Interferon-gamma restores immune competence after hemorrhagic shock

Hemorrhagic shock increases the susceptibility to infection in both clinical and laboratory settings. Hemorrhagic shock also is associated with a decreased production of interferon-gamma (IFN-gamma), a potent modulator of immune function. We investigated the effect of IFN-gamma both alone and in addition to antibiotic prophylaxis upon infection following hemorrhagic shock. Sprague-Dawley rats were bled to a mean arterial pressure of 45 mm Hg for 45 min and then were resuscitated with shed blood and normal saline. Abscess formation was induced 1 hr later by subcutaneous injection of 1 X 10(8) Staphylococcus aureus. Four treatments were investigated: (1) control; (2) recombinant rat IFN-gamma, 7500 units, 30 min after inoculation and daily for 3 days; (3) cefamandole (CEF) nafate, 30 mg/kg, 30 min before and 4 hr after inoculation; and (4) IFN-gamma + CEF as in (2) and (3). Abscess size, weight, and quantitative bacterial counts were measured 7 days after inoculation. Hemorrhagic shock increased mean abscess size from 11.7 +/- 2.8 to 14.1 +/- 1.9 mm (P less than 0.05), in untreated rats. IFN-gamma alone resulted in minor changes in abscess formation in both shocked and unshocked animals. Shock rendered CEF ineffective in reducing abscess size. IFN-gamma + CEF significantly reduced abscess size (14.1 +/- 1.9 to 8.1 +/- 1.8 mm) and weight (771 +/- 214 to 252 +/- 132 mg) and decreased bacterial count after shock to 12% of control (all P less than 0.05). These data demonstrate that hemorrhagic shock impairs antibiotic efficacy; however, the addition of IFN-gamma restores the ability of host defenses to combat bacterial infection.     

Bacterial CpG-DNA aggravates immune complex glomerulonephritis: role of TLR9-mediated expression of chemokines and chemokine receptors

Immune complex glomerulonephritis (GN) often deteriorates during infection with viruses and bacteria that, in contrast to mammals, have DNA that contains many unmethylated CpG motifs. Balb/c mice with horse apoferritin-induced GN (HAF-GN) were treated with either saline, CpG-oligodeoxynucleotides (ODN), or control GpC-ODN. Only CpG-ODN exacerbated HAF-GN with an increase of glomerular macrophages, which was associated with massive albuminuria and increased renal MCP-1/CCL2, RANTES/CCL5, CCR1, CCR2, and CCR5 mRNA expression. CpG-ODN induced a Th1 response as indicated by serum anti-HAF IgG(2a) titers, mesangial IgG(2a) deposits, and splenocyte IFN-gamma secretion. Messenger RNA for the CpG-DNA receptor Toll-like reeptor 9 (TLR9) was present in kidneys with HAF-GN but not in normal kidneys. The source of TLR9 mRNA in HAF-GN could be infiltrating macrophages or intrinsic renal cells, e.g., mesangial cells; but, in vitro, only murine J774 macrophages expressed TLR9. In J774 cells, CpG-ODN induced the chemokines MCP-1/CCL2 and RANTES/CCL5 and the chemokine receptors CCR1 and CCR5. It is concluded that CpG-DNA can aggravate preexisting GN via a shift toward a Th1 response but also by a novel pathway involving TLR9-mediated chemokine and chemokine receptor expression by macrophages, which may contribute to the enhanced glomerular macrophage recruitment and activation. This mechanism may be relevant during infection-triggered exacerbation of human immune-complex GN and other immune-mediated diseases in general.     

腹腔感染脓毒症时肺内主要模式识别受体表达变化及其与肺损伤的关系

目的观察腹腔感染脓毒症时肺组织内病原菌相关模式分子(内毒素、脂蛋白、DNA)的主要模式识别受体的表达变化及其意义。方法采用盲肠结扎穿孔(CLP)术建立30只昆明种小鼠腹腔感染脓毒症模型,将模型小鼠随机分为CLP组和假手术组,每组各15只鼠,分别于术后8,12和24h取5只鼠处死取右肺组织,采用逆转录聚合酶链反应(RT PCR)法检测肺组织内毒素受体[清道夫受体(SR)、白细胞分化抗原14(CD14)、TLR4]及细菌脂蛋白受体(TLR2)、细菌DNA受体(TLR9)mRNA的表达,酶联免疫吸附实验(ELISA)法检测肺组织内肿瘤坏死因子α(TNFα)含量,采用分光光度计法测定肺组织髓过氧化物酶(MPO)活性。结果与假手术组比较,术后8hCLP组肺组织内CD14mRNA(1.143±0.139,t=0.022,P<0.05),TLR2mRNA(0.418±0.102,t=0.021,P<0.05),TLR4mRNA(0.595±0.052,t=0.0001,P<0.01)和TLR9mRNA(0.743±0.178,t=0.0023,P<0.01)表达上调,其中TLR9mRNA呈持续上调变化,SRmRNA(0.659±0.159,t=0.029,P<0.05)呈持续下调改变;肺组织CD14、TLR4、SR、TLR2和TLR9mRNA的表达变化分别与MPO和TNFα变化呈明显的相关关系(P<0.05或P<0.01)。结论脓毒症发病过程中,肺组织内主要模式识别受体表达变化与肺损伤相关,除LPS外,其他细菌成分也可能参与了肺损伤过程。     

A monoclonal antibody against cytokine-induced neutrophil chemoattractant attenuates injury in the small intestine in a model of ruptured abdominal aortic aneurysm

PURPOSE: Ruptured abdominal aortic aneurysm (RAAA) continues to be a major source of aneurysm-related morbidity and mortality. Neutrophils have been implicated in RAAA repair-induced organ injury; however, the agents responsible for neutrophil activation and organ sequestration have not been identified. This study investigated the role of cytokine-induced neutrophil chemoattractant (CINC) in organ injury in an RAAA model. METHODS: Rats were subjected to 1 hour of hemorrhagic shock with resuscitation, followed by 45 minutes of lower torso ischemia and 2 hours of reperfusion, and randomly were selected to receive saline solution or anti-rat CINC monoclonal antibody at the start of hemorrhagic shock. Another group of animals underwent sham operation, and served as a control group. Intestinal and lung permeability, intestinal and lung myeloperoxidase (MPO) activity, intestinal and lung CINC, and tumor necrosis factor-alpha (TNF-alpha) levels, resuscitation fluid requirements, and histologic mucosal injury were evaluated in all groups. RESULTS: The RAAA model resulted in increased lung and intestinal permeability to radiolabeled albumin and lung MPO activity (P <.01), with increases in intestinal TNF-alpha (P <.001) and CINC (P <.01) levels, when compared with sham-operated animals. Treatment with anti-rat CINC monoclonal antibody attenuated the increases in intestinal permeability and histologic mucosal injury (P <.01), gut TNF-alpha level (P <.001), and resuscitation fluid volume required (P <.05), without significantly affecting lung and intestinal MPO activity, lung permeability, and intestinal CINC level (P = NS), compared with animals given saline solution. CONCLUSION: Neutralization of CINC by the anti-rat CINC monoclonal antibody attenuated intestinal injury and induction of intestinal TNF-alpha, but failed to significantly attenuate remote pulmonary injury in this model of RAAA.     

Toll-like receptor 9 ligand blocks osteoclast differentiation through induction of phosphatase.

CpG-ODN, in addition to stimulation of osteoclastogenic signals in early osteoclast precursors, also induces phosphatase, shifting the pattern of ERK phosphorylation from sustained to transient. This shift results in the degradation of c-fos, an essential molecule for osteoclast differentiation. Therefore, CpG-ODN blocks osteoclast differentiation. INTRODUCTION: Activation of either Toll-like receptor 9 (TLR9) or RANK induces similar responses in osteoclast precursors. Paradoxically, activation of TLR9 results in inhibition of RANKL-induced osteoclastogenesis. MATERIALS AND METHODS: We used bone marrow-derived osteoclast precursors. Analyses of signaling molecules phosphorylation were performed using Western blotting. Different levels of gene expression analyses were performed using RT-PCR, Northern, and run-on analyses (for RNA), and EMSA, Western, and pulse-chase experiments (for protein). Phosphatase activity was measured spectrophotometrically. RESULTS: We found that RANKL and TLR9 ligand, oligodeoxynucleotides containing unmethylated CpG dinucleotides (CpG-ODN), induce sustained and transient extracellular signal-regulated kinase (ERK) phosphorylation, respectively. Furthermore, together they induce a transient phosphorylation of ERK. The duration of ERK phosphorylation is a key factor in determining induction of c-fos, a protein critical for osteoclastogenesis. Indeed, we found that CpG-ODN does not induce c-fos and inhibits its induction by RANKL by enhancing c-fos mRNA and protein degradation. Our observation that CpG-ODN, but not RANKL, induces the expression of the phosphatase PP2A suggests that CpG-ODN exerts its inhibitory activity by induction of ERK dephosphorylation. Moreover, together with the phosphatase inhibitor okadaic acid, CpG-ODN induces sustained ERK phosphorylation and c-fos expression. CONCLUSIONS: Our findings suggest that the increased rate of c-fos degradation by the TLR9 ligand mediates the inhibition of RANKL-induced osteoclast differentiation. The TLR9 ligand, through induction of dephosphorylation, prevents the sustained ERK phosphorylation needed for maintaining high c-fos levels that are essential for osteoclast differentiation.     

Effects of two fluid resuscitations on the bacterial translocation and inflammatory response of small intestine in rats with hemorrhagic shock

Objective： To investigate the effects of two fluid resuscitations on the bacterial translocation and the inflammatory factors of small intestine in rats with hemorrhagic shock. Methods： Fifty SD healthy male rats were randomly divided into 5 groups （ n = 10 per group） ： Group A （ Sham group）, Group B （ Ringer＇ s solution for 1 h ）, Group C （Ringer＇ s solution for 24 h ）, Group D （ hydroxyethyl starch for 1 h ） and Group E （（ hydroxyethyl starch for 24 h）. A model of rats with hemorrhagic shock was established. The bacterial translocation in liver, content of tumor necrosis factor-α （TNF-α） and changes of myeloperoxidase enzyme （MPO） activities in small intestine were pathologically investigated after these two fluid resuscitations, respectively. Results ： The bacterial translocation and the expression of TNF-α in the small intestine were detected at 1 h and 24 h after fluid resuscitation. There were significant increase in the number of translocated bacteria, TNF-α and MPO activities in Group C compared with Group B, significant decrease in Group E compared with Group D and in Group B compared with Group D. The number of translocated bacteria and TNF-α expression significantly decreased in Group E as compared with Group C. Conclusions： The bacterial translocation and the expression of TNF-α in the small intestine exist 24 h after fluid resuscitation. 6 % hydroxyethyl starch can improve the intestinal mucosa barrier function better than the Ringer＇ s solution.     

The effect of L-tryptophan on hemorrhagic shock induced bacterial translocation

Hemorrhagic shock causes mucosal damage in intestine and it results in translocation of bacteria to distant organs. In this study, effects of various doses of L-Tryptophan on the prevention of bacterial translocation in hemorrhagic shock induced rabbits were investigated. This study was carried out on six groups, each was consisting of 10 rabbits. While any procedure was conducted on the rabbits in group 1 (as a control group), 1 x 10(10)Escherichia coli isolate were administered rabbits in the other groups by gavage. In groups 3, 4, 5, and 6, hemorrhagic shock was induced. After induction of hemorrhagic shock, 10, 50, and 200 mg/kg L-Tryptophan were intragastrically administered to animals in groups 4, 5, and 6, respectively. Blood and terminal ileum samples were taken to detect bacterial translocation by polymerase chain reaction and mucosal damage by histopathological examination at 24 h after hemorrhagic shock. The occurrence of bacterial translocation increased as well when intestinal bacterial intensity was increased (P < 0.05). The most intensive bacterial translocation was formed in group 3 as a result of the additive effect of hemorrhagic shock to bacterial augmentation. It was observed that bacterial translocation was significantly reduced in groups 5 and 6 that are 50 and 200 mg/kg L-Tryptophan were administered (P < 0.01). Histopathological changes on mucosa and submucosa support these results. As a result, we concluded that augmentation of intestinal bacterial intensity induces bacterial translocation, the addition of hemorrhagic shock to bacterial augmentation makes more excessive translocation and mucosal changes have effective roles in these events. L-Tryptophan decreased the intestinal mucosal damage and bacterial translocation induced by hemorrhagic shock, in a dose-dependent manner.     

New insights into the pathogenesis of IgA nephropathy: do toll like receptor 9-B cell activation factor-IgA class switching recombination signaling axis induce IgA hyper-production?

IgA nephropathy (IgAN) has become the most common form of primary glomerular disease worldwide. So far, it is still not very clear about the exact pathogenesis of IgAN, thus has no specific therapy. Generally mesangial deposition of IgA, especially polymeric IgA1 (pIgA1), suggests to be the initiating event in the pathogenesis of IgAN. In addition to decreased IgA clearance, IgA over production may also participate in the pathogenesis of IgAN. IgA class switching recombination (CSR) played key role during the process of IgA production. Stimulated with hemolytic streptococcus, tonsillar mononuclear cells (TMCs) of patients with IgAN presented with increased levels of Ia-Ca and activation-induced cytidine deaminase (AID), which are significant for IgA CSR. Human B cells and plasmacytoid dendritic cells express Toll-like receptor (TLR)-9, whose natural ligands are unmethylated cytosine–guanine dinucleotide (CpG) motifs characteristic of bacterial DNA (CpG-DNA). Unmethylated deoxycytidylic-deoxyguanosine oligodeoxynucleotide (CpG-ODN) is able to mimic the immunostimulatory activity of microbial DNA. Study found a significant increase in B cell activation factor (BAFF) production when tonsillar mononuclear cells stimulated with CpG-ODN in patients with IgAN. BAFF can induce germline Cα gene expression, AID expression, and IgA class switching in a CD40-independent manner. Therefore, it could be hypothesized that in IgAN there may exist TLR9-BAFF-IgA CSR axis, which induces excessive IgA production. If the hypothesis is correct, it could be of great significance for pathogenesis of IgAN elucidate and IgAN treatment.     

Role of nitric oxide in hemorrhagic shock-induced bacterial translocation

BACKGROUND: Hemorrhagic shock-induced bacterial translocation is an etiologic factor in the pathogenesis of multiple system organ damage. Excessive production of nitric oxide (NO) during hemorrhagic shock may lead to cellular injury and gut barrier failure that promotes bacterial translocation. We investigated the effect of aminoguanidine (AG) and N(G)-nitro-l-arginine methyl ester (l-NAME), both inhibitors of NO synthase, on hemorrhagic shock- induced bacterial translocation in the rat. MATERIALS AND METHODS: Anesthetized male Sprague-Dawley rats were subjected to a hemorrhagic shock protocol for 30 min followed by intravenous injection (1 mL/kg body wt) with normal saline, AG (100 mg/kg), or l-NAME (10 mg/kg). Tissues/organs were examined histologically for damage and bacterial translocation. Plasma nitrate/nitrite was measured using a procedure based on the Griess reaction, and nitric oxide synthase (NOS) expression was determined immunohistochemically. RESULTS: The shocked animals treated with saline died within 90 min, and deaths were associated with 100% bacterial translocation, increased tissue/organ damage, and elevated nitrate/nitrite production. In contrast, both AG and l-NAME increased the survival time of shocked rats to >72 h, abrogated bacterial translocation, reduced tissue/organ damage, and prevented excessive nitrate/nitrite production and upregulation of expression of endothelial NOS and inducible NOS. CONCLUSIONS: Prevention of bacterial translocation by pharmacologic agents such as aminoguanidine and l-NAME could be an important therapeutic approach to lessen mortality rates following hemorrhagic shock.     

Immune enhancement by tumor necrosis factor-alpha improves antibiotic efficacy after hemorrhagic shock

Immunomodulation with cytokines produced by recombinant DNA technology may be useful in combatting the increased susceptibility to infection seen after shock and trauma. We investigated the effect of tumor necrosis factor (TNF) alone and in combination with an antibiotic in a model of infection after shock. Interactions of TNF with another cytokine, interferon-gamma (IFN-gamma), were also examined. Sprague-Dawley rats were bled to maintain blood pressure at 45 mm Hg for 45 minutes and then resuscitated with shed blood and saline. Animals were inoculated with 10(8) S. aureus subcutaneously and placed into one of seven treatment groups: 1) control; 2) CEF-cefazolin, 30 mg/kg IP, 30 minutes before and 4 hours after inoculation; 3) IFN-recombinant rat IFN-gamma, 7,500 U SQ, 30 minutes after inoculation and daily for 3 days; 4) TNF-recombinant human TNF, 7,500 U SQ, 30 minutes after inoculation and daily for 3 days; 5) CEF + IFN as in 2 and 3; 6) CEF + TNF as in 2 and 4; 7) CEF + IFN + TNF as in 2, 3, and 4. Animals were sacrificed on day 7 and abscess number, diameter, and weight were measured. CEF reduced abscess diameter and weight following hemorrhagic shock, but did not affect abscess number. IFN-gamma and TNF alone did not reduce infection. The combination of either IFN-gamma or TNF with CEF significantly decreased infection following shock compared to control or CEF alone. CEF + TNF decreased abscess number compared to CEF + IFN (14/20 vs. 7/20;p less than 0.05) but did not alter abscess diameter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of gut microflora on mesenteric lymph cytokine production in rats with hemorrhagic shock

OBJECTIVE: The aim of the present study was to test the hypothesis that the resident gut microflora play a role in modulating gut cytokine production under normal circumstances and in response to tissue injury with or without hemorrhagic shock. METHODS: The postnodal lymph was collected from the main mesenteric lymphatic channel 1 hour before, during (1.5 hours), and hourly for 6 hours after 90 minutes of sham or actual hemorrhagic shock (30 mm Hg) in the following three groups of rats, all of which had laparotomies and vascular instrumentation: rats with a normal gut flora (NF), rats whose gut flora had been decontaminated with oral antibiotics (AD), and rats with Escherichia coli C25 intestinal overgrowth (MA). Interleukin (IL)-6 and TNF levels in the mesenteric lymph were measured using cytokine-dependent cellular assays. Endotoxin levels and endotoxin-neutralizing capacity in the lymph were also measured. RESULTS: Mesenteric lymph IL-6 levels in the laparotomized MA-sham animals were significantly elevated compared with NF-sham animals at 2 to 4 hours (p < 0.05) and at 5 and 6 hours after sham shock (p < 0.01). Similarly, IL-6 levels in laparotomized AD-shock animals were increased when compared with NF-shock animals 3 hours after shock (p < 0.001). Lymph tumor necrosis factor bioactivity, although present in all surgically manipulated groups, was scarcely detectable in untouched animals. Endotoxin-neutralizing capacity was significantly impaired in shocked animals compared with untouched animals. CONCLUSION: Changes in the gut microflora modulate the gut cytokine production after tissue injury with or without hemorrhagic shock, with intestinal bacterial overgrowth leading to the greatest increase in mesenteric lymph IL-6 levels.     

Influences of Intestinal Ligation on Bacterial Translocation and Inflammatory Response in Rats with Hemorrhagic Shock: Implications for Damage Control Surgery

Damage Control Surgery (DCS) treatment of rapid intestinal ligation to control the bowel spillage in severe abdominal trauma induces acute intestinal loop obstruction. The purpose of this study was to investigate the effects of intestinal ligation on bacterial translocation (BT) and inflammatory reaction under the condition of acute hemorrhagic shock and its probable pathophysiology. Escherichia coli TG1 labeled with green fluorescent protein was used to track BT by gavage to rats. Group Shock rats were subjected to hemorrhagic shock for 30 minutes. Group Shock+Ligation (Shock+L) rats were subjected to hemorrhagic shock and following intestinal ligation. We found that hemorrhagic shock alone or in combination with intestinal ligation caused not only morphological damage to ileal mucosa, but also induced BT and promoted release of TNF-α, IL-6, and IL-10 in serum and lymph. Ileal mucosa injuries and BT were significantly aggravated and cytokine levels in serum and lymph were significantly elevated in group Shock+L compared with group Shock. The positive proportions of bacterial culture and cytokine levels were significantly elevated in lymph compared with these in blood in both groups. By fluorescence microscope and XbaI restriction digestion analysis, we elucidated that the bacteria isolated from extraintestinal organs were the same bacteria we gavaged to the rats. We first confirmed that DCS treatment of rapid intestinal ligation under the condition of acute hemorrhagic shock leads to aggravated intestinal mucosa barrier injury and BT and elevated inflammatory response. The intestinal BT and immunoinflammatory factors may act through or mainly through lymph route.     

Differential contribution of osteoclast- and osteoblast-lineage cells to CpG-oligodeoxynucleotide (CpG-ODN) modulation of osteoclastogenesis.

CpG-ODNs modulate osteoclast differentiation through Toll-like receptor 9 (TLR9). Using TLR9-deficient mice, we found that activation of TLR9 on both osteoclast precursors and osteoblasts mediate the osteoclastogenic effect of CpG-ODN. Osteoclastic TLR9 is more important for this activity. INTRODUCTION: Bacterial infections cause pathological bone loss by accelerating differentiation and activation of the osteoclast. A variety of bacteria-derived molecules have been shown to enhance osteoclast differentiation through activation of Toll-like receptors (TLRs). We have shown that CpG-oligodeoxynucleotides (CpG-ODNs), mimicking bacterial DNA and exerting their cellular activities through TLR9, modulate osteoclast differentiation in a complex manner: the ODNs inhibit the activity of the physiological osteoclast differentiation factor RANKL in early osteoclast precursors (OCPs) but markedly stimulate osteoclastogenesis in cells primed by RANKL. MATERIALS AND METHODS: Osteoclast precursors and osteoblasts from TLR9-deficient (TLR9-/-) and wildtype (TLR9+/+) mice were used for in vitro analyses of osteoclast differentiation and modulation of signal transduction and gene expression. RESULTS: As expected CpG-ODN did not exert any activity in cells derived from TLR9-/-mice; these cells, however, responded in a normal manner to other stimuli. Using bone marrow/osteoblasts co-cultures from all possible combinations of TLR9-/- and TLR9+/+ mice-derived cells, we showed that TLR9 in the two lineages is required for CpG-ODN induction of osteoclastogenesis. CONCLUSIONS: CpG-ODN modulates osteoclastogenesis in a TLR9-dependent manner. Activation of TLR9 in bone marrow-derived osteoclasts precursors is more crucial to induction of osteoclastogenesis than activation of the osteoblastic TLR9.     

甘氨酸对出血性休克大白鼠生存率的影响

目的　研究甘氨酸对出血性休克大白鼠生存率的影响并探讨机制。方法　大白鼠经动脉放血 ,造成出血性休克 ,随后用自体血和生理盐水回输进行复苏。复苏前大白鼠分成 3组 :假休克组 ,休克组和甘氨酸治疗组。结果　 (1)复苏后 72h ,休克组与甘氨酸治疗组的生存率分别为 2 0 0 %和 77 8% ,差异有显著意义 (P <0 0 5 )。 (2 )复苏后 18h器官取材显示 :休克组大白鼠的肺、肾等组织出现水肿、变性、炎性细胞浸润等 ,甘氨酸治疗组上述的病理改变明显减轻。 (3)复苏后 18h ,肌酸磷酸激酶、肌酐、谷丙转氨酶、谷草转氨酶、碱性磷酸酶检测 ,休克组明显升高 ,而甘氨酸治疗组只有轻度增高 ,差异有非常显著性意义 (P <0 0 1)。 (4)复苏后 2h ,肝脏分离培养的枯否细胞用内毒素刺激后 ,休克组细胞内Ca2 浓度和TNF α产量明显高于甘氨酸治疗组 ,差异有非常显著性意义 (P <0 0 1)。结论　甘氨酸通过抑制枯否细胞内Ca2 浓度升高和抑制TNF α的过度产生 ,降低机体全身炎症反应程度 ,减轻出血性休克大白鼠脏器的损伤和降低病死率。     

Kupffer细胞CD14及Toll样受体4介导大鼠肝移植缺血再灌注损伤的机制

目的研究大鼠肝移植缺血再灌注后Kupffer细胞CD14和Toll样受体4(TLR4)的表达及其参与缺血再灌注损伤的机制。方法建立肝移植缺血再灌注模型,并分为正常对照组、缺血再灌注组、抗CD14抗体组,每组均为10只大鼠。分离培养大鼠肝移植缺血再灌注后的Kupffer细胞。检测Kupffer细胞CD14及TLR4的mRNA、蛋白表达、核转录因子κB(NFκB)活性以及培养上清TNFα的分泌量。结果再灌注后Kupffer细胞CD14及TLR4的mRNA和蛋白表达明显高于正常对照组(P<001),再灌注后核转录因子κB活性、培养上清TNFα表达量明显高于对照组(P<001)。用抗CD14抗体后NFκB活性,TNFα表达量明显下降(与再灌注组相比,P<005),但仍然高于对照组(P<001)。结论缺血再灌注后肠道内毒素(脂多糖)能够上调Kupffer细胞CD14及TLR4的表达,激活NFκB,启动细胞因子的转录和分泌,但除CD14和TLR4以外的其他信号途径参与了缺血再灌注损伤。     

Impact of prophylactic CpG Oligodeoxynucleotide application on implant-associated Staphylococcus aureus bone infection

TLR-9 ligand CpG oligodeoxynucleotide type B (CpG ODN) induces a proinflammatory environment. We evaluated the effects of a preoperative CpG ODN application in an implant-associated Staphylococcus aureus bone infection model by monitoring bacterial loads and cytokine and chemokine levels.A total of 95 rats were used in four different groups: CpG ODN group (group 1; n = 25), non-CpG-ODN group (group 2; n = 25); saline pretreatment (group 3; n = 25), and one uninfected group (group 4; n = 20). A single dose of CpG-ODN was administered to the left tibialis anterior muscle 3 days prior to surgery and the tibia midshaft was osteotomized, stabilized by an intramedullary implant and subsequently contaminated with 10³ colony forming units (CFUs) of S. aureus in groups 1–3. The osteotomy gap in animals of group 4 was not contaminated with S. aureus and those animals did not receive any pretreatment.CpG ODN administration resulted in significant reduction of the bacterial load in tibia tissue homogenate and on the implant surface on day 1 post-infection compared to non-CpG-ODN pretreatment (p < 0.05; p < 0.05). Reductions in bacterial CFUs, compared to non-treated (saline) controls, were approximately 67% and 77% for bone tissue homogenates and implants. No bacteria were detected in uninfected rats. Early reduction of bacterial CFUs in the tibia was accompanied by increased levels of proinflammatory mediators MIP-2, IL-1β and RANTES in bone tissue milieu of the CpG ODN treated group compared to controls.At day 42 post-infection, bone marrow tissue of rats pretreated with CpG ODN had comparable high bacterial CFU numbers as the non-CpG ODN or saline treated groups. Microbiological analysis of implants removed from CpG ODN treated rats showed high bacterial growth densities on their surfaces which were not different from those observed in controls. In histology, all animals of groups 1–3 showed established infected non-unions. Additionally, inflammatory mediator profiles in bone marrow homogenates of CpG ODN treated rats resembled those seen in infected controls.In this rat model, prophylactic administration of a single dose of CpG ODN, resulted in marked reduction of S. aureus load in the infected tibia during the initial stage of infection but failed to prevent development of chronic infection over time.     

Effect of tumor necrosis factor alpha,interferon gamma,and interleukin-4 on bacteria-enterocyte interactions

BACKGROUND: Little is known about the mechanisms involved in bacterial translocation from the intestinal lumen to extraintestinal sites. Because the cytokine cascade associated with sepsis, inflammation, and trauma has been shown to affect intestinal epithelial permeability, experiments were designed to clarify the effects of selected cytokines on bacterial adherence to and internalization by cultured HT-29 and Caco-2 enterocytes. METHODS: Mature, confluent enterocytes were pretreated 48 to 72 h with tumor necrosis factor alpha (TNF-alpha), interferon gamma, (IFN-gamma), or interleukin-4 (IL-4). Adherence of Listeria monocytogenes, Salmonella typhimurium, Proteus mirabilis, and Escherichia coli was measured by enzyme-linked immunosorbent assay and bacterial internalization was quantified by the gentamicin protection assay. Enterocyte permeability was measured by transepithelial electrical resistance and by flux of 40-kDa fluorescent dextran. Bacterial transmigration across confluent enterocytes was measured using enterocytes cultivated on permeable supports. RESULTS: TNF-alpha, IFN-gamma, and IL-4 had variable effects on bacterial adherence to HT-29 and Caco-2 enterocytes, although the most consistent finding was increased bacterial adherence associated with INF-gamma. However, none of these cytokines had a noticeable effect on bacterial internalization by either Caco-2 or HT-29 enterocytes. In addition, none of these cytokines had a noticeable effect on the permeability of confluent enterocytes as measured by transepithelial electrical resistance or dextran flux. Bacterial transmigration across confluent HT-29 enterocytes was not altered by TNF-alpha, IFN-gamma, or IL-4; however, IL-4 consistently decreased bacterial transmigration across confluent Caco-2 enterocytes. CONCLUSIONS: IFN-gamma may augment the epithelial adherence of selected species of enteric bacteria, and IL-4 may act as a barrier-sustaining agent to decrease bacterial migration across the intestinal epithelium.     

Human renal epithelial cells express iNOS in response to cytokines but not bacteria

BACKGROUND: Epithelial cells form the mucosal barriers that prevent the entry of mucosal pathogens, and respond to bacterial infections by producing various host defense molecules. In this study, we examined the inducible nitric oxide synthase (iNOS) response of primary human renal tubular epithelial cells (HRTEC) following infection with uropathogenic Escherichia coli Hu734, or stimulation with lipopolysaccharide (LPS) or cytokines. METHODS: Induction of iNOS was examined by RT-PCR, Western blot, immunohistochemistry and nitrite measurements. The effects of endogenously produced nitric oxide (NO), and exogenously applied DETA/NO, SIN-1 and H2O2 on cell viability were analyzed using a respiration assay. RESULTS: HRTEC did not produce NO following infection with E. coli Hu734, LPS alone, or in combination with interferon-gamma (IFN-gamma), even though these agents caused a marked increase in iNOS expression by RAW 264.7, a macrophage cell line. In contrast, iNOS protein and mRNA expression by HRTEC increased after exposure to a cytokine mixture consisting of interleukin (IL)-1beta, tumor necrosis factor-alpha (TNF-alpha) and IFN-gamma. This was due to the combination of IL-1beta and IFN-gamma, but the individual cytokines had no effect. Inducible NOS-expressing cell cultures showed reduced viability, and this effect was inhibited with the NOS inhibitor L-NMMA in RAW 264.7 cells, but not in HRTEC. HRTEC were more sensitive to oxidative stress induced by H2O2 than to nitrogen stress induced by DETA/NO. CONCLUSIONS: We conclude that uropathogenic E. coli that attach to HRTEC fail to directly activate iNOS expression, and that iNOS expression during bacterial infection is more likely to result from stimulation by local cytokines such as IL-1beta and IFN-gamma.