Complement-dependent acute-phase expression of C-reactive protein and serum amyloid P-component

The acute-phase response (APR) is regulated by TNF-alpha, IL-1beta, and IL-6 acting alone, in combination, or in concert with hormones. The anaphylotoxin C5a, generated during complement activation, induces in vitro the synthesis of these cytokines by leukocytes and of acute-phase proteins by HepG2 cells. However, there is no clear evidence for a role of C5a or any other complement activation product in regulation of the APR in vivo. In this study, using human C-reactive protein (CRP) transgenic mice deficient in C3 or C5, we investigated whether complement activation contributes to induction of the acute-phase proteins CRP and serum amyloid P-component (SAP). Absence of C3 or C5 resulted in decreased LPS-induced up-regulation of the CRP transgene and the mouse SAP gene. Also, LPS induced both the IL-1beta and IL-6 genes in normocomplementemic mice, but in complement-deficient mice it significantly induced only IL-6. Like LPS injection, activation of complement by cobra venom factor led to significant elevation of serum CRP and SAP in normocomplementemic mice but not in complement-deficient mice. Injection of recombinant human C5a into human CRP transgenic mice induced the IL-1beta gene and caused significant elevation of both serum CRP and SAP. However, in human CRP transgenic IL-6-deficient mice, recombinant human C5a did not induce the CRP nor the SAP gene. Based on these data, we conclude that during the APR, C5a generated as a consequence of complement activation acts in concert with IL-6 and/or IL-1beta to promote up-regulation of the CRP and SAP genes.     

Activation of caspases in lethal experimental hepatitis and prevention by acute phase proteins.

Lethal hepatitis can be induced by an agonistic anti-Fas Ab in normal mice or by TNF in mice sensitized to d -(+)-galactosamine or actinomycin D. In all three models, we found that apoptosis of hepatocytes is an early and necessary step to cause lethality. In the three models, we observed activation of the major executioner caspases-3 and -7. Two acute-phase proteins, alpha1-acid glycoprotein and alpha1-antitrypsin, differentially prevent lethality: alpha1-acid glycoprotein protects in both TNF models and not in the anti-Fas model, while alpha1-antitrypsin confers protection in the TNF/d -(+)-galactosamine model only. The protection is inversely correlated with activation of caspase-3 and caspase-7. The data suggest that activation of caspase-3 and -7 is essential in the in vivo induction of apoptosis leading to lethal hepatitis and that acute phase proteins are powerful inhibitors of apoptosis and caspase activation. Furthermore, Bcl-2 transgenic mice, expressing Bcl-2 specifically in hepatocytes, are protected against a lethal challenge with anti-Fas or with TNF/d -(+)-galactosamine, but not against TNF/actinomycin D. The acute-phase proteins might constitute an inducible anti-apoptotic protective system, which in pathology or disturbed homeostasis prevents excessive apoptosis.     

Sensitivity of metallothionein-null mice to LPS/D-galactosamine-induced lethality

Mice treated with lipopolysaccharide (LPS)/D-galactosamine (GalN) selectively develop hepatic failure. The acute-phase protein alpha(1)-acid glycoprotein (AGP) has been demonstrated to protect mice from LPS/GalN-induced lethality. Metallothionein (MT), which is a low-molecular weight, cysteine-rich, metal-binding protein, is also induced in the acute-phase reaction. However, the specific function of MT in acute-phase response remain to be elucidated. We showed that MT-null mice were more sensitive to LPS/GalN-induced lethality than wild-type mice. The increase in vital mediator levels, TNF-alpha and NO were of similar levels in wild-type and MT-null mice. A remarkable increase in plasma platelet-activating factor levels was not observed in our experimental conditions. On the other hands, the mRNA level of AGP in the response to LPS/GalN was decreased in MT-null mice compared to wild-type mice. These results indicated that MT may have the potential to prevent LPS/GalN-induced lethality, at least through the attenuation of AGP induction.     

Production of granulocyte colony-stimulating factor in the nonspecific acute phase response enhances host resistance to bacterial infection

Mice mounting an acute phase response, induced by sterile inflammation after a single s.c. injection of casein 24 h beforehand, were remarkably protected against lethal infection with Gram-positive or Gram-negative bacteria. This was associated with enhanced early clearance of bacteremia, greater phagocytosis and oxidative burst responses by neutrophils, and enhanced recruitment of neutrophils into tissues compared with control, nonacute phase mice. Casein-induced inflammation was also associated with increased concentrations of G-CSF in serum, and administration of neutralizing Ab to this cytokine completely abrogated protection against Escherichia coli infection after casein pretreatment. Injection of recombinant murine G-CSF between 3 and 24 h before infection conferred the same protection as casein injection. In contrast, the casein-induced acute phase response affected neither serum values of TNF-alpha, IL-1 beta, or IL-6 after E. coli infection nor susceptibility to LPS toxicity. Furthermore, protection against infection was unaffected in IL-1R knockout mice, which have deficient acute phase plasma protein responses, or after nonspecific inhibition of acute phase protein synthesis by D-galactosamine or specific depletion of complement C3 by cobra venom factor. Increased production of G-CSF in the acute phase response is thus a key physiological component of host defense, and pretreatment with G-CSF to prevent bacterial infection in at-risk patients now merits further study, especially in view of increasing bacterial resistance to antibiotics.     

Evidence for IFN-gamma as a mediator of the lethality of endotoxin and tumor necrosis factor-alpha.

Current evidence indicates that endogenously produced peptide cytokines, most notably TNF-alpha and IL-1, mediate the lethality of experimental endotoxemia. Because circulating serum levels of IFN-gamma can be detected soon after TNF-alpha and IL-1 in response to endotoxin, we investigated the role of IFN-gamma in endotoxin and TNF-alpha lethality. Specific neutralizing antibodies to murine TNF-alpha (anti-TNF-alpha Ab) or murine IFN gamma (anti-IFN-gamma Ab) produced in our laboratory protected mice against the lethality of Escherichia coli endotoxin (LPS) administered 6 h later. Serum IFN-gamma levels 2 h after i.v. LPS were lower in mice treated with anti-TNF-alpha Ab compared to mice that received nonimmune IgG (median less than 2.5 vs 3.0 U/ml, P2 less than 0.05). In contrast, serum TNF-alpha levels 1 h after i.v. LPS peaked more than fourfold higher in mice treated with anti-IFN-gamma Ab compared to controls (median greater than 6400 vs 1405 pg/ml, p2 less than 0.05). Doses of TNF-alpha (300 micrograms/kg) and IFN-gamma (50,000 U) which were well tolerated when given individually were synergistically lethal in combination (0% lethality vs 100% lethality, P2 less than 0.001), and were associated with higher serum levels of IL-6 than with either cytokine alone. Anti-IFN-gamma Ab provided complete protection against exogenous human rTNF-alpha at the LD100 dose (1400 micrograms/kg, p2 less than 0.001), and in fact prevented lethality at doses four- to fivefold greater than the LD100 human rTNF-alpha (up to 6000 micrograms/kg). We conclude that IFN-gamma is synergistic with TNF-alpha, is essential for the lethality of LPS and TNF-alpha, and may have modulating effects on the negative control of serum levels of TNF-alpha after LPS in mice.     

Critical role of lipopolysaccharide-binding protein and CD14 in immune responses against gram-negative bacteria

LPS-binding protein (LBP) and CD14 potentiate cell activation by LPS, contributing to lethal endotoxemia. We analyzed the contribution of LBP/CD14 in models of bacterial infection. Mice pretreated with mAbs neutralizing CD14 or LBP showed a delay in TNF-alpha production and died of overwhelming infection within 24 h, after a challenge with 250 CFU of virulent Klebsiella pneumoniae. Blockade of TNF-alpha also increased lethality, whereas pretreatment with TNF-alpha protected mice, even in the presence of LBP and CD14 blockade. Anti-LBP or anti-CD14 mAbs did not improve or decrease lethality with a higher inoculum (10(5) K. pneumoniae) and did not affect outcome following injections of low or high inocula of Escherichia coli O111. These results point to the essential role of LBP/CD14 in innate immunity against virulent bacteria.     

Therapeutic effects of lysophosphatidylcholine in experimental sepsis

Sepsis represents a major cause of death in intensive care units. Here we show that administration of lysophosphatidylcholine (LPC), an endogenous lysophospholipid, protected mice against lethality after cecal ligation and puncture (CLP) or intraperitoneal injection of Escherichia coli. In vivo treatment with LPC markedly enhanced clearance of intraperitoneal bacteria and blocked CLP-induced deactivation of neutrophils. In vitro, LPC increased bactericidal activity of neutrophils, but not macrophages, by enhancing H(2)O(2) production in neutrophils that ingested E. coli. Incubation with an antibody to the LPC receptor, G2A, inhibited LPC-induced protection from CLP lethality and inhibited the effects of LPC in neutrophils. G2A-specific antibody also blocked the inhibitory effects of LPC on certain actions of lipopolysaccharides (LPS), including lethality and the release of tumor necrosis factor-alpha (TNF-alpha) from neutrophils. These results suggest that LPC can effectively prevent and treat sepsis and microbial infections.     

Protective effect of C-reactive protein against the lethality induced by Vibrio vulnificus lipopolysaccharide

Vibrio vulnificus infection has attracted special interest because of its high mortality. A strong clinical association exists between hepatic dysfunction and increased morbidity and mortality from V. vulnificus infection. In this study, the effect of C-reactive protein (CRP), a typical hepatogenic acute phase protein, on the lethality induced by V. vulnificus lipopolysaccharide (LPS) was investigated in galactosamine-sensitized mice. The pretreatment of CRP, in a dose of at least 2 mg/kg, 2 hr before the challenge of LPS completely protected mice against the lethality by V. vulnificus LPS. The elevation of serum tumor necrosis factor-alpha (TNF-alpha) induced by LPS administration was not affected by CRP pretreatment. However, the LPS- or TNF-alpha-induced hepatotoxicity was completely prevented by CRP. These results indicate that CRP does not prevent the synthesis, but prevents the hepatotoxic action of TNF-alpha. The possibility that impaired production of acute phase proteins in patients with pre-existing hepatic dysfunction may predispose the higher risk of V. vulnificus infection needs to be evaluated further.     

Potent adjuvant action of lipopolysaccharides possessing the O-specific polysaccharide moieties consisting of mannans in antibody response against protein antigen

It was previously reported that Klebsiella O3 lipopolysaccharide (LPS) exhibits extraordinarily strong adjuvant activity in augmenting antibody response against protein antigens in mice compared with other kinds of LPS, for example, LPS from Escherichia coli O55, O111, and O127 and Salmonella enteritidis. The present study was undertaken to clarify the relationship between the strong adjuvant activity in augmenting antibody response against deaggregated bovine gammaglobulin and the chemical structure of LPS. Among LPS from Klebsiella O1, O4, O5, and O7, only O5 LPS exhibited nearly the same degree of the strong adjuvant activity as did O3 LPS. The adjuvant activity of the other LPS was very weak in a degree similar to that of LPS from E. coli O55 and O127. Even when the natural forms of Klebsiella O3 LPS and O1 LPS were converted to various defined uniform salt forms, their adjuvant activity did not significantly differ from that of the respective natural forms. It is therefore unlikely that the difference in strength of the adjuvant activity between Klebsiella O3 LPS and O1 LPS is due to the difference in their salt forms. The common feature in the structures of Klebsiella O3 LPS and O5 LPS is their O-specific polysaccharide chains consisting of the mannose homopolysaccharides (mannans). LPS from E. coli O8 and O9, the O-specific polysaccharide chains of which consist of the mannans, also exhibited much stronger adjuvant activity than do LPS from E. coli O55 and O127, and the strength of the adjuvant activities of the former two was comparable to that of LPS from Klebsiella O3 and O5. On the other hand, LPS from Klebsiella O3 and O5 and E. coli O8 and O9 showed the ability to activate B lymphocytes polyclonally in vivo in a degree similar to that of the other kinds of LPS. From the present results it can be concluded that LPS possessing the O-specific polysaccharide moieties consisting of the mannans exhibit extraordinarily strong adjuvant activity in augmenting antibody response against protein antigen.     

Reference maps of mouse serum acute-phase proteins: changes with LPS-induced inflammation and apolipoprotein A-I and A-II transgenes

We present reference maps of the mouse serum proteome (run under reducing and non-reducing conditions), from control animals, from mice injected with lipopolysaccharide (LPS) to induce systemic inflammation, and from mice transgenic for human apolipoproteins A-I and A-II. Seventy-seven spots/spot chains from the reducing gels were identified by HPLC MS/MS, representing 28 distinct proteins, including a species-specific protease inhibitor, contrapsin, and high levels of carboxylesterase. The concentrations of acute-phase reactants were monitored for 96 h after LPS challenge. The greatest changes (four-fold 48 h after LPS administration) were observed for haptoglobin and hemopexin. Orosomucoid/alpha(1)-acid glycoprotein and apolipoprotein A-I increased steadily, to 50-60% above baseline at 96 h from stimulation. In mice transgenic for human apolipoprotein A-I the levels of expression of orosomucoid/alpha(1)-acid glycoprotein, alpha(1)-macroglobulin, esterase, kininogen and contrapsin were altered compared to knockout mice lacking apolipoprotein A-I. In contrast, except for the presence of apolipoprotein A-II, no statistically significant difference was observed in mice transgenic for human apolipoprotein A-II.     

Hepcidin protects against lipopolysaccharide-induced liver injury in a mouse model of obstructive jaundice

Obstructive jaundice (OJ) increases the risk of liver injury and sepsis, leading to increased mortality. Cholestatic liver injury is associated with a downregulation of hepcidin expression levels. In fact, hepcidin has an important antimicrobial effect, especially against Escherichia coli. It is unknown whether supplementing recombinant hepcidin is effective in alleviating cholestasis-induced liver injury and mortality in mice with superimposed sepsis. A mouse model of cholestasis was developed using extrahepatic bile duct ligation for 3 days. In addition, sepsis due to E. coli 0111:B4 lipopolysaccharide (LPS) was induced in the model. The serum levels of total bilirubin, AST, ALT, and LDH and the mRNA levels of IL-1β, TNF-α, and MCP-1 in the liver were significantly higher in the OJ mice receiving LPS than in the sham-operated mice receiving LPS. Compared to the OJ mice receiving LPS, the hepcidin-pretreated OJ mice receiving LPS showed a significant decrease in the above mentioned parameters, as well as a reversal in the downregulation of LC3B-II and upregulation of cleaved caspase-3; this, in turn, led to significantly decreased lethality in 24h. In conclusion, these results indicate that hepcidin pretreatment significantly reduced hepatic proinflammatory cytokine expression and liver injury, leading to reduced early lethality in OJ mice receiving LPS. Enhanced autophagy and reduced apoptosis may account for the protective effects of hepcidin.     

Respiratory epithelial cells regulate lung inflammation in response to inhaled endotoxin

To determine the role of respiratory epithelial cells in the inflammatory response to inhaled endotoxin, we selectively inhibited NF-kappa B activation in the respiratory epithelium using a mutant I kappa B-alpha construct that functioned as a dominant negative inhibitor of NF-kappa B translocation (dnI kappa B-alpha). We developed two lines of transgenic mice in which expression of dnI kappa B-alpha was targeted to the distal airway epithelium using the human surfactant apoprotein C promoter. Transgene expression was localized to the epithelium of the terminal bronchioles and alveoli. After inhalation of LPS, nuclear translocation of NF-kappa B was evident in bronchiolar epithelium of nontransgenic but not of transgenic mice. This defect was associated with impaired neutrophilic lung inflammation 4 h after LPS challenge and diminished levels of TNF-alpha, IL-1 beta, macrophage inflammatory protein-2, and KC in lung homogenates. Expression of TNF-alpha within bronchiolar epithelial cells and of VCAM-1 within peribronchiolar endothelial cells was reduced in transgenic animals. Thus targeted inhibition of NF-kappa B activation in distal airway epithelial cells impaired the inflammatory response to inhaled LPS. These data provide causal evidence that distal airway epithelial cells and the signals they transduce play a physiological role in lung inflammation in vivo.     

Cutting edge: bacterial lipoprotein induces endotoxin-independent tolerance to septic shock

Tolerance to bacterial cell wall components is an adaptive host response. Endotoxin/LPS tolerance is characterized by a survival advantage against subsequent lethal LPS challenge. However, it is uncertain whether LPS tolerance can afford protection against other septic challenges. In this study, we show that tolerance induced by bacterial lipoprotein (BLP) protects mice against not only BLP-induced lethality, but also LPS-, live bacteria-, and polymicrobial sepsis-induced lethality. In contrast, LPS tolerance offers no survival benefit against the latter two challenges. Furthermore, induction of BLP tolerance results in overexpression of complement receptor type 3 and FcgammaIII/IIR on neutrophils (polymorphonuclear neutrophils) and peritoneal macrophages, with increased bacterial recognition and bactericidal activity, whereas LPS-tolerized mice exhibit an impaired ability to ingest and to kill bacteria. These results indicate that BLP tolerance is a novel adaptive host response associated with a unique protective effect during septic shock.     

Regulation of angiopoietin expression by bacterial lipopolysaccharide

Angiopoietins are ligands for Tie-2 receptors and play important roles in angiogenesis and inflammation. While angiopoietin-1 (Ang-1) inhibits inflammatory responses, angiopoietin-2 (Ang-2) promotes cytokine production and vascular leakage. In this study, we evaluated in vivo and in vitro effects of Escherichia coli lipopolysaccharides (LPS) on angiopoietin expression. Wild-type C57/BL6 mice were injected with saline (control) or E. coli LPS (20 mg/ml ip) and killed 6, 12, and 24 h later. The diaphragm, lung, and liver were excised and assayed for mRNA and protein expression of Ang-1, Ang-2, and Tie-2 protein and tyrosine phosphorylation. LPS injection elicited a severalfold rise in Ang-2 mRNA and protein levels in the three organs. By comparison, both Ang-1 and Tie-2 levels in the diaphragm, liver, and lung were significantly attenuated by LPS administration. In addition, Tie-2 tyrosine phosphorylation in the lung was significantly reduced in response to LPS injection. In vitro exposure to E. coli LPS elicited cell-specific changes in Ang-1 expression, with significant induction in Ang-1 expression being observed in cultured human epithelial cells, whereas significant attenuation of Ang-1 expression was observed in response to E. coli LPS exposure in primary human skeletal myoblasts. In both cell types, E. coli LPS elicited substantial induction of Ang-2 mRNA, a response that was mediated in part through NF-kappaB. We conclude that in vivo endotoxemia triggers functional inhibition of the Ang-1/Tie-2 receptor pathway by reducing Ang-1 and Tie-2 expression and inducing Ang-2 levels and that this response may contribute to enhanced vascular leakage in sepsis.     

Enhanced susceptibility to lipopolysaccharide-induced arthritis and endotoxin shock in interleukin-32 alpha transgenic mice through induction of tumor necrosis factor alpha

Introduction

The present study assessed the potential functions of interleukin (IL)-32α on inflammatory arthritis and endotoxin shock models using IL-32α transgenic (Tg) mice. The potential signaling pathway for the IL-32-tumor necrosis factor (TNF)α axis was analyzed in vitro.

Methods

IL-32α Tg mice were generated under control of a ubiquitous promoter. Two disease models were used to examine in vivo effects of overexpressed IL-32α: Toll-like receptor (TLR) ligand-induced arthritis developed using a single injection of lipopolysaccharide (LPS) or zymosan into the knee joints; and endotoxin shock induced with intraperitoneal injection of LPS and D-galactosamine. TNFα antagonist etanercept was administered simultaneously with LPS in some mice. Using RAW264.7 cells, in vitro effects of exogenous IL-32α on TNFα, IL-6 or macrophage inflammatory protein 2 (MIP-2) production were assessed with or without inhibitors for nuclear factor kappa B (NFκB) or mitogen-activated protein kinase (MAPK).

Results

Single injection of LPS, but not zymosan, resulted in development of severe synovitis with substantial articular cartilage degradation in knees of the Tg mice. The expression of TNFα mRNA in inflamed synovia was highly upregulated in the LPS-injected Tg mice. Moreover, the Tg mice were more susceptive to endotoxin-induced lethality than the wild-type control mice 48 hours after LPS challenge; but blockade of TNFα by etanercept protected from endotoxin lethality. In cultured bone marrow cells derived from the Tg mice, overexpressed IL-32α accelerated production of TNFα upon stimulation with LPS. Of note, exogenously added IL-32α alone stimulated RAW264.7 cells to express TNFα, IL-6, and MIP-2 mRNAs. Particularly, IL-32α -induced TNFα, but not IL-6 or MIP-2, was inhibited by dehydroxymethylepoxyquinomicin (DHMEQ) and U0126, which are specific inhibitors of nuclear factor kappa B (NFκB) and extracellular signal regulated kinase1/2 (ERK1/2), respectively.

Conclusions

These results show that IL-32α contributed to the development of inflammatory arthritis and endotoxin lethality. Stimulation of TLR signaling with LPS appeared indispensable for activating the IL-32α-TNFα axis in vivo. However, IL-32α alone induced TNFα production in RAW264.7 cells through phosphorylation of inhibitor kappa B (IκB) and ERK1/2 MAPK. Further studies on the potential involvement of IL-32α-TNFα axis will be beneficial in better understanding the pathology of autoimmune-related arthritis and infectious immunity.     

Protection from Streptococcus pneumoniae infection by C-reactive protein and natural antibody requires complement but not Fc gamma receptors

Streptococcus pneumoniae is an important human pathogen and the most common cause of community-acquired pneumonia. Both adaptive and innate immune mechanisms provide protection from infection. Innate immunity to S. pneumoniae in mice is mediated by naturally occurring anti-phosphocholine (PC) Abs and complement. The human acute-phase reactant C-reactive protein (CRP) also protects mice from lethal S. pneumoniae infection. CRP and anti-PC Ab share the ability to bind to PC on the cell wall C-polysaccharide of S. pneumoniae and to activate complement. CRP and IgG anti-PC also bind to Fc gamma R. In this study, Fc gamma R- and complement-deficient mice were used to compare the mechanisms of protection conferred by CRP and anti-PC Ab. Injection of CRP protected wild-type, FcR gamma-chain-, Fc gamma RIIb-, and Fc gamma RIII-deficient mice from infection. Complement was required for the protective effect of CRP as cobra venom factor treatment eliminated the effect of CRP in both gamma-chain-deficient and wild-type mice, and CRP failed to protect C3- or C4-deficient mice from infection. Unexpectedly, gamma-chain-deficient mice were extremely sensitive to pneumococcal infection. This sensitivity was associated with low levels of natural anti-PC Ab. Gamma-chain-deficient mice immunized with nonencapsulated S. pneumoniae produced both IgM- and IgG PC-specific Abs, were protected from infection, and were able to clear the bacteria from the bloodstream. The protection provided by immunization was eliminated by complement depletion. The results show that in this model of systemic infection with highly virulent S. pneumoniae, protection from lethality by CRP and anti-PC Abs requires complement, but not Fc gamma R.     

Yohimbine Enhances Protection of Berberine against LPS-Induced Mouse Lethality through Multiple Mechanisms

Sepsis remains a major cause of mortality in intensive care units, better therapies are urgently needed. Gram-negative bacterial lipopolysaccharide (LPS) is an important trigger of sepsis. We have demonstrated that berberine (Ber) protects against lethality induced by LPS, which is enhanced by yohimbine (Y) pretreatment, and Ber combined with Y also improves survival in septic mice. However, the precise mechanisms by which Y enhances protection of Ber against LPS - induced lethality remain unclear. The present study confirmed that simultaneously administered Y also enhanced protection of Ber against LPS-induced lethality. Ber or/and Y attenuated liver injury, but not renal injury in LPS-challenged mice. Ber or/and Y all inhibited LPS-stimulated IκBα, JNK and ERK phosphorylation, NF-κB activation as well as TNF-α production. Ber also increased IL-10 production in LPS-challenged mice, which was enhanced by Y. Furthermore, Ber or/and Y all suppressed LPS-induced IRF3, TyK2 and STAT1 phosphorylation, as well as IFN-β and IP-10 mRNA expression in spleen of mice at 1 h after LPS challenge. Especially, Y enhanced the inhibitory effect of Ber on LPS-induced IP-10 mRNA expression. In vitro experiments further demonstrated that Y significantly enhanced the inhibitory effect of Ber on TNF-α production in LPS-treated peritoneal macrophages, Ber combined with Y promoted LPS-induced IL-10 production and LPS-stimulated IκBα, JNK, ERK and IRF3 phosphorylation and NF-κB activation were also suppressed by Ber or/and Y pretreatment in peritoneal macrophages. Taken together, these results demonstrate that Y enhances the protection of Ber against LPS-induced lethality in mice via attenuating liver injury, upregulating IL-10 production and suppressing IκBα, JNK, ERK and IRF3 phosphorylation. Ber combined with Y may be an effective immunomodulator agent for the prevention of sepsis.     

Characterization of calves exhibiting a novel inheritable TNF-alpha hyperresponsiveness to endotoxin: associations with increased pathophysiological complications.

A subpopulation of calves, herein termed "hyperresponders" (HPR), was identified and defined by the patterns of plasma TNF-alpha concentrations that developed following two challenges with endotoxin (LPS, 0.8 mug Escherichia coli 055:B5 LPS/kg(0.75) live body wt) separated by 5 days. The principle characteristic of HPR calves was a failure to develop tolerance to repeated LPS challenge that was evident in the magnitude of the TNF-alpha concentrations and prolonged severity of pathological sequellae. Whereas calves failing to develop LPS tolerance were identified on the basis of their excessive in vivo plasma TNF-alpha concentration responses, in vitro TNF-alpha responses of peripheral blood mononuclear cells isolated from each calf and challenged with LPS or PMA did not correlate or predict the magnitude of in vivo plasma TNF response of the calf. Intentional breeding to obtain calves from bulls and/or cows documented as HPR resulted in offspring displaying the HPR character when similar progeny calves were tested with LPS in vivo, with extensive controls in place to account for sources of variability in the general TNF-alpha response to LPS that might compromise interpretation of the data. Feed intake, clinical serology and hematology profiles, and acute-phase protein responses of HPR calves following LPS were significantly different from those of calves displaying tolerance. These results suggest that the pattern of plasma TNF-alpha changes that evolve from a low-level double LPS challenge effectively reveal the presence of a genetic potential for animals to display excessive or prolonged pathological response to LPS-related stress and compromised prognosis for recovery.     

特异性卵黄抗体(IgY)对小鼠大肠杆菌败血症的保护作用

目的:研究特异性IgY对小鼠大肠杆菌败血症的保护作用.方法:以灭活的E.coli O111免疫产蛋母鸡,抗体经水稀释及盐析分离纯化.ELISA法检测大肠杆菌特异性IgY对大肠杆菌及LPS的结合活性.腹腔注射E.coli O111(1011cfu/mL)建立小鼠败血症模型,攻毒剂量为0.1mL/10g体重.小鼠随机分为5组,分别给药保护:空白组(生理盐水)、阴性对照组(非特异性IgY,20mg/mL)、阳性对照组(头孢哌酮20mg/mL)、高剂量组(特异性IgY,40mg/mL),低剂量组(特异性IgY,20mg/mL).给药剂量为:攻毒前,0.15mL/10g体重,每天一次,共两天;攻毒后,0.25mL/10g体重,每天一次,共七天.观察小鼠临床表现、体重变化、白细胞(WBC)和血小板(PLT)数变化及各组小鼠的死亡率.结果:特异性IgY与E.coli O111和LPS均有体外结合活性.大肠杆菌攻毒后,小鼠体重下降,各组小鼠外周血中WBC和PLT数均有不同程度的下降.特异性IgY保护组各项指标较快恢复到正常水平,其他组恢复缓慢.各组小鼠七天内的死亡率分别为:空白组与阴性对照组都为100%;阳性对照组60%;低剂量IgY组30%;高剂量IgY组10%.结论:特异性IgY对小鼠大肠杆菌败血症有保护作用.