were admixed at a ratio of 95:5. Elution lasted for 28 days. Eluted concentrations peaked on days 1 and 11, when the mean values were 1,320.1 and 949.2 μg/ml, respectively. The lowest eluted concentration was detected on day 28. These results support the application of the system described in experimental models of osteomyelitis.Chronic osteomyelitis is an infection difficult to treat due both to multidrug resistance of common pathogens and to poor penetration of antibiotics into bone (16). Carriers for local delivery of antimicrobials have been developed, attempting to provide locally high concentrations of antibiotics (5). Some newly developed biodegradable carriers have been shown to be very potent for the eradication of experimental osteomyelitis (6, 7). The semihydrate form of calcium sulfate (CaSO₄), commonly known as plaster of Paris, may be applied as a biodegradable system for local drug delivery. It has been used for decades to fill bone cavities resulting from disease, trauma, or surgery (11). It was recently shown to be potent in vitro for the release of vancomycin, teicoplanin, gentamicin, and clindamycin (15). The antimicrobial applied in such an elution system should be active against the most commonly involved pathogens of chronic bone infections, namely, methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative staphylococci (CoNS) (5). Daptomycin, which is a novel lipopeptide antimicrobial with excellent in vitro activity against these isolates (4), may be a candidate for application in a system for local drug delivery.Stimulan (Biocomposites, Keele Science Park, Staffordshire, United Kingdom) is a synthetic biocompatible bone graft material made of calcium sulfate. It is completely reabsorbed and replaced with new bone. It is synthesized at 100% purity with no traces of potentially toxic impurities that have been associated with naturally occurring mineral sources of calcium sulfate. It has been recently shown by our group that moxifloxacin and fusidic acid may be eluted at high concentrations in vitro by using Stimulan as a carrier (11). The purpose of the present study was to develop an in vitro system for daptomycin elution by using Stimulan as a delivery system.Stimulan was mixed with daptomycin (Novartis, Basel, Switzerland) at a ratio 95:5 to a total weight of 3 g. This was put into sterile vials (160 by 100 mm) and left at room temperature for 15 to 30 min for solidification. Five similar vials were prepared. One milliliter of Mueller-Hinton broth (Trec Diagnostic Systems, West Sussex, United Kingdom) was added over the free surface of each mixture and replaced every 24 h. The vials were incubated at 37°C. On each consecutive day, the eluent was removed, transferred to a sterile plastic tube, and replaced with 1 ml of broth. That procedure was repeated until the optical degradation of the prepared mixture. Samples were stored at −70°C until analysis.Concentrations of daptomycin were estimated in duplicate by a modification of the method already described (3). Briefly, 300 μl of sample was mixed with 20 μl of 99% methanol and centrifuged for 10 min at 5,000 rpm and 4°C. Twenty microliters of the supernatant was injected into a high-pressure liquid chromatography system (1100 series; Agilent, Waldbronn, Germany) with the following elution characteristics: a Zorbax Eclipse XDB-C₈ (4.6 by 150 mm, 5 μm) separating column (Agilent Technologies) warmed at 37°C, a mobile phase of 32.6% acetonitrile and 67.4% 0.5% ammonium phosphate buffer at a flow rate of 1.5 ml/min, and UV detection at 214 nm. The retention time of daptomycin was 3.3 min, and it was estimated as micrograms per milliliter by a standard curve created with known concentrations of daptomycin. The lower detection limit was 6.25 μg/ml, and the interday coefficient of variation of the assay was 4.7%. Results were expressed as means ± standard errors. The area under curve (AUC) for each vial was determined by the linear trapezoidal rule.Elution of daptomycin lasted 28 days. Then the prepared mixture was destroyed. Eluted concentrations peaked on days 1 and 11, when the mean concentrations were 1,320.1 and 949.2 μg/ml, respectively. The lowest eluted concentration was detected on day 28, and the mean value was 233.9 μg/ml (Fig. ​(Fig.1).1). This gradual step-down rate of release over the 28 days was consistent for all five replicates. The mean ± standard deviation AUC of daptomycin elution over this 28-day period was 14,542.7 ± 1,925.9 μg · day/ml. Short peaks of daptomycin release were apparent on days 5 and 11. These may be related to the properties of Stimulan.Open in a separate windowFIG. 1.Elution of daptomycin by Stimulan.Daptomycin is a new lipopeptide that has been recently licensed for the management of skin and soft-tissue infections (13). It has excellent intrinsic activity against MRSA and methicillin-resistant CoNS but also vancomycin-resistant enterococci (10). The MICs for 90% of the strains of MRSA, methicillin-resistant CoNS, and vancomycin-resistant enterococcal isolates tested are reported to be 0.78, 0.44, and 0.5 μg/ml, respectively (1, 4). Although the eluted daptomycin concentrations obtained with the system described here are much greater than the above-mentioned MICs for 90% of the strains tested, results should be interpreted with caution, since the kinetics of release apply to an in vitro system and not to in vivo conditions. It should, however, be underscored that eluted levels are considerably greater than the concentrations that are required to eliminate the growth of small-colony variants of S. aureus that are often implicated in chronic bone infections (1).The estimated AUC of elution may be considered indirect evidence of the total amount of drug released (2). The AUC for daptomycin by the elution system presented here is much greater than the AUC required for the management of an experimental thigh infection with MRSA in mice (9).Calcium sulfate has been applied as a carrier for daptomycin in two previous studies. The drug was admixed with CaSO₄ in pellet form. The total time of drug elution was 28 days, but the eluted concentrations were 10- to 100-fold lower than those achieved here (12). Even at a low rate of elution, the drug amounts released inhibited the growth of 10⁴ CFU of two different locally instilled isolates, one of S. aureus and another of S. epidermidis (14).Although the effectiveness of daptomycin has not been evaluated in any prospective randomized clinical trial for the therapy of staphylococcal osteomyelitis, retrospective data favor its application for the management of such infections (8). These favorable clinical prospects, along with the successful in vitro elution of daptomycin from Stimulan reported in the present study, support the application of the biodegradable system described here in experimental models of osteomyelitis.     

The impact of multidrug resistance on the pathogenicity of Escherichia coli: an experimental study

Based on the controversial findings of clinical studies regarding the influence of multidrug resistance on mortality, 10 susceptible and 10 multidrug-resistant (MDR) and extended-spectrum beta-lactamase-producing isolates of Escherichia coli were applied to stimulate monocytes isolated from healthy donors. Immune mediators were estimated in supernatants. Four susceptible isolates (Group A) and four MDR isolates (Group B) were used to initiate acute pyelonephritis in 48 rabbits following inoculation of the pathogen into the right renal pelvis. Survival was recorded and blood monocytes were isolated and incubated to estimate the ex vivo release of tumour necrosis factor-alpha (TNFalpha). Release of TNFalpha, interleukin (IL)-6 and IL-8 was higher after 2 h and 4 h of stimulation by MDR isolates compared with susceptible isolates. The opposite occurred for the release of IL-12. Death occurred in 22 rabbits in Group A (91.7%) compared with 12 in Group B (50.0%) (P=0.003). Monocytes isolated at 24 h from Group A rabbits released significantly higher TNFalpha than monocytes from Group B. Tissue bacterial load after animal death was significantly higher in the kidneys of Group A rabbits. It is concluded that susceptible and MDR E. coli stimulate monocytes resulting in a different pattern of release of pro-inflammatory cytokines, which is accompanied by prolonged survival following experimental sepsis by MDR isolates.     

In vitro elution of moxifloxacin and fusidic acid by a synthetic crystallic semihydrate form of calcium sulphate (Stimulan)

Stimulan was evaluated in vitro as a biodegradable carrier for local delivery of moxifloxacin and fusidic acid. Moxifloxacin or fusidic acid was mixed with calcium sulphate at a ratio of 95:5 to prepare five replicas per antibiotic. In vitro elution was estimated daily using a high-performance liquid chromatography (HPLC) system. Elution of moxifloxacin lasted for 31 days. Eluted concentrations reached their peak on Day 13 (mean level 745 microg/mL); the lowest eluted concentration was detected on Day 30 (mean level 367 microg/mL). Elution of fusidic acid lasted for 14 days. Eluted concentrations reached their peak on Day 6 (mean value 249.5 microg/mL); the lowest eluted concentration was detected on Day 13 (mean value 10.9 microg/mL). The presented results revealed that Stimulan may allow adequate in vitro elution of moxifloxacin and fusidic acid. The latter results support the application of this system in experimental models of osteomyelitis.