B4 LPS Research Articles

The Role of O-antigen in P1 Transduction of Shigella flexneri and Escherichia coli with its Alternative S' Tail Fibre

Enterobacteria phage P1 expresses two types of tail fibre, S and S'. Despite the wide usage of phage P1 for transduction, the host range and the receptor for its alternative S' tail fibre was never determined. Here, a ΔS-cin Δpac E. coli P1 lysogenic strain was generated to allow packaging of phagemid DNA into P1 phage having either S or S' tail fibre. P1(S') could transduce phagemid DNA into Shigella flexneri 2a 2457O, Shigella flexneri 5a M90T and Escherichia coli O3 efficiently. Mutational analysis of the O-antigen assembly genes and LPS inhibition assays indicated that P1(S') transduction requires at least one O-antigen unit. E. coli O111:B4 LPS produced a high neutralising effect against P1(S') transduction, indicating that this E. coli strain could be susceptible to P1(S')-mediated transduction. Mutations in the O-antigen modification genes of S. flexneri 2a 2457O and S. flexneri 5a M90T did not cause significant changes to P1(S’) transduction efficiency. A higher transduction efficiency of P1(S') improved the delivery of a cas9 antimicrobial phagemid into both S. flexneri 2457O and M90T. These findings provide novel insights into P1 tropism-switching, by identifying the bacterial strains which are susceptible to P1(S')-mediated transduction, as well as demonstrating its potential for delivering a DNA sequence-specific Cas9 antimicrobial into clinically relevant S. flexneri.

Endotoxin activity and leukocytic STAT3 mRNA alterations differ according to age in lipopolysaccharide-challenged calves

The objective of this study was to investigate the age difference in the response to endotoxin in calves related to the plasma endotoxin activity and mRNA expression of cytokines. Fifteen calves were divided into three groups: control (191 ± 21 days), weaning (162.4 ± 17.5 days), and calf (22.4 ± 8.2 days). The weaning and calf groups received 2.5 μg/kg of ultrapure O111:B4 LPS in 10 mL of each autologous serum, whereas the control calves received a similar volume of saline. Blood samples were collected at 0, 0.5, 1, 2, 4, 8, 12, and 24 h. Liver samples were collected by ultrasound-guided liver biopsy at 0, 2, 4, and 24 h. Plasma endotoxin activity was measured by the limulus amebocyte lysate kinetic turbidimetric assay. The mRNA expression level of GAPDH, TLR-4, NF-κB2, TNF-α, IL-6, and STAT3 in leukocytes and the liver was measured by real-time PCR.Following LPS challenge, the maximal plasma endotoxin activity, and leukocytic expression of TLR4, NF-κB2, TNF-α, and STAT3 were reached at 0.5, 4, 2–4, 2–4, and 4 h, respectively, in weaning and calf groups. The endotoxin activity in calf remained high until 2 h. Furthermore, the expression of leukocytic STAT3 mRNA in calf was not significantly different from the pre-value. In contrast, STAT3 mRNA in weaning markedly increased at 2 and 4 h. Therefore, this study provides new information to the literature of immune and inflammatory responses in calves.

An In Vitro Assessment of Immunostimulatory Responses to Ten Model Innate Immune Response Modulating Impurities (IIRMIs) and Peptide Drug Product, Teriparatide.

Understanding, predicting, and minimizing the immunogenicity of peptide-based therapeutics are of paramount importance for ensuring the safety and efficacy of these products. The so-called anti-drug antibodies (ADA) may have various clinical consequences, including but not limited to the alteration in the product’s distribution, biological activity, and clearance profiles. The immunogenicity of biotherapeutics can be influenced by immunostimulation triggered by the presence of innate immune response modulating impurities (IIRMIs) inadvertently introduced during the manufacturing process. Herein, we evaluate the applicability of several in vitro assays (i.e., complement activation, leukocyte proliferation, and cytokine secretion) for the screening of innate immune responses induced by ten common IIRMIs (Bacillus subtilis flagellin, FSL-1, zymosan, ODN2006, poly(I:C) HMW, poly(I:C) LMW, CLO75, MDP, ODN2216, and Escherichia coli O111:B4 LPS), and a model biotherapeutic Forteo™ (teriparatide). Our study identifies cytokine secretion from healthy human donor peripheral blood mononuclear cells (PBMC) as a sensitive method for the in vitro monitoring of innate immune responses to individual IIRMIs and teriparatide (TP). We identify signature cytokines, evaluate both broad and narrow multiplex cytokine panels, and discuss how the assay logistics influence the performance of this in vitro assay.

The Effect of Water-Soluble Polysaccharide from Jackfruit (Artocarpus heterophyllus Lam.) on Human Colon Carcinoma Cells Cultured In Vitro.

Various phytochemical studies have revealed that jackfruit (Artocarpus heterophyllus Lam.) is rich in bioactive compounds, including carotenoids, flavonoids, volatile acids, tannins, and lectins. The aim of the study was to analyze the biological activity of water-soluble polysaccharide (WSP) isolated from jackfruit and to assess its immunomodulatory, cytotoxic, and anti-oxidative effects on human colon carcinoma cells in vitro. The neutral red (NR) uptake assay revealed no toxic influence of the polymer on the viability of tumor cells (HT29 and SW620). After 24 h and 48 h of incubation, the cellular viability was not lower than 94%. The metabolic activity of the cells (MTT) at the compound concentration of 250 µg/mL was higher than 92% in comparison to the control. WSP (250 µg/mL) exerted no significant effect on the morphology of the cells was determined by May-Grünwald-Giemsa staining. WSP changed nitric oxide (NOx) production by the tumor cells depending on the time of incubation and prior 2-h stimulation of the cells with E. coli 0111:B4 LPS. It significantly stimulated IL-1β production by the tumor cells. The IL-6 level increased but that of IL-10 decreased by a WSP concentration-dependent manner. No such effect was detected in SW620. The WSP had antioxidant properties. In conclusion, water-soluble polysaccharide isolated from A. heterophyllus exhibits significant biological activity towards many types of both normal and cancerous cells. Therefore, it may be considered as a useful agent in the protection of human health or in functional and dietary nutrition.

In vitro immune response of chinook salmon (Oncorhynchus tshawytscha) peripheral blood mononuclear cells stimulated by bacterial lipopolysaccharide

We investigated cellular functional and targeted immune cytokine responses of farmed Chinook salmon (Oncorhynchus tshawytscha) peripheral blood mononuclear cells (PBMCs) in vitro to LPS from Escherichia coli (E. coli) serotypes O111: B4 and O55: B5, and a phorbol ester phorbol 12-myristate 13-acetate (PMA). Bacterial LPS and PMA significantly (p < 0.05) induced reactive oxygen species (ROS) production in O. tshawytscha PBMCs, and enhanced by interferon (IFN)-inducible cytokine production. Cellular phagocytosis was significantly enhanced with PMA and E. coli serotype O111: B4 LPS after 1 and 2 h respectively. At the molecular level, LPS and PMA significantly (p < 0.05) upregulated pro-inflammatory cytokine gene transcripts for IFNγ, TNF-α, and anti-inflammatory IL-10, 24 h post-stimulation. This response is postulated to be mediated via the MyD88 and TRIF pathways in TLR4, or synergistic TLR1 and TLR2 receptors. This is the first report of LPS induced immune related in vitro responses in farmed O. tshawytscha PBMCs.

Protective Effect of Citropin 1.1 and Tazobactam-Piperacillin Against Oxidative Damage and Lethality in Mice Models of Gram-Negative Sepsis

Gram-negative sepsis ranks as the leading cause of death in intensive care units, and its incidence is increasing steadily and mortality rates has not changed much over recent decades. We investigated the efficacy of the amphibian peptide, citropin 1.1 alone and in combination with tazobactam-piperacillin (TZP) in two experimental mice models of gram-negative sepsis. Animals were given an intraperitoneal injection of (1) 1 mg Escherichia coli 0111:B4 LPS, and (2) 2×10(10) CFU of E. coli ATCC 25922. For each model, all animals were randomized to receive intraperitoneally isotonic sodium chloride solution, 1 mg/Kg citropin 1.1 and 120 mg/Kg of TZP, and finally 1 mg/Kg citropin 1.1 plus 60 mg/Kg of TZP. Lethality, bacterial growth in blood and peritoneum, and oxidative stress indices in plasma were evaluated. All compounds reduced the lethality compared with controls. Treatment with citropin 1.1 resulted in significant decrease in plasma endotoxin and cytokine levels, while TZP exerted opposed effect. The combination between citropin 1.1 and TZP proved to be the most effective treatment in reducing all variables measured. Due to its multifunctional properties, citropin 1.1 may become an important future consideration to treat conditions in which oxidative organ failure may be present.

Cellular Recognition of Trimyristoylated Peptide or Enterobacterial Lipopolysaccharide via Both TLR2 and TLR4

Evidence for specific and direct bacterial product recognition through toll-like receptors (TLRs) has been emphasized recently. We analyzed lipopeptide analogues and enterobacterial lipopolysaccharide (eLPS) for their potential to activate cells through TLR2 and TLR4. Whereas bacterial protein palmitoylated at its N-terminal cysteine and N-terminal peptides derived thereof are known to induce TLR2-mediated cell activation, a synthetic acylhexapeptide mimicking a bacterial lipoprotein subpopulation for which N-terminal trimyristoylation is characteristic (Myr(3)CSK(4)) activated cells not only through TLR2 but also through TLR4. Conversely, highly purified eLPS triggered cell activation through overexpressed TLR2 in the absence of TLR4 expression if CD14 was coexpressed. Accordingly, TLR2(-/-) macrophages prepared upon gene targeting responded to Myr(3)CSK(4) challenge, whereas TLR2(-/-)/TLR4(d/d) cells were unresponsive. Through interferon-gamma (IFNgamma) priming, macrophages lacking expression of functional TLR4 and/or MD-2 acquired sensitivity to eLPS, whereas TLR2/TLR4 double deficient cells did not. Not only TLR2(-/-) mice but also TLR4(-/-) mice were resistant to Myr(3)CSK(4) challenge-induced fatal shock. d-Galactosamine-sensitized mice expressing defective TLR4 or lacking TLR4 expression acquired susceptibility to eLPS-driven toxemia upon IFNgamma priming, whereas double deficient mice did not. Immunization toward ovalbumin using Myr(3)CSK(4) as adjuvant was ineffective in TLR2(-/-)/TLR4(-/-) mice yet effective in wild-type, TLR2(-/-), or TLR4(-/-) mice as shown by analysis of ovalbumin-specific serum Ig concentration. A compound such as Myr(3)CSK(4) whose stimulatory activity is mediated by both TLR2 and TLR4 might constitute a preferable adjuvant. On the other hand, simultaneous blockage of both of the two TLRs might effectively inhibit infection-induced pathology.

Escherichia coli lipopolysaccharides produce serotype-specific hypothermic response in biotelemetered rats

We investigated whether LPS-induced hypothermia develops in a serotype-specific manner in biotelemetered conscious rats. Two different Escherichia coli serotypes of LPSs were injected at a dose of 250 mug/kg ip. E. coli O55:B5 LPS elicited an initial hypothermia and subsequent fever, but E. coli O111:B4 LPS caused more potent monophasic hypothermia. Serum tumor necrosis factor (TNF)-alpha levels were dramatically elevated at the initial phase of the hypothermia induced by both LPSs. This elevation tended to subside at the nadir of E. coli O55:B5 LPS-induced response but progressively increased at the nadir of E. coli O111:B4 LPS hypothermia. Serum IL-10 levels were moderately elevated at the initial phase of the hypothermia and persisted at the same level at the nadir of each LPS-induced response. No change was observed at the serum IL-18 levels. A selective cyclooxygenase (COX)-1 enzyme inhibitor, valeryl salicylate (20 mg/kg sc), abolished the hypothermia without any effect on the elevated cytokine levels. Another COX-1-selective inhibitor, 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole (SC-560; 1 mg/kg sc) inhibited hypothermic responses as well. Meanwhile, cytokine levels were also reduced by SC-560 treatment. These findings suggest that LPS-induced hypothermia may have serotype-specific characteristics in rats. E. coli O111:B4 LPS has more potent hypothermic activity than E. coli O55:B5 LPS; that may presumably be related to its higher or sustained capability to release antipyretic cytokines, such as TNF-alpha. COX-1 enzyme may be involved in the generation of the hypothermia, regardless of the type of LPS administered.

TLR2 and TLR4 agonists synergistically up-regulate SR-A in RAW264.7 through p38

It is known that macrophage scavenger receptor A (SR-A) can protect mice from endotoxemia. In addition, Escherichia coli O111:B4 LPS from Sigma (sLPS), which contains both TLR4 and TLR2 agonists, was previously reported to be able to induce SR-A expression on murine macrophage cell line RAW264.7. However, the relative role of both TLR4 and TLR2 agonists from Sigma (sLPS) in the up-regulation of SR-A on RAW264.7 is still undefined. Here, we found that sLPS could only slightly up-regulate SR-A on RAW264.7 following removing its TLR4 and TLR2 agonists, respectively. In contrast, the combination of TLR4 agonist uLPS (re-extracted sLPS) and TLR2 agonist Pam3CSK4 dramatically induced SR-A expression, and synergistically promoted RAW264.7 to bind and internalize FITC-LPS specifically through SR-A. The combination had no such effect either on TLR2 or TLR4 expression, and incubation with IL-6, IL-10, IL-12 or TNF-α alone could not induce SR-A expression on RAW264.7. In addition, treatment with a NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) could only weakly suppress the up-regulation of SR-A by the combination. However, the combination synergistically promoted MAPK p38 phosphorylation, and p38 specific inhibitor SB203580 completely suppressed its inducible effect on SR-A expression. Hence, we demonstrated that up-regulation of SR-A by sLPS was resulted from the cooperation of its TLR4 and TLR2 agonists through p38, and we also presented a novel synergy effect of TLR2 and TLR4 agonists.

THE CATHELICIDIN-DERIVED TRITRPTICIN ENHANCES THE EFFICACY OF ERTAPENEM IN EXPERIMENTAL RAT MODELS OF SEPTIC SHOCK

Sepsis remains a serious clinical problem despite intense efforts to improve survival. In this study, the efficacy of ertapenem combined with the cathelicidin tritrpticin was investigated in two rat models of septic shock. Main outcome measures were bacterial growth in blood, peritoneum, spleen, liver, and mesenteric lymph nodes; endotoxin, interleukin 6, and tumor necrosis factor alpha concentrations in plasma; and lethality. Adult male Wistar rats were given (1) an intraperitoneal injection of 1 mg Escherichia coli serotype 0111:B4 LPS or (2) intra-abdominal sepsis induced via cecal ligation and puncture. For each model, all animals were randomized to receive intraperitoneally isotonic sodium chloride solution, 1 mg/kg tritrpticin, 15 mg/kg ertapenem, and 1 mg/kg tritrpticin combined with 15 mg/kg ertapenem. Each group included 20 animals. All compounds significantly reduced bacterial growth and lethality as compared with saline treatment. Treatment with tritrpticin resulted in significant decrease in plasma endotoxin and cytokine levels, whereas ertapenem exerted opposite effect. The combination between tritrpticin and ertapenem proved to be the most effective treatment in reducing all variables measured. In conclusion, tritrpticin enhances ertapenem efficacy in gram-negative septic shock rat models.

LL-37 Protects Rats against Lethal Sepsis Caused by Gram-Negative Bacteria

We investigated the efficacy of LL-37, the C-terminal part of the only cathelicidin in humans identified to date (termed human cationic antimicrobial protein), in three experimental rat models of gram-negative sepsis. Adult male Wistar rats (i) were given an intraperitoneal injection of 1 mg Escherichia coli 0111:B4 LPS, (ii) were given 2 x 10(10) CFU of Escherichia coli ATCC 25922, or (iii) had intra-abdominal sepsis induced via cecal ligation and puncture. For each model, all animals were randomized to receive intravenously isotonic sodium chloride solution, 1-mg/kg LL-37, 1-mg/kg polymyxin B, 20-mg/kg imipenem, or 60-mg/kg piperacillin. Lethality; growth of bacteria in blood, peritoneum, spleen, liver, and mesenteric lymph nodes; and endotoxin and tumor necrosis factor alpha (TNF-alpha) concentrations in plasma were evaluated. All compounds reduced lethality compared to levels in controls. Endotoxin and TNF-alpha plasma levels were significantly higher in conventional antibiotic-treated rats than in LL-37- and polymyxin B-treated animals. All drugs tested significantly reduced bacterial growth compared to saline treatment. No statistically significant differences between LL-37 and polymyxin B were noted for antimicrobial and antiendotoxin activities. LL-37 and imipenem proved to be the most effective treatments in reducing all variables measured. Due to its multifunctional properties, LL-37 may become an important future consideration for the treatment of sepsis.

Effects of pexiganan alone and combined with betalactams in experimental endotoxic shock

To investigate the efficacy of pexiganan, a 22-residue magainin analog, alone and combined with betalactmas antibiotics in three experimental rat models of Gram-negative septic shock. Adult male Wistar rats were given (i) an intraperitoneal injection of 1 mg Escherichia coli 0111:B4 LPS; (ii) 2 × 10 10 CFU of E. coli ATCC 25922; and (iii) intra-abdominal sepsis induced via cecal ligation and puncture. For each model, all animals were randomized to receive intraperitoneally isotonic sodium chloride solution, 1 mg/kg pexiganan, 1 mg/kg polymyxin B, 20 mg/kg imipenem, 60 mg/kg piperacillin alone and combined with 1 mg/kg pexiganan. Each group included 15 animals. Lethality, bacterial growth in blood or intra-abdominal fluid, endotoxin and TNF-α concentrations in plasma. All compounds reduced the lethality when compared to controls. Piperacillin and imipenem significantly reduced the lethality and the number of E. coli in abdominal fluid compared with saline treatment. Pexiganan showed a slightly lower antimicrobial activity than betalactams even though it achieved a substantial higher decrease in endotoxin and TNF-α plasma concentrations than imipenem and piperacillin. No statistically significant differences were noted for antimicrobial and antiendotoxin activities between pexiganan and polymyxin B. Combination between pexiganan and betalactams showed to be the most effective treatment in reducing all variables measured. The use of a novel antimicrobial compound able to bind to LPS associated to potent antibiotics such as betalactams may become an important future consideration for sepsis treatment.

Therapeutic efficacy of the magainin analogue MSI-78 in different intra-abdominal sepsis rat models.

This study was designed to investigate the antimicrobial and anti-endotoxin activity of MSI-78, a synthetic cationic peptide analogue of magainin 2. The in vitro antimicrobial activity of MSI-78 was investigated against the commercially available quality control strain Escherichia coli ATCC 25922. In addition, three rat models of septic shock were investigated: (i) rats were injected intraperitoneally with 1 mg Escherichia coli 0111:B4 LPS; (ii) rats were given an intraperitoneal injection of 2 x 10(10) cfu of Escherichia coli ATCC 25922; (iii) intra-abdominal sepsis was induced via caecal ligation and puncture. All animals were randomized to receive after 360 min intravenously isotonic sodium chloride solution, 1 mg/kg MSI-78, or 60 mg/kg piperacillin. Main outcome measures were: abdominal exudate and plasma bacterial growth, plasma endotoxin and tumour necrosis factor alpha (TNF-alpha) concentrations, and lethality. Our in vitro data showed that MSI-78 possesses a strong activity against Escherichia coli. The in vivo studies showed that all compounds reduced the lethality when compared to controls. MSI-78 showed a slightly higher antimicrobial activity than piperacillin and achieved a substantial decrease in endotoxin and TNF-alpha plasma concentrations than the beta-lactam. Because of its strong double anti-endotoxin and antimicrobial activities MSI-78 could be an interesting compound for Gram-negative septic shock treatment.

Increased iNOS activity is essential for hepatic epithelial tight junction dysfunction in endotoxemic mice.

We tested the hypothesis that increased production of nitric oxide (NO*) by inducible NO* synthase (iNOS) is a key factor responsible for alterations in the expression, localization, and function of key tight junction (TJ) proteins in mice challenged with lipopolysaccharide (LPS, endotoxin). Endotoxemia was associated with hepatobiliary epithelial barrier dysfunction, as evidenced by increased plasma-to-bile leakage of FITC-labeled dextran (relative molecular mass 40 kDa) and increased circulating levels of bile acids and conjugated bilirubin. Immunoblotting revealed decreased expression of zonula occludens (ZO)-1, ZO-2, ZO-3, and occludin in liver after injection of C57Bl/6J mice with 2 mg/kg Escherichia coli 0111:B4 LPS. Nonidet P-40-insoluble (i.e., TJ-associated) occludin and ZO-1 were virtually undetectable 12 and 18 h after injecting LPS. Immunofluorescence microscopy also revealed deranged subcellular localization of ZO-1 and occludin in endotoxemic mice. Pharmacological inhibition of iNOS activity using l-N6-(1-iminoethyl)lysine (5 mg/kg) or genetic ablation of iNOS ameliorated LPS-induced changes in hepatobiliary barrier function, and these strategies partially preserved TJ protein expression and localization. Steady-state levels of occludin and ZO-3 transcripts decreased transiently after injecting LPS but returned toward normal by 12 and 24 h after induction of endotoxemia, respectively. These results support the view that iNOS-dependent NO* production is an important factor contributing to hepatobiliary epithelial barrier dysfunction resulting from systemic inflammation and suggest that iNOS induction may play a role in the development of cholestatic jaundice in patients with severe sepsis.

Human C-reactive protein does not protect against acute lipopolysaccharide challenge in mice.

The physiological and pathophysiological functions of C-reactive protein (CRP), the classical acute-phase protein, are not well established, despite many reports of biological effects of CRP in vitro and in model systems in vivo. Limited, small scale experiments have suggested that rabbit and human CRP may both protect mice against lethal toxicity of Gram-negative bacterial LPS. However, in substantial well-controlled studies in C57BL/6 mice challenged with Escherichia coli O111:B4 LPS, we show in this work that significant protection against lethality was conferred neither by an autologous acute-phase response to sterile inflammatory stimuli given to wild-type mice 24 h before LPS challenge, nor by human CRP, whether passively administered or expressed transgenically. Male mice transgenic for human CRP, which mount a major acute-phase response of human CRP after LPS injection, were also not protected against the lethality of LPS from either E. coli O55:B5 or Salmonella typhimurium. Even when the acute-phase human CRP response was actively stimulated in transgenic mice before LPS challenge, no protection against LPS toxicity was observed. Indeed, male mice transgenic for human CRP that were pretreated with casein to stimulate an acute-phase response 24 h before LPS challenge suffered significantly greater mortality than unstimulated human CRP transgenic controls. Rather than being protective in this situation, human CRP may thus have pathogenic proinflammatory effects in vivo.

Cathelicidin peptide sheep myeloid antimicrobial peptide-29 prevents endotoxin-induced mortality in rat models of septic shock.

The present study was designed to investigate the antiendotoxin activity and therapeutic efficacy of sheep myeloid antimicrobial peptide (SMAP)-29, a cathelicidin-derived peptide. The in vitro ability of SMAP-29 to bind LPS from Escherichia coli 0111:B4 was determined using a sensitive limulus chromogenic assay. Two rat models of septic shock were performed: (1) rats were injected intraperitoneally with 1 mg E. coli 0111:B4 LPS and (2) intraabdominal sepsis was induced via cecal ligation and single puncture. All animals were randomized to receive parenterally isotonic sodium chloride solution, 1 mg/kg SMAP-29, 1 mg/kg polymyxin B or 20 mg/kg imipenem. The main outcome measures were: abdominal exudate and plasma bacterial growth, plasma endotoxin and tumor necrosis factor-alpha concentrations, and lethality. The in vitro study showed that SMAP-29 completely inhibited the LPS procoagulant activity at approximately 10 microM peptide concentration. The in vivo experiments showed that all compounds reduced the lethality when compared with control animals. SMAP-29 achieved a substantial decrease in endotoxin and tumor necrosis factor-alpha plasma concentrations when compared with imipenem and saline treatment and exhibited a slightly lower antimicrobial activity than imipenem. No statistically significant differences were noted between SMAP-29 and polymyxin B. SMAP-29, because of its double antiendotoxin and antimicrobial activities, could be an interesting compound for septic shock treatment.

Effect of Intramammary Escherichia coli Endotoxin in Early- vs. Late-Lactating Dairy Cows

We investigated the differences in the effect of intramammarily infused endotoxin for cows in early (EL) and late (LL) lactation. In this crossover study, nine cows were challenged twice with 100μg of intramammarily infusedEscherichia coli 0111:B4 LPS, each cow serving its own control. Systemic and local signs were recorded throughout the experiment to assess the severity of each cow's response. Daily milk yield and indicators of inflammation in blood and milk were also recorded. The response was significantly more severe in the EL period. Before the challenge, milk yield and serum free fatty acid concentration were higher and serum urea concentration lower in EL compared with LL cows. No significant differences were present in other parameters. After the challenge, milk SCC and leukocyte function, measured as chemiluminescence, increased, more markedly in EL. Blood neutrophil content varied from neutropenia to neutrophilia. Simultaneously with neutropenia, the function of neutrophils increased in LL but decreased in EL. Serum cortisol peaked 4h after challenge in both groups. In conclusion, endotoxin challenge resulted in more severe response in EL. During that time cows also had impaired neutrophil function postchallenge.

Inhibition of lung phosphodiesterase improves responsiveness to inhaled nitric oxide in isolated-perfused lungs from rats challenged with endotoxin.

To investigate the ability of phosphodiesterase (PDE) selective inhibitors to improve responsiveness to inhaled nitric oxide (NO) in isolated-perfused lungs of rats pretreated with endotoxin/lipopolysaccharide (LPS). Prospective, controlled animal study in the animal research facility of a university hospital. Sixteen hours after adult Sprague-Dawley rats were injected intraperitoneally with 0.4 mg/ kg E. coli 0111:B4 LPS administration, lungs were isolated and perfused, and the thromboxane mimetic U46619 was employed to increase the mean pulmonary artery pressure by 5-7 mmHg. The lungs were then ventilated with or without 0.4 ppm NO, and erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA; PDE type 2 inhibitor), milrinone (PDE type 3 inhibitor), or zaprinast (inhibitor of PDE types 5 and 9) were added to the perfusate. In the presence of EHNA (12.5, 25, 50 microM) the vasodilator response to inhaled NO was not greater than in its absence (0.25 +/- 0.25, 0.5 +/- 0.25, 0.75 +/- 0.25 mmHg vs. 0.25 +/- 0.25, 0.5 +/- 0.25, 0.75 +/- 0.25 mmHg, respectively). In the presence of milrinone (125, 250, 500 nM), the vasodilator response to inhaled NO was also not improved. In contrast, zaprinast (3.7, 7.4, 14.8 microM) augmented the pulmonary vasodilatory effect of inhaled NO in lungs from LPS-pretreated rats from 0.25 +/- 0.25, 0.5 +/- 0.25, 0.75 +/- 0.25 mmHg to 0.75 +/- 0.25, 1.5 +/- 0.5, 1.75 +/- 0.75 mmHg, respectively (p < 0.05). Our results demonstrate that inhibition of pulmonary PDE enzyme activity with zaprinast increases vasodilator responsiveness to inhaled NO in lungs obtained from rats 16 h after LPS challenge.

Characterization of the simultaneous binding of Escherichia coli endotoxin to Kupffer and endothelial liver cells by flow cytometry

BACKGROUND The triggering of cellular responses during endotoxic shock is initiated for the binding of endotoxin (lipopolysaccharide; LPS) to the cell surface. Kupffer and endothelial liver cells, involved in the removal of endotoxin from blood circulation, show in vitro a rapid response to LPS in the absence of serum. METHODS A double-labeling fluorescent assay was designed to evaluate the binding properties of Escherichia coli O111:B4 LPS to individual endothelial and Kupffer cells in suspension, where both populations occurred in the same relative proportion as in liver. After immunolabeling of the Kupffer cell population with the monoclonal antibody ED1 conjugated to R. phycoerythrin, the binding characteristics of LPS labeled with fluorescein to both endothelial and Kupffer cells were simultaneously studied by flow cytometry in serum-free conditions. RESULTS Specific and saturable binding of endotoxin was observed with both populations, showing properties of a receptor-mediated process. The Kupffer cell population showed a faster capacity and a higher affinity for LPS binding. The Hill coefficients indicated positive cooperativity in the LPS interaction with both populations. CONCLUSIONS Specific endotoxin binding to liver sinusoidal cells occurs in a serum-independent manner, particularly at high LPS concentrations. Flow cytometry is a fast, precise, and efficient technique to evaluate the simultaneous interaction of a ligand with two different cell types.

Characterization of the simultaneous binding of Escherichia coli endotoxin to Kupffer and endothelial liver cells by flow cytometry.

The triggering of cellular responses during endotoxic shock is initiated for the binding of endotoxin (lipopolysaccharide; LPS) to the cell surface. Kupffer and endothelial liver cells, involved in the removal of endotoxin from blood circulation, show in vitro a rapid response to LPS in the absence of serum. A double-labeling fluorescent assay was designed to evaluate the binding properties of Escherichia coli O111:B4 LPS to individual endothelial and Kupffer cells in suspension, where both populations occurred in the same relative proportion as in liver. After immunolabeling of the Kupffer cell population with the monoclonal antibody ED1 conjugated to R. phycoerythrin, the binding characteristics of LPS labeled with fluorescein to both endothelial and Kupffer cells were simultaneously studied by flow cytometry in serum-free conditions. Specific and saturable binding of endotoxin was observed with both populations, showing properties of a receptor-mediated process. The Kupffer cell population showed a faster capacity and a higher affinity for LPS binding. The Hill coefficients indicated positive cooperativity in the LPS interaction with both populations. Specific endotoxin binding to liver sinusoidal cells occurs in a serum-independent manner, particularly at high LPS concentrations. Flow cytometry is a fast, precise, and efficient technique to evaluate the simultaneous interaction of a ligand with two different cell types.