Immunosuppression of the burned patient

The burn patient is highly susceptible to infection due to the loss of the skin as a barrier to microorganisms. Immune defenses are activated in response to the burn injury; however, some of these defenses are altered. Neutrophil chemotaxis is compromised by decreased perfusion caused by hypovolemia and the formation of microthrombi. Chemotaxis and phagocytosis are dependent on complement components that are reduced in a large burn wound. Neutrophil intracellular killing power is reduced as oxygen delivery to the wound is decreased. Humoral immunity is altered with the drop in IgG levels. Cell-mediated immunity is depressed and T cell lymphocyte counts are deceased. Suppressor T cells are generated. Specific sources of infection for the burn patient include the patient's own bacterial flora; hospital personnel; respiratory equipment; and catheters, both urinary and intravascular. The best control for burn wound infection is the closure of the wound by early excision and grafting. When lack of donor sites prohibits this surgical therapy, control centers on the environment and wound care techniques. The selection of wound topical antibiotics on the basis of visual inspection and surface culturing assists in the prevention of burn wound sepsis. When wound sepsis does occur, systemic antibiotics are instituted. Although burn wound sepsis is an obvious cause of death for the burn patient, it is not the primary cause. Increasing sophistication in fluid resuscitation and in intensive care therapy has resulted in patients living beyond the initial insult and the following few days. Burn patient mortality is now associated with a syndrome presenting clinically as sepsis but without any identifiable septic source.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chronic plasminogen activator inhibitor-1 (PAI-1) overexpression dampens CD25+ lymphocyte recruitment after lipopolysaccharide endotoxemia in mouse lung

BACKGROUND: Plasma plasminogen activator inhibitor-1 (PAI-1) level rises during sepsis and confers a worse prognosis. PAI-1 participation to sepsis has been poorly documented and was mainly associated with fibrin deposits. Beside fibrin deposits, increased tissue PAI-1 expression may contribute to the poor outcome of endotoxemia through other mechanisms. OBJECTIVE AND METHODS: During lipopolysaccharide (LPS) challenge, the role of PAI-1 in the early phase of inflammation was examined in the lungs of transgenic mice that either overexpress or lack the PAI-1 gene (PAI-1Tg or PAI-1(-/-)). RESULTS: Analysis of leukocytes revealed that neutrophil and macrophage infiltrations did not differ for PAI-1Tg and wild-type (WT) mice. Remarkably, CD25+ lymphocyte infiltration was totally blunted in PAI-1Tg lungs and inversely correlated with fibrin depositions. In parallel, mRNA levels of the regulatory T cell (Treg) markers FoxP3, CTLA-4, and GITR were significantly lower in PAI-1Tg than in WT lungs after LPS challenge. These data are supported by opposite results in PAI-1(-/-) lungs. The systemic compartments (spleen and peripheral blood) showed no decrease in CD25+, CD4+ CD25+ lymphocytes, and Treg markers in PAI-1Tg mice after LPS injection compared with WT mice. In addition, plasma and lung concentrations of interleukin-6 (IL-6) and macrophage inflammatory protein-1alpha (MIP-1alpha) were significantly higher in PAI-1Tg mice than WT mice. CONCLUSION: Our results suggest that chronic tissue PAI-1 overexpression influences the early phase of the inflammatory response during endotoxemia through the control of T lymphocyte traffic.     

Human endotoxemia and human sepsis: limits to the model

Sepsis remains the most common cause of death in intensive care units of the developed world. Accurate models of this disease syndrome are crucial for to the understanding of the complex pathophysiology of this disorder. The administration of a small dose of lipopolysaccharide to healthy volunteers is one such model of spontaneous human sepsis. Although this human endotoxemia model appears to be reasonably effective in mimicking early biochemical, metabolic, hematologic and cardiovascular septic responses in septic shock, the ability to mimic other aspects of human sepsis is open to question. The current study demonstrates that human experimental endotoxemia fails to generate evidence of increased vascular permeability within the relatively short time frame of the study.     

Human endotoxemia and human sepsis: limits to the model

Sepsis remains the most common cause of death in intensive care units of the developed world. Accurate models of this disease syndrome are crucial for to the understanding of the complex pathophysiology of this disorder. The administration of a small dose of lipopolysaccharide to healthy volunteers is one such model of spontaneous human sepsis. Although this human endotoxemia model appears to be reasonably effective in mimicking early biochemical, metabolic, hematologic and cardiovascular septic responses in septic shock, the ability to mimic other aspects of human sepsis is open to question. The current study demonstrates that human experimental endotoxemia fails to generate evidence of increased vascular permeability within the relatively short time frame of the study.     

Evaluation of antiinflammatory and antiadhesive effects of heparins in human endotoxemia

OBJECTIVE: Cytokines and adhesion molecules have a decisive role in the development of early inflammatory response as well as the late sequelae of sepsis. Because L-selectin-deficient mice are protected from lethal endotoxemia, blockade of L-selectin may provide a useful therapeutic option in human sepsis. Heparin has immunomodulatory properties and effectively inhibits L- and P-selectin binding in vitro. We therefore investigated whether clinically applied doses of unfractionated or low-molecular-weight heparin affect early inflammatory response in human endotoxemia. DESIGN: The study was randomized, double-blinded, placebo-controlled, in three parallel groups consisting of 30 healthy male volunteers. SETTING: University medical center. INTERVENTIONS: All subjects received a 2-ng/kg intravenous bolus of lipopolysaccharide and 10 mins later unfractionated heparin, low-molecular-weight heparin, or placebo as bolus primed continuous infusion for 6 hrs. MEASUREMENTS AND MAIN RESULTS: Lipopolysaccharide infusion induced similar increases of tumor necrosis factor-alpha, interleukin-6, interleukin-8, C-reactive protein, and soluble E-selectin levels in all treatment groups. CD11b expression increased by approximately 400%, but L-selectin decreased by 41% in the placebo arm 6 hrs after lipopolysaccharide infusion. Interestingly, both heparins (in particular unfractionated heparin) decreased L-selectin down-regulation as compared with placebo. Similarly, the decrease in lymphocyte counts was significantly less in the unfractionated heparin group during the first 24 hrs (p <.05 vs. placebo) CONCLUSIONS:Heparins displayed little effects on cytokine production and endothelial cell activation in endotoxemia. Of note, however, unfractionated heparin reduced L-selectin down-regulation and lymphocytopenia. These could present novel mechanisms of action of unfractionated heparin.     

Release of the mitochondrial enzyme carbamoyl phosphate synthase under septic conditions

To identify sepsis-related dysregulations of protein expression in the liver, we used a baboon model of acute endotoxemia and performed comparative proteome analysis. Treatment with lipopolysaccharide (LPS) was followed by an early but long-lasting (5-48 h) generation of N-terminal fragments of carbamoyl phosphate synthase-1 (CPS-1), an abundant enzyme of the hepatic urea cycle, which is normally located in the mitochondrial matrix. In addition, we developed a new sandwich immunoassay to determine circulating CPS-1 in human and baboons. We found CPS-1 to be induced by LPS and to be released into the circulation of healthy humans and baboons as early as 4 to 5 h after stimulation. Similarly, CPS-1 levels increased after injection of gram-positive bacteria in another baboon model. Enhanced CPS-1 levels were also detected in serum of patients with sepsis. Our data demonstrate fragmentation of CPS-1 in the liver and early increase in circulating CPS-1 levels under septic conditions. We suggest that circulating CPS-1 might serve as a novel serum marker indicating mitochondrial impairment of the liver and/or the small intestine in critically ill patients.     

Control of endotoxemia in burn patients by use of polymyxin B

A group of patients with severe burns were entered into two sequential prospective randomized trials for reduction of endotoxemia by the use of intravenous polymyxin B. The first group underwent polymyxin administration during the first week after burn injury in a bell-shaped dosage form constructed to resemble the level of endotoxemia as previously documented. This group showed a statistically highly significant reduction in endotoxin levels and a suggestive, but not statistically significant, reduction in wound infection and mortality in the treated group compared with controls. The second group of patients underwent treatment with perioperative polymyxin B given in conjunction with an excisional procedure of the burn wound. In this group, polymyxin B also accomplished a reduction in endotoxemia from preoperative to postoperative cases, but there was no significant reduction in clinical complication rate or mortality. In the dosages used, polymyxin B is nontoxic and promises to be a useful part of the surgeon's armamentarium in dealing with severe complications of gram-negative sepsis.     

The role of Kupffer cell inhibition in porcine endotoxemia

The selective Kupffer cell inhibitor gadolinium chloride (GdCl3) has been demonstrated to protect animals from lethality in experimental endotoxemia and sepsis in rodent models. This study was designed to investigate the effect of Kupffer cell blockade on the early response to endotoxin in a large animal model. Using a porcine endotoxemia model, animals were randomized to receive either GdCl3 (10 mg/kg or 30 mg/kg; n = 8 in each group) or vehicle saline (n = 8) 24 h before exposure to endotoxin. Pretreatment with GdCl3 resulted in a dose dependent reduction in early hepatic oxygen consumption as well as oxygen extraction ratio in response to continuous infusion of endotoxin. At 5 h there was significant lower serum AST level in animals given 30 mg/kg of GdCl3 as compared to the two other groups. Pretreatment with GdCl3 induced a dose dependent reduction of Kupffer cells in the liver sinusoids. Despite this, all animals deteriorated with continuous infusion of endotoxin as evidenced by the progressive reduction in cardiac output, mean arterial pressure and total liver blood flow. Also, increases in pulmonary arterial pressure, portal venous pressure and systemic, pulmonary and hepatic vascular resistance were seen. This is consistent with activation of other cell populations and defense mechanisms by endotoxin, perpetuating the septic response. However, modulation of reticuloendothelial cell function seems feasible also in larger animals, and our results stimulate to further research on potential immunomodulatory tools in early sepsis.     

Endotoxemia after burn injury: effect of early excision on circulating endotoxin levels

Serial circulating endotoxin measurements (quantitative chromogenic limulus assay) were performed in sera from 19 burned patients to determine the profile of circulating serial circulating endotoxin after burn and the effect of early wound excision on serial circulating endotoxin level. Results indicate an early endotoxemia with the peak serial circulating endotoxin levels 7 to 12 hours and 4 days after burn. More importantly, the level of circulating serial circulating endotoxin can be diminished by early excision; late wound excision was associated with a transient increase of serial circulating endotoxin level. Early excision, therefore, may play a critical role in limiting endotoxemia after burn injury.     

感染性休克患者外周血T淋巴细胞凋亡和影响因素分析

目的观察感染性休克患者的外周血T淋巴细胞的凋亡情况,并对可能诱导T淋巴细胞凋亡的原因进行探讨。方法采集南京市第一医院ICU自2006年1月至2009年1月收治的32例感染性休克患者的外周血,用Annexin-V法和流式细胞仪测定其中的T淋巴细胞的凋亡情况,同时用酶联免疫法（ELISA）测定血浆中肿瘤坏死因子α（TNF-α）、白细胞介素10（IL-10）、白细胞介素1β（IL-1β）的浓度,用化学发光法检测血浆中的游离皮质醇浓度,将上述的结果和脓毒血症病患者及健康对照（n=22）相比。结果在初始状态下,外周血T淋巴细胞CD4＋亚群在健康对照组、脓毒血症组及感染性休克组分别为（4.41±1.45）%、（7.87±3.82）%及（11.01±4.52）%,感染性休克患者的外周血T淋巴细胞CD4＋亚群的凋亡比例高于脓毒血症患者（P〈0.05）,脓毒血症患者高于健康对照组（P〈0.05）;而初始状态下健康对照组、脓毒血症组及感染性休克组患者外周血T淋巴细胞CD8＋亚群的凋亡为（9.62±8.32）%、（13.09±15.84）%及（11.33±19.62）%,3组比较差异无统计学意义（P〉0.05）。在基础状态下TNF-α的浓度在健康对照组,脓毒血症组及感染性休克组中分别为（16.44±9.55）ng/L、（29.58±13.6）ng/L及（47.99±25.63）ng/L,感染性休克组TNF-α的浓度高于脓毒血症组（P〈0.05）,脓毒血症组高于健康对照组（P〈0.05）,持续72小时;IL-10的浓度在健康对照组,脓毒血症组及感染性休克组中分别为（6.38±7.5）ng/L、（9.67±4.88）ng/L及（15.01±5.36）ng/L,感染性休克组中的IL-10的浓度高于脓毒血症组（P〈0.05）,脓毒血症组高于健康对照组（P〈0.05）,持续72小时;游离皮质醇的浓度在健康对照组,脓毒血症组及感染性休克组中分别为（0.45±0.42）μmol/L、（0.87±0.49）μmol/L及（1.39±0.84）μmol/L,感染性休克组中的游离皮质醇浓度高于脓毒血症组（P〈0.05）,脓毒血症组高于健康对照组（P〈0.05）,持续72小时;IL-1β在健康对照组、脓毒血症组及感染性休克组中分别为（13.12±5.07）ng/L、（25.21±14.76）ng/L及（22.94±22.01）ng/L,感染性休克组和脓毒血症组的浓度高于健康对照组（P〈0.05）,但是感染性休克组和脓毒血症组的浓度差异无统计学意义（P〉0.05）。结论感染性休克患者的外周血T淋巴细胞中CD4＋亚群可发生明显的凋亡,TNF-α、IL-10及皮质醇可能参与了诱导CD4＋亚群的凋亡。     

Changes in blood lymphocyte populations after multiple trauma: association with posttraumatic complications.

OBJECTIVE: To study the frequency of several lymphocyte subsets, circulating cytokines, and prostaglandin plasma values at their time course over a period of 14 days in severely injured trauma patients in relation to the development of sepsis and multiple organ failure (MOF). DESIGN: Prospective study. SETTING: An operative intensive care unit (ICU) of a university hospital. PATIENTS: Sixty-eight consecutive severely injured trauma patients. INTERVENTIONS: Patients were separated into patients without sepsis and MOF (group 1, n = 51), and patients who developed sepsis and MOF (group 2, n = 17) during their stay in the ICU. Therapy was adjusted to the standards of modern intensive care management by physicians who were not involved in the study. MEASUREMENTS AND MAIN RESULTS: In arterial blood samples, the profile of lymphocyte subset frequencies was performed by flow cytometry and, together with interleukin (IL)-1, IL-10, tumor necrosis factor (TNF)-alpha soluble TNF-alpha receptor 1 (sTNF-alpha r1 [p55]), and prostaglandin E2 (PGE2alpha)-alpha, serially measured after arrival in the ICU (baseline value) and during the next 14 days. Mean plasma IL-1 (29.3 +/- 5.8 [SD] pg/mL), TNF-alpha (138.5 +/- 22.4 pg/mL), and soluble TNF-alpha r1 (6.1 +/- 0.3 ng/mL) values were significantly higher in group 2 patients before clinical evidence of sepsis and MOF. With the onset of severe infections in group 2 patients, IL-1, TNF-alpha, and sTNF-alpha r1 values decreased, while immunosuppressive IL-10 (191.7 +/- 29.1 pg/mL) and PGE2alpha (87.7 +/- 20.4 pg/mL) values further increased and remained elevated during the time course. Analysis of lymphocyte subsets revealed a fall in total lymphocyte levels, in CD4+ T lymphocytes, and natural killer (NK) cells, but no change in CD8+ T lymphocyte subset. Despite a marked change in the T helper (CD4+) to T suppressor (CD8+) ratio (from 1:1.72 to 1:1.10), patients without MOF (group 1) had no significant difference in any of the markers tested compared with baseline values. In addition to the inverse CD4+/CD8+ T cell ratio (from 1:1.75 to 1:0.91) and increased activated T cells, each of these markers was significantly elevated and peaked before the onset of MOF in group 2 patients. CONCLUSIONS: A severely depressed cellular immune response associated with increased suppressive mediators might be closely related to the development of severe sepsis and MOF in trauma patients. Therefore, an in-depth understanding of the deficits in host defense following multiple trauma will provide the basis for therapeutic interventions.     

A protective role of interleukin 11 on hepatic injury in acute endotoxemia

The liver is one of the major target organs affected in sepsis, and its failure always results in critical consequences. It has been reported that recombinant human interleukin 11 (rhIL-11), a pleiotropic cytokine, may be useful in the treatment of sepsis. However, the effect of IL-11 specifically on the hepatic failure in sepsis has not been evaluated. In the present study, we examined the effect of rhIL-11 on the hepatic injury in a rat endotoxemia model. Acute endotoxemia was induced in male Sprague-Dawley rats by intraperitoneal injection (i.p.) of bacterial lipopolysaccharide (LPS, 20 mg/kg). Immediately after injection of LPS, rats were treated with rhIL-11 (150 microg/kg, i.p.) or the vehicle. LPS treatment induced severe hepatic injury as revealed by marked increases in serum alanine transaminase (ALT) and aspartate transaminase (AST) activities, extensive hepatocyte necrosis, tumor necrosis factor-alpha (TNF-alpha) mRNA, inducible nitric oxide synthase (iNOS) mRNA, and DNA-binding activity of nuclear factor-kappaB (NF-kappaB). In contrast, rhIL-11 treatment significantly ameliorated the LPS-induced hepatic injury, as judged by marked improvement in all these indices. In addition, rhIL-11 treatment markedly decreased LPS-induced mortality. These results indicate that rhIL-11 plays a significant protective role in LPS-induced hepatic injury in acute endotoxemia.     

Impaired T lymphocyte trafficking in mice deficient in an actin-nucleating protein, mDia1

Trafficking of immune cells is controlled by directed migration of relevant cells toward chemotactic signals. Actin cytoskeleton undergoes continuous remodeling and serves as machinery for cell migration. The mDia family of formins and the Wiskott-Aldrich syndrome protein (WASP)-Arp2/3 system are two major actin nucleating-polymerizing systems in mammalian cells, with the former producing long straight actin filaments and the latter producing branched actin meshwork. Although much is known about the latter, the physiological functions of mDia proteins are unclear. We generated mice deficient in one mDia isoform, mDia1. Although mDia1(-/-) mice were born and developed without apparent abnormality, mDia1(-/-) T lymphocytes exhibited impaired trafficking to secondary lymphoid organs in vivo and showed reduced chemotaxis, little actin filament formation, and impaired polarity in response to chemotactic stimuli in vitro. Similarly, mDia1(-/-) thymocytes showed reduced chemotaxis and impaired egression from the thymus. These results suggest that mDia1 plays a distinct role in chemotaxis in T lymphocyte trafficking.     

Transcutaneous vagus nerve stimulation reduces serum high mobility group box 1 levels and improves survival in murine sepsis

OBJECTIVE: Electrical vagus nerve stimulation inhibits proinflammatory cytokine production and prevents shock during lethal systemic inflammation through an alpha7 nicotinic acetylcholine receptor (alpha7nAChR)-dependent pathway to the spleen, termed the cholinergic anti-inflammatory pathway. Pharmacologic alpha7nAChR agonists inhibit production of the critical proinflammatory mediator high mobility group box 1 (HMGB1) and rescue mice from lethal polymicrobial sepsis. Here we developed a method of transcutaneous mechanical vagus nerve stimulation and then investigated whether this therapy can protect mice against sepsis lethality. DESIGN: Prospective, randomized study. SETTING: Institute-based research laboratory. SUBJECTS: Male BALB/c mice. INTERVENTIONS: Mice received lipopolysaccharide to induce lethal endotoxemia or underwent cecal ligation and puncture to induce polymicrobial sepsis. Mice were then randomized to receive electrical, transcutaneous, or sham vagus nerve stimulation and were followed for survival or euthanized at predetermined time points for cytokine analysis. MEASUREMENTS AND MAIN RESULTS: Transcutaneous vagus nerve stimulation dose-dependently reduced systemic tumor necrosis factor levels during lethal endotoxemia. Treatment with transcutaneous vagus nerve stimulation inhibited HMGB1 levels and improved survival in mice with polymicrobial sepsis, even when administered 24 hrs after the onset of disease. CONCLUSIONS: Transcutaneous vagus nerve stimulation is an efficacious treatment for mice with lethal endotoxemia or polymicrobial sepsis.     

Exhaled nitric oxide as a marker for serum nitric oxide concentration in acute endotoxemia

The main aim of this study was to assess the correlation between exhaled nitric oxide (NO) and serum NO concentrations during the course of endotoxemia. We also assessed whether or not the inducible isoform of NO synthase is responsible for the increase in NO production in endotoxemia animals.

Anesthetized and mechanically ventilated dogs were injected with either saline (control) or Escherichia coli endotoxin (LPS [Lipopolysaccharides]), and the animals were sacrificed 150 minutes later. We measured hemodynamics, exhaled NO, and serum arterial and mixed venous NO concentrations. Western blotting was performed on lung, pulmonary artery, aorta, and kidney tissue samples using anti-inducible NO synthase antibody.

Arterial pressure, cardiac output, and pulmonary arterial pressure in the control group remained unchanged, whereas a significant decline in these parameters was observed in the LPS group. Exhaled NO and serum arterial NO concentrations rose significantly within 30 minutes of endotoxin injection and remained higher than baseline values, whereas mixed venous serum NO did not change from baseline values. There was a significant linear relationship between exhaled NO and arterial serum NO concentrations. By comparison, exhaled NO, and arterial and mixed venous serum NO levels remained unchanged in the control group. Western blotting showed no expression of inducible NO synthase (iNOS) isoform in the control or LPS groups.

These results suggest that exhaled NO accurately reflects changes in arterial serum NO concentration and that the source of enhanced NO release in acute endotoxemia is not the iNOS isoform.     

The value of correlation of serum 20S proteasome concentration and percentage of lymphocytic apoptosis in critically ill patients: a prospective observational study

Introduction

Sepsis in critically ill patients is almost associated with bad prognosis and its early detection may improve the prognosis. However, it is difficult to monitor the immunological state of these patients depending on the traditional markers of infection or inflammatory mediators. Accelerated lymphocyte death may reflect good idea about the prognosis especially when combined with 20S proteasome determinations, a recently discovered marker for muscle degradation in patients with sepsis. The hypothesis of the present study is to evaluate the role of serum 20S proteasome at early diagnosis of sepsis and its correlation with lymphocyte apoptosis to predict prognosis and consequently the early interference in critically ill patients suffering from a broad range of diseases in the intensive care unit.

Methods

Sixty-seven critically ill adult intensive care patients were divided into two groups, 32 septic critically ill patients (sepsis group) and 35 non-septic critically ill patients (non-sepsis group), in addition to 33 apparently healthy subjects from the out patient clinic (control group). Patients were tested for serum values of 20S proteasome using ELISA and for percentage of lymphocyte death using annexin V and 7-aminoactinomycin D dye by flow cytometry.

Results

Measured median value of serum 20S proteasome was significantly higher in septic patients compared with both the non-septic and control groups. A significant increase in the percentage of apoptotic lymphocytes was detected in septic patients when compared with the non-sepsis and control groups. The correlation of both 20S proteasome and percentage of apoptotic lymphocytes was found to be significantly positive in both septic and non-septic patients.

Conclusions

The correlation of median values of 20S proteasome and the percentage of apoptotic lymphocyte median values could be a good indicator of patient prognosis and survival in critically ill patients.     

内毒素血症大鼠脑内TRH及其受体对脑作用的研究

目的：探讨内毒素血症对大鼠脑内促甲状腺激素释放激素(TRH)与其受体含量及分布情况的作用。方法：建立内毒素血症加失血性休克双重打击模型，用放射免疫法测定模型动物脑内TRH及其受体含量。结果：在内毒素血症早期，脑内TRH含量显著减少，TRH受体总量增加，但与TRH亲和力下降，表现为可结合TRH的受体无显著性变化。结论：脑内TRH含量与外周不平行；内毒素血症早期脑内TRH减少及有活性TRH受体含量稳定可能与机体对脑组织的保护作用及遏制多器官功能障碍综合征进展有关。     

Regulated progression of B lymphocyte differentiation from cultured fetal liver

Lymphoid fetal liver cultures (LFLC) are long-term, nontransformed cultures of early B lymphoid lineage cells which appear developmentally blocked at the pre-B stage in vitro. When injected into severe combined immunodeficient (SCID) mice, cells from LFLC could reconstitute splenic B lymphocytes and serum IgM. T lymphocyte reconstitution was not observed and serum IgG levels were very low. IgG3 was the predominant gamma subisotype in the serum of the LFLC-reconstituted mice, indicating impaired class switching in these B lymphocytes. When thymocytes were coinjected with LFLC, the B lymphocytes were able to class switch fully and respond to T-dependent antigens. These serological responses were heterogeneous. This experimental system allows separation of three B lymphocyte developmental stages: early differentiation in vitro, progression to IgM secretion in vivo, and late differentiation dependent upon mature T lymphocytes in vivo. The unique advantage of this system is the ability to regulate the B lymphocyte developmental pathway in a defined, stepwise manner.