Lipopolysaccharide-binding Protein mRNA Research Articles

Liver lipopolysaccharide binding protein prevents hepatic inflammation in physiological and pathological non-obesogenic conditions

Lipopolysaccharide binding protein (LBP) knockout mice models are protected against the deleterious effects of major acute inflammation but its possible physiological role has been less well studied. We aimed to evaluate the impact of liver LBP downregulation (using nanoparticles containing siRNA- Lbp) on liver steatosis, inflammation and fibrosis during a standard chow diet (STD), and in pathological non-obesogenic conditions, under a methionine and choline deficient diet (MCD, 5 weeks). Under STD, liver Lbp gene knockdown led to a significant increase in gene expression markers of liver inflammation (Itgax, Tlr4, Ccr2, Ccl2 and Tnf), liver injury (Krt18 and Crp), fibrosis (Col4a1, Col1a2 and Tgfb1), endoplasmic reticulum (ER) stress (Atf6, Hspa5 and Eif2ak3) and protein carbonyl levels. As expected, the MCD increased hepatocyte vacuolation, liver inflammation and fibrosis markers, also increasing liver Lbp mRNA. In this model, liver Lbp gene knockdown resulted in a pronounced worsening of the markers of liver inflammation (also including CD68 and MPO activity), fibrosis, ER stress and protein carbonyl levels, all indicative of non-alcoholic steatohepatitis (NASH) progression. At cellular level, Lbp gene knockdown also increased expression of the proinflammatory mediators (Il6, Ccl2), and markers of fibrosis (Col1a1, Tgfb1) and protein carbonyl levels. In agreement with these findings, liver LBP mRNA in humans positively correlated with markers of liver damage (circulating hsCRP, ALT activity, liver CRP and KRT18 gene expression), and with a network of genes involved in liver inflammation, innate and adaptive immune system, endoplasmic reticulum stress and neutrophil degranulation (all with q-value<0.05). In conclusion, current findings suggest that a significant downregulation in liver LBP levels promotes liver oxidative stress and inflammation, aggravating NASH progression, in physiological and pathological non-obesogenic conditions.

Effects of dietary oil sources (sunflower and fish) on fermentation characteristics, epithelial gene expression and microbial community in the rumen of lambs fed a high-concentrate diet

ABSTRACT The feeding of high-concentrate diets commonly results in lowered pH and ruminal dysbiosis which cause shifts in uptake dynamics of short-chain fatty acids (SCFA) and altered epithelial function. Therefore, the current study evaluated the effect of dietary polyunsaturated fatty acids (PUFA) on ruminal fermentation products, gene expression in the ruminal epithelium and the associated changes in ruminal microorganisms in lambs fed a high-concentrate diet. Twenty-six Afshari lambs adapted to a high-concentrate diet during a completely randomised design were fed with a basal diet supplemented with 100 g oil supplement (OS; 60 g sunflower oil and 40 g fish oil) for 10 (OS10), 20 (OS20) and 30 (OS30) d, respectively (n = 6). Lambs with no oil supplementation (OS0, n = 8) were considered as control and slaughtered at d 0 of the experiment, and the remaining lambs were slaughtered at 10, 20 and 30 d on feed. After slaughter, ruminal digesta was collected for evaluating fermentation and microbial community. Ruminal papillae were taken for assessment of epithelial gene expression. Compared with OS0 lambs, supplemental PUFA in OS30 lambs tended to decrease total SCFA concentration with decreased acetic and increased propionic acid concentrations. Acetate:propionate ratios were decreased and ruminal pH was increased in OS20 and OS30 lambs compared to OS0. All groups with included OS had decreased concentrations of iso-valeric and valeric acids compared to OS0. Relative mRNA abundance of monocarboxylate transporter isoforms 1 and 4, insulin-like growth factor binding protein 3, sterol regulatory element-binding proteins 1 and 2 decreased with increasing OS duration. The relative abundance of 3-hydroxy-3-methylglutaryl-CoA synthase 1 mRNA transcript was higher for OS10 and OS20 lambs relative to OS0 lambs. OS20 and OS30 showed a decrease of lipopolysaccharide binding protein mRNA expression compared with OS0. Feeding supplemental PUFA decreased Ciliate protozoa and increased Butyrivibrio fibrisolvens in OS20 and OS30 lambs, whereas Megasphaera elsdenii was increased in OS30 lambs. In conclusion, combined supplementation of sunflower and fish oil to a high-concentrate diet affects the ruminal microbial community with prominent decreases in ruminal ciliate protozoa and increases in B. fibrisolvens and M. elsdenii. These results lead to a more stabilised ruminal pH and a fermentation shift towards more propionate generation. Consideration of nutrients digestion will help to fully understand the benefits of feeding PUFA with a high-concentrate diet.

Oral exposure of pigs to the mycotoxin deoxynivalenol does not modulate the hepatic albumin synthesis during a LPS-induced acute-phase reaction.

The sensitivity of pigs to deoxynivalenol (DON) might be influenced by systemic inflammation (SI) which impacts liver. Besides following acute-phase proteins, our aim was to investigate both the hepatic fractional albumin (ALB) synthesis rate (FSR) and the ALB concentration as indicators of ALB metabolism in presence and absence of SI induced by LPS via pre- or post-hepatic venous route. Each infusion group was pre-conditioned either with a control diet (CON, 0.12 mg DON/kg diet) or with a DON-contaminated diet (DON, 4.59 mg DON/kg diet) for 4 wk. A depression of ALB FSR was observed 195 min after LPS challenge, independent of feeding group or LPS application route, which was not paralleled by a down-regulated ALB mRNA expression but by a reduced availability of free cysteine. The drop in ALB FSR only partly explained the plasma ALB concentrations which were more depressed in the DON-pre-exposed groups, suggesting that ALB levels are influenced by further mechanisms. The abundances of haptoglobin, C-reactive protein, serum amyloid A, pig major acute-phase protein, fibrinogen and LPS-binding protein mRNA were up-regulated upon LPS stimulation but not accompanied by increases in the plasma concentrations of these proteins, pointing at an imbalance between synthesis and consumption.

Tissue-specific expression and correlation with promoter DNA methylation of the LBP gene in pigs

Lipopolysaccharide binding protein (LBP) is a key factor in the recognition of lipopolysaccharide (LPS) and the initiation of immune response, thus regulating the body's resistance to pathogenic infection. To investigate the tissue-specific expression characteristics of the LBP gene and its transcriptional regulation in pigs, we detected LBP expression in different tissues of 35-day-old Meishan weaned piglets, determined LBP core promoter region using bioinformatics prediction combined with dual luciferase activity assay, and finally detected methylation levels by pyrosequencing. The results showed that LBP expression in the liver tissue was significantly higher (P>0.01) than that in other tissues, followed by the intestinal tissues. The core promoter region of LBP was located at –500–(–206) bp (chr.17: g.46837534–g.46837828), containing three CpG sites (CpG1, CpG2 and CpG3). Of the three CpG sites, CpG2 and CpG3 were variously methylated (P>0.01) in different tissues. Moreover, LBP mRNA levels were negatively correlated (P>0.01) with methylation levels of the CpG2 and CpG3 sites in the YY1 transcription factor binding sequence. It is speculated that the methylation of CpG2 and CpG3 sites might inhibit YY1 binding to the promoter sequences, thereby regulating the tissue-specific expression of LBP. This study demonstrated the distinct patterns of LBP expression and promoter methylation in the tissues of Meishan pigs and indicated the potential roles of DNA methylation in regulating LBP expression, which may contribute to further investigations on pig LBP gene expression and function.

The Kinetic Expression of Lipopolysaccharide-Binding Protein and CD14 Gene in Obstructive Jaundice

ABSTRACTBackground: Binding lipopolysaccharide (LPS) with high-affinity, lipopolysaccharide-binding protein (LBP) and CD14 lower the threshold stimulatory concentrations of LPS dramatically and enhance the rate of cytokine production markedly. This study aimed to investigate the kinetic expression of LBP/CD14 and its possible relationship with tumor necrosis factor alpha (TNF-α) to better understand the pathophysiology of obstructive jaundice. Materials and Methods: The tissues (liver, spleen, intestine, and lung) of male Sprague-Dawley rats were harvested at pre-bile duct ligation in controls and at specific time points (24, 48, 72, 96, and 120 hr) after bile duct ligation. LBP, CD14, and TNF-α mRNA expression were measured in tissues harvested from controls and at the specific time points. Results: Hepatic LBP mRNA expression increased significantly at five days after bile duct ligation. CD 14 mRNA expression increased significantly after five days of bile duct ligation in liver, lung, spleen, and ileum. TNF-α mRNA expression increased significantly in all four organs (liver, lung, spleen, and ileum) after four days of bile duct ligation. Conclusion: Five days of bile duct ligation upregulated CD 14 mRNA expression in liver, lung, spleen, and ileum and increased TNF-α mRNA expression simultaneously in the liver, lung, spleen, and ileum. In addition, five days of bile duct ligation also upregulated LBP mRNA expression in the liver and increased hepatic TNF-α mRNA expression simultaneously. The kinetic expressions of LBP and CD 14 in obstructive jaundice are intriguing and further evaluation is warranted.

Porphyromonas gingivalis LPS stimulates the expression of LPS-binding protein in human oral keratinocytes in vitro

LPS-binding protein (LBP) functions as a crucial molecule in innate immune responses to bacterial challenge. Our study has shown the expression of LBP in human gingiva and its significant association with periodontal health and disease. Porphyromonas gingivalis is a key pathogen of periodontal disease. P. gingivalis LPS as a main virulence factor is strongly involved in periodontal pathogenesis and it displays a significant lipid A structural heterogeneity. Currently, it remains unknown whether, and to what extent, the lipid A structural heterogeneity of P. gingivalis LPS affects LBP expression. The present study investigated the expression profile of LBP in human oral keratinocytes (HOKs) stimulated by two isoforms of P. gingivalis LPS [tetra- (LPS(1435/1449)) and penta-acylated (LPS(1690))] and Escherichia coli LPS, and the involvement of TLRs in LBP expression. The results showed that the expression of LBP mRNA and peptide was significantly up-regulated by P. gingivalis LPS(1690) and E. coli LPS, while P. gingivalis LPS(1435/1449) did not affect LBP expression. Blocking assay and siRNA gene silencing revealed that P. gingivalis LPS(1690)-induced LBP expression was through both TLR2 and TLR4. This in vitro study demonstrates that P. gingivalis LPS with a lipid A structural heterogeneity differentially modulates LBP expression in HOKs.

Serum LBP levels reflect the impaired synthetic capacity of the remnant liver after partial hepatectomy in rats

Lipopolysaccharide (LPS) binding protein (LBP) is up-regulated in inflammation and infection. Its use as a biomarker of severe infection and sepsis is currently discussed controversially. Here a novel LBP-ELISA was established to assess serum LBP-levels after various degree of in a rat partial hepatectomy (PH) model. The LBP enzyme-linked immunosorbent assay (ELISA) was designed based on the binding between LPS and LBP. LPS was employed as capture molecule. An anti-LBP antibody was used as detection antibody. Serum LBP levels were measured in serum obtained 24h after 30% PH, 70% PH and 90% PH in rats using newly established LBP ELISA method. Expression of hepatic LBP mRNA was measured by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). The detection range of the ELISA was from 0.1μg/ml to 60μg/ml. The correlation between ELISA and western blotting was strong (r=0.885, p<0.0001). Hepatic LBP mRNA expression was upregulated after PH. Of note, elevations of serum LBP protein levels were positively correlated to the remnant liver mass (R=0.821, p<0.0001), serum albumin levels (R=0.532, p<0.05) and total protein levels(R=0.813, p<0.0001). In conclusion, we established an economic and rapid ELISA assay for rat serum LBP quantification. Our results speak against using LBP as diagnostic marker of the acute phase response after liver resection as its elevation is related to the size and thereby the synthetic capacity of the small remnant liver.

Toll‐like receptor 4 is involved in bacterial endotoxin‐induced endothelial cell injury and SOC‐mediated calcium regulation

Bacterial endotoxins may lead to vascular endothelial cell injury. Our study explored the role of TLR4 (Toll-like receptor 4) and STIM1 (stromal interaction molecule 1) in bacterial endotoxin-induced calcium overload and inflammatory reactions in HUVECs (human umbilical vein endothelial cells). It showed that under LPS (lipopolysaccharide) stimulation, LBP (LPS-binding protein) mRNA levels peaked at 24 h, TLR4 levels at 12 h and NF-κB (nuclear factor κB) levels at 6 h (all P<0.01). LBP levels increased gradually and peaked at 24 h of LPS treatment. TLR4 protein levels increased significantly at 1 h and peaked at 12 h. NF-κB protein levels markedly increased at 1 h and peaked at 6 h. Knockdown of STIM1 alone, TLR4 alone or both STIM1 and TLR4 together, markedly abolished LPS-induced increase in calcium influx into cells (P<0.05, P<0.01 and P<0.01 respectively). LBP-TLR4 and STIM-NF-κB interactions were detected without LPS treatment, enhanced by LPS stimulation, and markedly reduced by knocking down TLR4 and STIM respectively. Both the NF-κB inhibitor, PDTC (pyrrolidine dithiocarbamate) and TLR4 knockdown could block LPS induction of NF-κB, STIM, TNFα (tumour necrosis factor α) and IL-6 (interleukin 6). The data indicate LPS-LBP may activate TLR4 signalling and downstream transcription factor NF-κB, which further can activate STIM1 and eventually lead to calcium influx and injury of HUVECs. Inhibition of TLR4 effectively reverses LPS induction of inflammatory mediator generation and extracellular calcium influx mediated by STIM1.

Distribution of acute phase proteins in the bovine forestomachs and abomasum

Acute phase proteins (APPs) are produced mainly by the liver and their concentration is increased during the systemic inflammatory response. Expression of haptoglobin (Hp), serum amyloid A (SAA), lipopolysaccharide-binding protein (LBP) and α-1 acid glycoprotein (AGP) was determined in the mucosa of the normal bovine forestomachs and abomasum by qualitative and quantitative reverse transcriptase-PCR for mRNA and by Western blot analysis and immunohistochemistry for proteins. Although expression of SAA mRNA was evident in the forestomachs and abomasum, SAA protein was identified only in the abomasum. Expression of Hp protein was high in the forestomachs and abomasum, even though expression of Hp mRNA was negligible. The main site of expression of LBP mRNA was the omasum, whereas the highest protein expression was evident in the abomasum. AGP was expressed at low levels in the bovine forestomachs. Western blot analysis revealed a heterogeneous electrophoretic pattern for AGP, LBP and Hp, indicating that different stomach compartments produce isoforms that are different to those expressed by the liver. Expression of APPs by the bovine forestomachs and abomasum may contribute to regulation of the innate immune response against pathogens.

Lipopolysaccharide-binding protein: Local expression in bovine extrahepatic tissues

Lipopolysaccharide-binding protein (LBP) is an acute phase protein involved in host response to Gram-negative and Gram-positive pathogens. It is synthesized by hepatocytes and released as 60–65 kDa glycoprotein in plasma. Little is known about the distribution of LBP in non-pathological bovine tissues. The aim of the present study was to investigate the extra hepatic expression of LBP in different bovine tissues by qualitative and quantitative real time (RT) PCR. The presence of the protein was also confirmed by immunohistochemistry using an anti-human LBP antibody preliminarily validated by cross-reactivity in bovine tissues. While a wide panel of organs and tissues was investigated, the attention was focused on the digestive tract and mammary gland. Moderate amount of mRNA was detected in most of the tissues involved in this study. Extra hepatic LBP mRNA expression was particularly high in parotid and submandibular salivary glands. Remarkably, LBP mRNA was found in rumen, reticulum and omasum. High expression was also found in the mammary gland. Intensity of protein staining paralleled mRNA expression in most tissues, with the exception of lung, ovary and thyroid gland. The presence of LBP throughout epithelial mucosal tissues is indicative of an important role of LBP in mucosal immunity at sites of bacterial exposure. These results suggest that ruminant forestomachs may mount a local acute phase reaction.

THE SYSTEMIC AND PULMONARY LPS BINDING PROTEIN RESPONSE TO INTRATRACHEAL LIPOPOLYSACCHARIDE

LPS binding protein (LBP) is an acute-phase glycoprotein that facilitates LPS activation of immune cells through interactions with CD14 and Toll-like receptor 4. Initially, LBP production was thought to occur exclusively in the liver in response to stimulation with TNF-alpha, IL-1, and IL-6. More recently, it has been shown that type II pneumocytes are also capable of LBP production. Little is known, however, regarding the regulation and or distribution of this protein in response to localized intrapulmonary infection. We performed time-course experiments challenging C3H mice intratracheally with LPS (10 mug). In separate experiments, mice deficient in IL-6 were given the same dose of intratracheal LPS and euthanized 8 h later. Despite the intratracheal route of LPS administration, an increase in plasma LBP concentrations occurred earlier and was of greater magnitude than the increase observed in bronchoalveolar lavage fluid. Liver LBP mRNA increased to a greater extent than did lung LBP mRNA. Whereas the TNF-alpha response remained localized within the alveolar space, IL-6 was increased both locally and in plasma. Of several tissues analyzed, the lung was the greatest producer of IL-6 mRNA. Plasma LBP was significantly decreased in the IL-6-deficient mice compared with wild-type controls challenged with intratracheal LPS. We conclude that lung-derived IL-6 is an important mediator of hepatic LBP up-regulation. We speculate that the disruption of these lung-liver signaling pathways may be important to host response efforts to confine infection to the lung. If impaired, this may be one mechanism underlying the increased mortality observed in patients with liver disease who develop pneumonia.

G-CSF Administration in a Small-for-Size Liver Model

Background: Extended liver resection is applied in the treatment of liver tumors and during living-related liver transplantation. This procedure can lead to transient, in some patients even lethal liver failure. Administration of G-CSF is associated with an increased survival after toxic liver damage. It is the aim of this study to test the effect of G-CSF administration in the rat 90% extended liver resection model. Materials: Rats with and without G-CSF treatment were subjected to 90% partial hepatectomy using a mass ligation technique. Animals were sacrificed 6 hr, 24 hr, and 7 days after resection. Read-out parameters were liver damage in terms of liver enzymes and histomorphological alterations. Hepatic regeneration was determined by measuring liver weight recovery and calculating the BrdU proliferation index of hepatocytes. G-CSF-receptor expression was visualized in both groups by immunohistochemistry. Expression of lipopolysaccharide-binding-protein (LBP) mRNA was evaluated by quantitative PCR. Results: Survival rate was increased in the G-CSF-treatment group. Full liver weight recovery within one week was only achieved in the treatment group and was accompanied by reduced liver damage. G-CSF-receptor upregulation subsequent to administration of G-CSF may indicate a direct receptor mediated effect of G-CSF on the liver. Upregulation of LBP mRNA in the liver of G-CSF treated animals—reported to be associated with an increased host defense—could demonstrate a mode of action of G-CSF.

Effects of Traditional Chinese Medicine on Endotoxin and Its Receptors in Rats with Non-Alcoholic Steatohepatitis

The aim of this research is to study the effects of traditional Chinese medicine on endotoxin and its receptors in rats with nonalcoholic steatohepatitis (NASH). Fifty-six SD rats were divided into seven groups. All the animals were fed high fatty diet for 12 weeks. Rats with non-alcoholic steatohepatitis (NASH) were treated with traditional Chinese medicine according to low-dose, middle-dose, high-dose and Lipitor from fifth week. All rats were killed at the end of 12th week. The liver pathology changes were observed under light microscope. The levels of serum lipoid, alanine aminotransferase (ALT), endotoxin (ET), tumor necrosis factor-alpha (TNF-alpha) and interleukine-1beta (IL-1beta) were determined. The expressions of CD14 and nuclear transcriptional factor kappaB (NF-kappaB) were observed by immunohistochemistry. The expressions of lipopolysaccharide binding protein (LBP), toll-like receptor-4 (TLR-4), myeloid differentiation-2 (MD-2) and induced nitric oxide synthase (iNOS) mRNA were detected by the reverse transcription polymerase chain reaction (RT-PCR). The levels of serum endotoxin in the middle dose group (0.0225 +/- 0.0112 EU/l) were lower than those in high fatty diet model group (0.2249 +/- 0.0982 EU/l) at 12th week, the difference was significant (P < 0.01). In the middle dose group, mean values of serum TNF-alpha and IL-1beta levels decreased dramatically (1.604 +/- 0.302 ng/ml and 0.052 +/- 0.024 ng/ml) compared with those in the high fatty diet model group (4.029 +/- 1.180 ng/ml and 14.944 +/- 0.491 ng/ml; P < 0.01 and P < 0.01). The expressions of CD14 and NF-kappaB in the middle dose group decreased compared with those in the high fatty diet model group. The expressions of LBP mRNA (0.284 +/- 0.105) and TLR-4 mRNA (0.290 +/- 0.123) in the middle dose group down regulated compared with those in the high fatty diet model group (1.060 +/- 0.158 and 1.261 +/- 0.368; P < 0.01 and P < 0.01). In the middle dose group MD-2 and iNOS gene expressions were 0.132 +/- 0.058 and 0.164 +/- 0.061, respectively, which were significantly lower compared with the high fatty diet model group (0.795 +/- 0.294 and 1.029 +/- 0.388; P < 0.01 and P < 0.01). The mechanism of non-alcoholic steatohepatitis (NASH) maybe related to increasing the levels of serum endotoxin, upregulating endotoxin receptors of hepatic tissue and enhancing liver inflammatory injury. Traditional Chinese medicine is a good treatment for non-alcoholic steatohepatitis (NASH). It can produce a marked effect via relieving LPS-induced liver injury.

IL-22 Induces Lipopolysaccharide-Binding Protein in Hepatocytes: A Potential Systemic Role of IL-22 in Crohn’s Disease

Crohn's disease (CD) is a common, chronic, inflammatory bowel disease characterized by intestinal infiltration of activated immune cells and distortion of the intestinal architecture. In this study, we demonstrate that IL-22, a cytokine that is mainly produced by activated Th1 and Th17 cells, was present in high quantities in the blood of CD patients in contrast to IFN-gamma and IL-17. In a mouse colitis model, IL-22 mRNA expression was elevated predominantly in the inflamed intestine but also in the mesenteric lymph nodes. IL-22BP, the soluble receptor for IL-22, demonstrated an affinity to IL-22 that was at least 4-fold higher than its membrane-bound receptor, and its strong constitutive expression in the intestine and lymph nodes was decreased in the inflamed intestine. To investigate the possible role of systemic IL-22 in CD, we then administered IL-22 to healthy mice and found an up-regulation of LPS-binding protein (LBP) blood levels reaching concentrations known to neutralize LPS. This systemic up-regulation was associated with increased hepatic but not renal or pulmonary LBP mRNA levels. IL-22 also enhanced the secretion of LBP in human primary hepatocytes and HepG2 hepatoma cells in vitro. This increase was mainly transcriptionally regulated and synergistic with that of other LBP inducers. Finally, elevated LBP levels were detected in CD patients and the mouse colitis model. These data suggest that systemic IL-22 may contribute to the prevention of systemic inflammation provoked by LPS present in the blood of CD patients through its induction of hepatic LBP.

Upregulation of TNF-alpha and IL-6 mRNA in mouse liver induced by bacille Calmette-Guerin plus lipopolysaccharide

To investigate the mechanism of immunological liver injury induced by bacille Calmette-Guerin (BCG) plus lipopolysaccharide (LPS). Mice were injected via the tail vein with 125 mg/kg BCG, and 12 d later, the mice were injected intravenously with different doses of LPS (125, 250, or 375 microg/kg). Serum alanine aminotransferase (ALT) activity and liver pathological changes were examined. The expression of tumor necrosis factor (TNF)- alpha, interleukin (IL)-6, lipopolysaccharide binding protein (LBP) and CD14 mRNA, and NF-kappaB and IkappaB-alpha protein in mouse liver at different time points after BCG and LPS injection were measured using RT-PCR, immunohistochemistry and Western blotting analysis, respectively. The activity of serum ALT in mice treated with BCG and LPS was significantly increased. Different degrees of liver injury, such as inflammatory cell infiltration, spotty necrosis, piecemeal necrosis, even bridging necrosis, could be seen in liver sections from mice after BCG and LPS administration. Furthermore, the levels of TNF-alpha and IL-6 mRNA in mouse liver were significantly elevated after administration of BCG plus LPS (P<0.05). The levels of LBP and CD14 mRNA in mouse liver were markedly upregulated after treatment with BCG and LPS, and treatment with BCG alone led to an increase in CD14 mRNA in mouse liver. Finally, immunoreactivity for NF-kappaB p65 was predominantly detected in hepatocyte nuclei from mice treated with BCG plus LPS, compared with the normal group. Protein levels of IkappaB-alpha were strikingly decreased by LPS or BCG plus LPS treatment, compared with the normal group or BCG group. TNF-alpha and IL-6 mRNA were partially involved in early immunological liver injury induced by challenge with small doses of LPS after BCG priming. Upregulation of TNF-alpha and IL-6 mRNA might be related to increases in LBP and CD14 mRNA expression and activation of NF-kappaB. Furthermore, BCG priming in immunological liver injury may occur via upregulation of CD14 mRNA expression in mononuclear cell infiltration into the liver.

PROTECTIVE EFFECT OF PAEONIFLORIN ON IMMUNOLOGICAL LIVER INJURY INDUCED BY BACILLUS CALMETTE‐GUERIN PLUS LIPOPOLYSACCHARIDE: MODULATION OF TUMOUR NECROSIS FACTOR‐α AND INTERLEUKIN‐6 mRNA

1. Paeoniflorin is one of the main effective components of the total glucosides of paeony (TGP) extracted from the root of Paeonia lactiflora which has been used for gynaecological problems and for cramp, pain and giddiness for over 1,500 years in Chinese medicine. Anti-inflammatory, antioxidative, antihepatic injury and immunoregulatory activities of TGP have been extensively proved in our laboratory for many years. Our present study investigates the effects and mechanisms of paeoniflorin on immunological liver injury in mice. 2. A model of immunological liver injury was induced by tail vein injection of bacillus Calmette-Guérin (BCG) and lipopolysaccharide (LPS) in mice. Activities of serum alanine aminotransferase (ALT) were measured by biochemical methods. Hepatic tissue sections were stained with haematoxylin and eosin and examined under a light microscope. Tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, lipopolysaccharide binding protein (LBP) and CD14 mRNA (messenger ribonucleic acid) expression in mouse liver were determined by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) analysis. 3. Immunological liver injury induced by BCG plus LPS was successfully duplicated. Serum ALT activities were significantly decreased by paeoniflorin. (25, 50, 100 mg/kg). Histological examination demonstrated that paeoniflorin could attenuate the area and extent of necrosis and reduce the immigration of inflammatory cells. The increase in TNF-alpha, LBP and CD14 mRNA expression in mouse liver after BCG and LPS injection was significantly decreased by paeoniflorin (100 mg/kg) and was changed by paeoniflorin (25, 50 mg/kg) at different time-point. The augmentation of IL-6 mRNA in mouse liver was markedly increased by paeoniflorin at 1 h and 3 h after LPS injection. 4. Paeoniflorin could significantly protect against immunological liver injury in mice. TNF-alpha, IL-6, LBP and CD14 mRNA expression in mouse liver may be involved in BCG plus LPS induced liver injury. The protective mechanism of paeoniflorin might be partially related to modulation of TNF-alpha, IL-6, LBP and CD14 mRNA expressions in mouse liver.

Local expression of lipopolysaccharide-binding protein in human gingival tissues.

Lipopolysaccharide-binding protein (LBP) functions as a crucial molecule in innate host defense responses to bacterial challenge through neutralization of bacterial lipopolysaccharide (LPS) and activation of cellular responses. This study was to investigate the expression profile and levels of LBP in gingival tissues and their associations with periodontal health and disease. Gingival biopsies were collected from 44 chronic periodontitis patients, including periodontal pocket tissues (PoTs) and the adjacent healthy gingival tissues (HT-Ps), as well as from 15 periodontally healthy subjects as controls (HT-Cs). The peptide and mRNA of LBP were detected by semi-quantitative immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR), respectively. LBP peptide was detected in 90.9% of PoTs (20/22), 84.6% of HT-Ps (11/13) and all HT-Cs (7/7). The expression of LBP was mainly confined to the cytoplasm of granular and keratinized layers of gingival epithelium, spreading from the oral sulcular epithelium to oral epithelium with the expression density decreasing gradually from coronal to apical portion. LBP peptide was also found on endothelial surfaces and/or inside the lumens of blood vessels in connective tissues. The mean LBP expression levels in HT-Cs were significantly higher than those in HT-Ps and PoTs. LBP mRNA was detected in 55% of PoTs (11/20), 55% of HT-Ps (11/20) and 75% of HT-Cs (6/8). We for the first time found the expression of LBP peptide and mRNA in human gingival tissues. Local expression of LBP in gingival tissues might contribute to periodontal homeostasis.

Tissue lipopolysaccharide-binding protein expression in rats after thermal injury: potential role of TNF-α

Recent studies have suggested that levels of lipopolysaccharide-binding protein (LBP) might play a harmful role by up-regulating the host’s sensitivity to endotoxin. Our previous studies demonstrated that local endotoxin could up-regulate LBP expression after acute insults, however, the definite molecular mechanisms downstream of endotoxin action remain unclear. This study investigates whether tumor necrosis factor (TNF-α) might be responsible for the LBP formation during endogenous endotoxemia postburn. Wistar rats were anesthetized, and a 35% TBSA full-thickness burn was created. Animals were randomly divided into normal control, thermal injury and anti-TNF-α mAb treatment group. A significant elevation of plasma endotoxin concentration was observed after acute insults. TNF-α levels in plasma also rapidly increased after thermal injury. Meanwhile, LBP mRNA expression markedly increased in liver, lungs, kidneys and intestine postburn. There was no detectable TNF-α in the plasma of anti-TNF-α mAb treated animals. Treatment with anti-TNF-α mAb also resulted in significantly lower concentrations of LBP mRNA in local tissues. Additionally, several organ function parameter levels in plasma significantly decreased in treatment group. These results demonstrated that an increase of plasma TNF-α levels caused by burns might be associated with a marked elevation of tissue LBP mRNA expression, which could contribute to the development of multiple organ damage.

Hyperlipoproteinemic low-density lipoprotein receptor-deficient mice are more susceptible to sepsis than corresponding wild-type mice

High circulating concentrations of lipoproteins have been shown to modify the cytokine response and reduce mortality after endotoxin or live bacterial challenge. Sepsis, however, is more complex than endotoxemia, and it is not clear whether elevated plasma lipoproteins will be protective. Previous studies have shown that the low-density-lipoprotein receptor deficient (LDLR —/— ) mice with increased circulating LDL are protected against the lethal effects of endotoxemia and Gram-negative infection. We evaluated whether the LDLR —/— mice would be protected against the effects of sepsis induced by cecal ligation and puncture (CLP). Mortality was greater in LDLR—/ — mice than in control C57Bl/6J mice. At 120 h after inducing sepsis, 20% of the control mice survived whereas none of the LDLR—/— mice were alive. Prior to inducing sepsis, serum concentrations of amyloid A protein and lipopolysaccharide binding protein (LBP) were significantly elevated in the LDLR —/— mice in comparison to the C57Bl/6J mice. Protein expression of sCD14 was also greater in the serum from the LDLR —/ — mice than the C57Bl/6J mice. The elevated serum concentrations of LBP and CD14 were not associated with increases in the levels of liver CD14 mRNA and LBP mRNA. After inducing sepsis, serum concentration of interleukin (IL)-1β was also significantly higher in LDLR — /— mice than in the control C57Bl/6J mice. These findings indicate that the LDLR — /— mice were more susceptible to the lethal effects of sepsis induced by CLP. The LDLR — /— mice also had higher serum concentrations of baseline, acute phase response proteins, SAA and LBP, and increased production of IL-1β in response to CLP.

The effects of thermal environment and spray-dried plasma on the acute-phase response of pigs challenged with lipopolysaccharide.

Forty barrows (TR4 x C22) were weaned at 17 d of age (BW = 6.27 +/- 0.30 kg), housed (two pigs/pen) in a thermal-neutral environment (TN; constant 26.7 degrees C), and fed diets with or without 7% (as-fed basis) spray-dried plasma (SDP). On d 7, one pig/ pen was moved into a cold environment (CE; constant 15.6 degrees C). Pigs were fitted with jugular catheters on d 11. On d 12, 16 pigs per environment (eight pigs per dietary treatment) were challenged i.v. with 75 microg of lipopolysaccharide (LPS)/kg of BW. Blood samples were collected over a 4.5-h period. Pigs were then killed and tissue samples were harvested for messenger RNA (mRNA) analysis. From d 0 to 7, pigs fed SDP diets had a lower gain:feed ratio (G/F) than pigs fed no SDP (533 +/- 14 vs. 585 +/- 17 g/kg; P < 0.03). Pigs housed in the CE consumed more feed and had a lower G/F than pigs housed in TN from d 7 to 11 (P < 0.001). There were no environment x diet interactions from d 7 to 11 (P > 0.78). Baseline concentrations of serum ACTH and cortisol were lower in the TN pigs than in the CE pigs (P < 0.001). Pigs fed diets without SDP had lower serum cortisol concentrations over the 4.5-h period than pigs fed SDP (time x diet, P < 0.001). Serum concentrations of tumor necrosis factor-alpha (TNF-alpha) were highest for pigs consuming SDP in the CE, whereas there were no differences among the other treatments (time x diet x environment, P < 0.02). Pigs housed in the CE had higher serum interleukin-1beta (IL-1beta) (P < 0.001) and interleukin-6 (IL-6; P < 0.001) than TN pigs. Pigs fed SDP also had slightly higher serum IL-1beta concentrations (P < 0.10) and higher (P < 0.001) IL-6 concentrations than pigs fed no SDP. Pigs fed SDP had 9% lower liver and 13% lower thymus mRNA expression of tumor necrosis factor-alpha (TNF-alpha) than pigs that consumed no SDP (P < 0.06). Liver IL-1beta, IL-6, and LPS-binding protein mRNA were higher in the CE than in the TN (P < 0.03, P < 0.001, and P < 0.05; respectively). In addition, spleen TNF-alpha (P < 0.03) and IL-6 (P < 0.01) mRNA levels were higher in the CE than in the TN. Pigs consuming SDP and challenged with LPS responded with elevated serum concentrations of cortisol and cytokines compared with pigs fed diets with no SDP. Housing pigs in a CE increased the baseline concentrations of ACTH and cortisol, and when coupled with an LPS challenge, resulted in elevated serum and tissue mRNA levels of cytokines. Cold stress and feeding SDP during a LPS challenge may result in increased stress and immune responses in young pigs.