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Injury to endothelial cells (ECs) often results in cell retraction and gap formation. When caused by antigen aggregation or complement, this injury can be prevented by pretreatment of the ECs with IL-4, suggesting that IL-4 modifies the intercellular junction. Therefore, we investigated the effects of IL-4 on expression of intercellular junction proteins and whether such effects are required for IL-4-induced resistance of ECs against complement-mediated injury. We found that IL-4 induces up-regulation of the junction protein claudin-5 in porcine ECs through activation of Jak/STAT6 and phosphorylation and translocation of FoxO1 from the nucleus to the cytoplasm. Increased claudin-5 expression resulted in increased transmembrane electrical resistance of the endothelial monolayer and participated in IL-4-induced protection of the ECs from complement injury. Down-regulation of FoxO1 using siRNA by itself caused up-regulation of claudin-5 expression and partial protection from cytotoxicity. This protection was enhanced by stimulation with IL-4. We previously reported that increased phospholipid synthesis and mitochondrial protection were required for IL-4-induced resistance of ECs against complement injury and now we demonstrate a contribution of claudin-5 expression in IL-4-induced protection.

Background Despite ongoing advances in treatment, thousands of patients still die annually from complications due to hemorrhagic shock, a condition causing dramatic physiologic and metabolic changes as cells switch to anaerobic metabolism in response to oxygen deprivation. As the shift from aerobic to anaerobic metabolism occurs in the peripheral tissues during shock, the liver must increase production of endogenous glucose as well as process excess lactate produced in the periphery. This places the liver at the center of metabolic regulation in the body during hemorrhagic shock. Therefore, we hypothesized that liver tissue from pigs during an in vivo model of hemorrhagic shock ( n = 6) would reflect resultant metabolic changes. Materials and Methods The in vivo model of shock consisted of 45 min of shock followed by 8 h of hypotensive resuscitation (80 mmHg) and subsequent normotensive resuscitation (90 mmHg) ending 48 h after the shock period. Control groups of pigs ( n = 3) (1) shock with no resuscitation, and (2) only anesthesia and instrumentation, also were included. Metabolic changes within the liver after shock and during resuscitation were investigated using both proton ( 1 H) and phosphorous ( 31 P) nuclear magnetic resonance (NMR) spectroscopy. Results Concentrations of glycerylphosphorylcholine (GPC) and glycerylphosphorylethanolamine (GPE) were significantly lower at 8 h after shock, with recovery to baseline by 23 and 48 h after shock. Uridine diphosphate-glucose (UDP-glucose), and phosphoenolpyruvate (PEP) were elevated 23 h after shock. Conclusions These results indicate that 1 H and 31 P NMR spectroscopy can be used to identify differences in liver metabolites in an in vivo model of hemorrhagic shock, indicating that metabolomic analysis can be used to elucidate biochemical events occurring during this complex disease process.

Introduction Hyaluronan (HA) and its biosynthetic enzymes hyaluronan synthases (HAS2 and HAS3) mediate Matrigel invasion by SW620 colon carcinoma cells. Because matrix metalloproteinases (MMPs) have been implicated in cancer invasion, we hypothesized that changes in HAS expression would alter MMP expression and activity in these cells. Methods To determine whether an MMP was involved in invasion, Matrigel invasion assays with SW620 cells were performed in the presence or absence of the MMP inhibitors GM6001 or TIMP2. HAS isozymes were inhibited by stably transfecting SW620 cells with vectors that contained antisense HAS2 and/or -3 cDNA; transfection with an empty vector served as a control. MMP-7 transcription was assessed by quantitative reverse transcription polymerase chain reaction (RT-PCR). MMP-7 protein was detected by enzyme-linked immunosorbent assay (ELISA) and enzymatic activity compared using zymography. Results GM6001 and TIMP2 decreased Matrigel invasion, which confirms that an MMP played a key role in this process. MMP-7 expression was then detected in SW620 cells. Finally, MMP-7 expression, protein, and enzymatic activity were significantly lower in antisense HAS tranfectants than in SW620 or vector control cells. Conclusion We have demonstrated previously that inhibition of HAS expression and HA production in SW620 colon carcinoma cells inhibits Matrigel invasion. In the studies presented here, we have demonstrated that SW620 cells express high levels of MMP-7 and that inhibition of HAS isozymes dramatically decreases MMP-7 expression, protein, and enzymatic activity. Taken together, these findings suggest that HAS and HA may mediate cellular invasion via changes in MMP-7 expression.

Background and Aims: Acute pancreatitis is an inflammatory disease involving acinar cell injury, and the rapid production and release of inflammatory cytokines, which play a dominant role in local pancreatic inflammation and systemic complications. Toll-like receptor 4 (TLR4) initiates a complex signalling pathway when it interacts with lipopolysaccharide (LPS), which ultimately results in a proinflammatory response. We hypothesised that TLR4 is important in the pathophysiology of acute pancreatitis, independently of LPS. Using two different models of acute pancreatitis, we investigated how genetic deletion of TLR4 or its co-receptor CD14 effects its progression and severity. Methods: We induced acute pancreatitis by administering either caerulein or l-arginine to wild-type, TLR4 −/− , and CD14 −/− mice. Control mice received normal saline injections. The severity of acute pancreatitis was determined by measuring serum amylase activity, quantifying myeloperoxidase (MPO) activity in the pancreatic tissue, and histologically assessing acinar cell injury. Results: It was found that administering caerulein and l-arginine to wild-type mice resulted in acute pancreatitis (as assessed by hyperamylasaemia, oedema, increased pancreatic MPO activity, and pancreatic necrosis) and associated lung injury. The same treatment to TLR4 −/− or CD14 −/− mice resulted in significantly less severe acute pancreatitis, and reduced lung injury. We found no evidence of either bacteria or LPS in the blood or in pancreatic tissue. Conclusions: The severity of acute pancreatitis is ameliorated in mice that lack either TLR4 or CD14 receptors. Furthermore, these results indicate that TLR4 plays a significant pro-inflammatory role independently of LPS in the progression of acute pancreatitis.

Steatotic mice are particularly susceptible to hepatic ischemia/reperfusion injury compared with their lean littermates. We have previously demonstrated that livers of mice having a spontaneous mutation in the leptin gene (ob/ob), resulting in global obesity and liver steatosis, are ATP depleted, are endotoxin sensitive, and do not survive (I/R) injury. We hypothesize that administration of an anti-LPS monoclonal antibody (mAb) prior to initiation of I/R would be protective from that insult. Steatotic mice (ob/ob) were subjected to 15 min of ischemia via complete porta-hepatis occlusion and varying lengths of reperfusion with or without pre-treatment with an anti-LPS mAb. There was 14-31% survival of isotype matched control mAb treated ob/ob mice after 15 min of ischemia and 24 h of reperfusion. In contrast, 75-83% of ob/ob mice pre-treated with an anti-LPS mAb prior to initiation of I/R survived both ischemia and 24 h of reperfusion. Furthermore, there was a decrease in ALT and circulating endotoxin levels when treated with an anti-LPS mAb compared with control antibodies. Attenuation of the endotoxin load with anti-LPS mAb, prior to initiation of I/R, was cytoprotective and improved survival. Consequently, these studies might offer a solution to the problems associated with using steatotic livers in clinical transplantation.

Injury to endothelial cells (ECs) often results in cell retraction and gap formation. When caused by antigen aggregation or complement, this injury can be prevented by pretreatment of the ECs with IL-4, suggesting that IL-4 modifies the intercellular junction. Therefore, we investigated the effects of IL-4 on expression of intercellular junction proteins and whether such effects are required for IL-4-induced resistance of ECs against complement-mediated injury. We found that IL-4 induces upregulation of the junction protein claudin-5 in porcine ECs through activation of Jak/STAT6 and phosphorylation and translocation of FoxO1 from the nucleus to the cytoplasm. Increased claudin-5 expression resulted in increased transmembrane electrical resistance of the endothelial monolayer and participated in IL-4-induced protection of the ECs from complement injury. Down-regulation of FoxO1 using siRNA by itself caused up-regulation of claudin-5 expression and partial protection from cytotoxicity. This protection was enhanced by stimulation with IL-4. We previously reported that increased phospholipid synthesis and mitochondrial protection were required for IL-4-induced resistance of ECs against complement injury and now we demonstrate a contribution of claudin-5 expression in IL-4-induced protection.

This chapter focuses on the ethical principles that have been established and applied to the field of biomedical research and development. At the conclusion of this chapter, we describe a pathway for the development of a new prosthetic heart valve which will be used to illustrate the application of biomedical ethics as it applies today.

The purpose of using animal models in preclinical evaluation of biomaterials is to provide a preliminary assessment of human safety and efficacy relative to the manufacturer claims and comparable technology that can be used as an appropriate control standard. The development of new technologies, including in vitro methods and computer modeling, that has occurred in the last few decades has enhanced the approach to the study of physiology. However, the use of in vivo models still remains a necessity in investigations into certain physiologic phenomena, especially those related to the pathophysiologic mechanisms of disease, as in vitro studies are limited to the assessment of durability of the test material and hemodynamic performance. In this chapter, we will focus on three commonly used animal models for in vivo biomaterials research and testing in cardiac and vascular surgery: pigs; cows; and sheep. The chapter begins with current recommendations for in vivo preclinical evaluation of cardiac and vascular devices and continues with a short discussion on responsible use of animals in the assessment of biomaterials and biomedical devices, followed by specific considerations and examples of existing animal models for each species. Finally, we discuss conventionally-placed devices and percutaneously-placed devices, as well as potential future directions.

Background Hemorrhagic shock can lead to multiple organ failure and death. We have previously shown that noninvasive measurement of tissue oxygen saturation (StO 2 ) has predictive value for outcomes in patients suffering hemorrhagic shock. Our study objectives were twofold: (1) to compare invasive and noninvasive measurements of local and systemic tissue hemoglobin oxygenation and (2) to compare the effects of various physiologic conditions seen in patients in hemorrhagic shock on tissue hemoglobin oxygenation. Materials and Methods We studied pigs in controlled conditions mimicking shock induced by one of the following: hypothermia, isovolemic hemodilution, or manipulations of vascular tone. We obtained both invasive and noninvasive measurements in a hind limb of StO 2 , tissue hemoglobin index, femoral artery and venous flows, blood pressures, temperature, pH, pO 2 , pCO 2 , oxygen saturation, lactate, hemoglobin, and base excess. In all cases, we measured baseline values in both experimental and control hind limbs. Results We found that tissue hemoglobin oxygenation did not vary significantly over relevant physiologic temperatures. Under all physiologic conditions tested, we found supply-dependent oxygen consumption at oxygen levels less than 7 mL O 2 /min/kg. Similarly, we found that local oxygen delivery in animals subjected to varying degrees of isovolemic hemodilution or altered vascular tone was correlated with supply-dependent oxygen consumption, as measured by local noninvasive StO 2 . Conclusions Noninvasive StO 2 measurements are valid and durable over a wide range of physiologic conditions and correlate with invasively-measured oxygen delivery.

Hyaluronan mediates growth of SW620 colon cancer cells. Because hyaluronan is the active ingredient in Seprafilm, we hypothesized that Seprafilm would affect intraperitoneal tumor growth in a mouse model of peritoneal seeding.Immunodeficient mice underwent laparotomy and intraperitoneal inoculation of 10(5) SW620 cells. Seprafilm (n = 22), Vicryl mesh (foreign body control; n = 24), or no material (sham; n = 19) was placed under the incision. Mice were killed after four weeks and tumors were dissected, counted, and weighed.Ninety-five percent of mice in the sham group and 96 percent in the Vicryl group developed intraperitoneal tumors. In contrast, only 64 percent of mice in the Seprafilm group developed tumors (P = 0.024), and these tumors were smaller than those in the sham group; (Seprafilm = 42 +/- 9 mg vs. sham = 82 +/- 17 mg; P = 0.05). In contrast, tumors in the Vicryl group were dramatically larger (349 +/- 49 mg; P0.001 vs. sham or Seprafilm).Despite previous data that suggested that hyaluronan increases colon cancer cell growth, we found that Seprafilm decreased tumor formation and tended to decrease size in this model. In contrast, Vicryl mesh increased tumor formation and size. Our results suggest that Seprafilm does not promote intraperitoneal tumor growth, especially compared with Vicryl mesh.

Toll-like receptor 4 (TLR4) mediates the innate immune response to lipopolysaccharide (LPS) by a complex intracellular signaling pathway that activates the transcription factor nuclear factor (NF)-kappaB, with subsequent production of inflammatory cytokines. The precise mechanisms involved have not been delineated. We hypothesized that specific regulatory elements exist within the TLR4 promoter.We cloned regions of the murine TLR4 promoter (0.25 kb to 2 kb; -2000 bp to +244 bp) into the pGL3-Basic plasmid containing the firefly luciferase gene and used the resulting constructs to transfect HEK293 or RAW 264.7 cells. After 24 h, we used LPS to stimulate RAW 264.7 cells. We quantified relative light units (RLUs) of cell lysates and secreted tumor necrosis factor (TNF)-alpha. For pairwise comparison, we used Student's t-test. Sequence analysis of the promoter revealed several putative Sp1 sites. We used two constructs showing increased promoter activity, TLR4-750/-1 and TLR4-500/-1, to transfect cells in the presence of a specific Sp1 inhibitor, mithramycin A.Of the six promoter constructs, four showed greater transcriptional activity (p0.05 vs. pGL3-Basic), as measured by luciferase activity. However, we did not observe differences in transcriptional activity of the promoter in five of those six constructs when we stimulated transfected RAW 264.7 cells with 10 ng of LPS. This finding suggests that transcriptional regulation of the promoter is unaffected by cellular changes caused by LPS. Both TLR4-750/-1 and TLR4-500/-1 showed dose-dependent reductions in transcriptional activity in the presence of increasing concentrations of the specific Sp1 inhibitor mithramycin A in both HEK293 and RAW 264.7 cells (p0.05 vs. no mithramycin A).At least one Sp1 transcriptional regulatory element is present within the murine TLR4 promoter (range -750 bp to -250 bp). This finding holds promise for manipulating this fundamental inflammatory response.

Limulus anti-lipopolysaccharide (LPS) factor (LALF) is a 102-amino acid LPS-binding protein from the horseshoe crab, Limulus polyphemus. The peptide includes the LPS-binding domain of holoLALF, yet it lacks the loop structure stabilized by disulfide or other covalent bonds that is a common motif in the LPS-binding regions of holo- LALF and several other LPS-binding proteins. Although it neutralizes LPS and is bactericidal against Pseudomonas aeruginosa, the LALF 28-54 portion of LALF is not protective in a murine model of intraperitoneal sepsis compared with holoLALF. We examined the effects of cyclizing this linear peptide to determine if this action would recapitulate the stable loop-type structure and enhance its LPS-neutralization and bactericidal activity in vitro.Cyclic LALF 28-54 was produced by oxidizing linear LALF 28-54. Each peptide, along with appropriate controls, was assayed for LPS neutralization using the chromogenic Limulus amebocyte lysate (LAL) assay and a bioassay to measure inhibition of LPS-stimulated production of tumor necrosis factor-alpha (TNF-alpha) by murine macrophages. Bactericidal activity against Pseudomonas aeruginosa was also assessed. Data were analyzed using a two-tailed Student t-test.Polymyxin B and holoLALF exhibited potent endotoxin antagonism in both assays, as well as bactericidal activity. Concordant with prior studies, the linear form of LALF 28-54 exhibited either similar or a slightly lesser degree of activity in all assays. However, cyclization was associated with significantly diminished endotoxin neutralization in both assays (p0.05) and decreased bactericidal activity (p0.05) compared with linear LALF 28-54.Whereas a synthetic linear peptide based on the endotoxin-binding region of holoLALF retained activity, cyclization was associated with a diminution in potency in vitro. We postulate that cyclization does not constrain the peptide in a manner that recreates the loop structure necessary for potent endotoxin antagonism.

Interaction between lipopolysaccharide (LPS), LPS-binding protein, and the CD14 receptor at the surface of LPS-responsive cells results in inflammatory cytokine release and internalization and detoxification of LPS. Monoclonal antibodies (mAbs) raised against the deep-core lipid A or the O-linked polysaccharide moieties of LPS accelerate internalization and detoxification of LPS without stimulating cytokine release. This study was conducted to test the hypothesis that the antibody-mediated internalization of LPS is an Fc receptor (FcR)--mediated process.Fluoroisothiocyanate (FITC)-conjugated Escherichia coli O111:B4 LPS was incubated with RAW 264.7 cells and allowed to internalize for 2 hours in the presence and absence of anti-LPS, anti-CD14, and isotype control mAbs, and Fab fragments from the anti-CD14, anti--Fc receptor, and control mAbs. Tumor necrosis factor--alpha (TNF-alpha) release was measured by WEHI 164 cell bioassay. FITC-LPS uptake was measured by flow cytometry. Statistical analysis was by analysis of variance and Fisher exact test.Addition of anti-LPS antibodies resulted in a 30- to 40-fold acceleration of LPS internalization (P.01) in agreement with previous studies. This increase was blunted by anti-CD14 and also by isotype control holo-antibody (P.01), but not by Fab fragments from anti-CD14 or isotype control antibody. Both anti-FcR antibodies and Fab fragments blocked anti-LPS antibody--stimulated uptake of FITC-LPS. Both intact anti-CD14 holo-antibody and Fab fragments blocked TNF-alpha release (P.01).Clearance and detoxification of LPS are thought to be essential to the host response to endotoxin. It has been shown that antibodies to LPS accelerate its internalization by monocytic cell lines without increasing the elaboration of cytokines. We found that specific blockade of CD14 by Fab fragments could block TNF-alpha release but not alter the accelerated internalization of LPS produced by anti-LPS antibodies. In contrast, a nonspecific blockade of internalization was produced by competing antibody, which suggests a mechanistic role for the FcR. Specific blockade of FcR by either holo-antibody or Fab fragments blocked accelerated internalization, which confirms a FcR mechanism. We conclude that the accelerated internalization of LPS produced by anti-LPS antibody is an Fc receptor--mediated process. These results have significance for the development of adjuvant immunotherapy for gram-negative bacterial sepsis.

The deep core/lipid A (DCLA) region of gram-negative bacterial lipopolysaccharide (LPS) is common to most gram-negative pathogens and contains anionic phosphoryl groups plus numerous acyl chains as part of the toxic lipid A moiety. Several disparate agents that antagonize the effects of LPS exhibit extensive physicochemical similarities (hydrophobicity, cationic charge) within their binding domains. It is presumed that binding to the DCLA region by each of these antagonists-cross-reactive anti-LPS monoclonal antibodies (mAbs), polymyxin B (PmB), plus bactericidal permeability-increasing protein (BPI) and Limulus anti-LPS factor (LALF)-may be related to these properties. Therefore, we hypothesized that in addition to secondary and tertiary protein conformation, electrostatic interactions involving the negatively charged phosphoryl groups, hydrophobic interactions involving the acyl chains of lipid A, or both might be important factors that promote LPS antagonism. Binding of PmB, BPI, LALF, or anti-DCLA mAb 1B6 to Salmonella minnesota monophosphoryl lipid A (MPLA), diphosphoryl lipid A (DPLA), and Salmonella minnesota Re (which possess a common structural moiety, but vary considerably in structure and charge) was examined. Highly phosphorylated DNA and bovine serum albumin served as unrelated structural controls. BPI bound MPLA, which is hydrophobic and minimally charged, while mAb 1B6 bound anionic DNA; neither PmB nor LALF were reactive with MPLA or DNA. We surmised that hydrophobic interactions play a role in BPI binding to LPS, and although electrostatic interactions appear to be important for binding of mAb 1B6 to DCLA, they may not contribute to as great an extent for PmB, BPI, or LALF. Thus our data support the contention that the contribution of these specific physicochemical factors varies among endotoxin antagonists.

Release of lipopolysaccharide (endotoxin, LPS) is a critical inciting event in the development of sepsis syndrome due to gram-negative bacteria, and mortality associated with this entity remains approximately 40%. Limulus anti-LPS factor (LALF) is a naturally occurring horseshoe crab derived protein that, unlike antibiotics, is both bactericidal for gram-negative bacteria and capable of neutralizing LPS. We hypothesized that a peptide derived from the active domain of LALF (LALF #28-54) would exhibit potent biologic activity similar to that of LALF itself and could potentially be useful as a therapeutic agent.The effects of LALF, synthetic peptide LALF #28-54, polymyxin B (PmB), and a biologically inactive synthetic peptide were examined in several models. In vitro bactericidal activity was determined against Pseudomonas aeruginosa, and LPS-neutralizing capacity was determined via inhibition of LPS-induced tumor necrosis factor-alpha (TNF-alpha) secretion by RAW 264.7 cells. In vivo biologic activity was determined via pretreatment following which P aeruginosa endotoxemia or bacteremia was induced; serum TNF-alpha levels, bacterial clearance, and survival were assessed.LALF and LALF #28-54 exhibited potent in vitro bactericidal and LPS-neutralizing activity comparable to PmB (P.01). However, although LALF #28-54 diminished systemic TNF-alpha production and aided bacterial clearance similar to that observed for LALF (P.01), it did not provide significant protective capacity (P.1).These data demonstrate that peptide LALF #28-54 retained the LPS-neutralizing and bactericidal biologic activity of LALF but failed to protect during overwhelming P aeruginosa bacteremia, perhaps due to short serum half-life.

The purpose of this study was to determine whether antiendotoxin agents exhibit molecular homology within their lipopolysaccharide (LPS) binding domains, suggesting a common mechanism of action. We hypothesized that the presence of positively charged basic amino acids or a paucity of negatively charged acidic amino acids, or both, would be a critical characteristic of that portion of the molecule that binds to the highly negatively charged deep core/lipid A (DCLA) region of LPS.We analyzed the amino acid sequences of the variable light (VL) and heavy (VH) chain complementarity-determining regions (CDRs) of anti-DCLA monoclonal antibodies (mAbs) 1B6, 5A5, and 7C5 and compared them with (1) the CDRs of three irrelevant control mAbs and (2) the LPS binding region of bactericidal permeability-increasing protein (BPI). We purified and amplified the specific nucleotide sequences of the variable regions using reverse transcriptase polymerase chain reaction. DNA was sequenced by dideoxy termination, and protein sequences were deduced and analyzed. The percentages of acidic, basic, polar, and hydrophobic amino acids within VH and VL chain CDRs were determined.We identified a paucity of negatively charged acidic amino acids exclusively within VL chain CDRs of anti-DCLA mAbs (P0.005). Although increased, the number of positively charged basic residues was not statistically significantly different; neither was the number of polar or hydrophobic amino acids. Conclusions. Our data suggest that the near absence of negatively charged acidic residues is critical for LPS binding. This characteristic appears to reside exclusively in the VL chain CDRs of anti-DCLA mAbs.

Background. Bactericidal permeability increasing protein (BPI) binds to and neutralizes lipopolysaccharide (LPS, endotoxin). Small synthetic peptides based on the amino add sequence of the LPS binding domain of BPI neutralize LPS, albeit inefficiently. Although the LPS binding domain of native BPI possesses a β-turn secondary structure, this structure is not present in small derivative peptides. The purpose of this study was to determine whether the addition of a β-turn to a BPI-derived peptide is associated with more potent endotoxin antagonism. Methods. We generated a hybrid peptide (BU3) on the basis of (1) a portion of the LPS binding domain from BPI and (2) amino acids known to initiate a β-turn. BU3 folds with a β-turn, and we tested its effects on LPS neutralization and LPS-induced tumor necrosis factor-α secretion, comparing it with BPI-derived peptide BG22 that lacks a β-turn and to an irrelevant peptide (BG16). Results. Compared with BG22, BU3 demonstrated enhanced LPS neutralization and inhibition of LPS-induced tumor necrosis factor-α secretion in vitro and a similar diminution of endotoxemia and tumor necrosis factor-α secretion in a murine model of endotoxemia. Conclusions. These data demonstrate the potential for enhancing the biologic activity of a BPI-derived peptide endotoxin antagonist via manipulation of its conformational structure.

Introduction: Lipopolysaccharide-binding protein (LBP) is a serum acute phase protein that catalyzes the inflammatory response to lipopolysaccharide (LPS, endotoxin). Loss of the carboxyl-terminus of LBP ablates the catalysis of pro-inflammatory signaling. This study was designed to further localize the catalytic activity of LBP by direct mutational analysis. Methods: Murine LBP cDNA was cloned and successive deletions of the C-terminus were obtained and expressed in COS-7 cells. The ability of secreted LBP protein constructs to catalyze LPS-responsiveness was determined by incubating lysate with P.aeruginosa LPS and RAW 264.7 macrophages, quantifying TNF-α release by WEHI cell bioassay. Statistical analysis was by ANOVA and Fisher’s Exact test. Results: Both purified and expressed full-length LBP (LBP-1446) catalyzed the production of TNF-α by 4–5 fold in response to LPS compared to LPS alone, while the deletion construct LBP-1327 catalyzed TNF-α production by 1.5–2 fold (p Download : Download high-res image (43KB) Download : Download full-size image

Introduction: The endotoxin-binding domain of Limulus anti-lipopolysaccharide factor (LALF 31–52) contains eight positively-charged amino acids. We hypothesized that adding more positively-charged residues to this domain would further enhance its anti-endotoxin properties. Methods: A series of peptides (PV11 to PV42) was created based upon the endotoxin-binding region of LALF (PV00). Neutral or negatively-charged amino acids were sequentially replaced with one to four positively-charged (lysine+1) residues at various positions. The peptides were then tested for LPS neutralization by two distinct assays and for bactericidal activity against P. aeruginosa. Results for each peptide were compared to the native sequence using Student’s t-test. Results: Most peptides with at least two additional lysine residues displayed significantly increased bactericidal activity versus the native peptide sequence (p < 0.05; left graph). Many lysine-addition peptides also demonstrated significantly increased activity versus the native peptide in a Limulus amebocyte lysate (LAL) endotoxin neutralization assay (p < 0.05; right graph). In both assays, the maximum increase resulted from the addition of three lysine residues to the amino-terminus of the domain (PV31, PV32). These peptides also showed significantly better endotoxin neutralization than the native peptide as exhibited by inhibition of TNF-α production by murine macrophages (p < 0.05; not shown). Conclusions: The addition of positively-charged amino acids to the LALF endotoxin-binding region augments its ability to antagonize endotoxin in vitro. This effect is most pronounced when three additions are made at the amino-terminus of the endotoxin-binding domain. Download high-res image (127KB) Download full-size image

Introduction: The protein TIRAP (Toll-Interleukin Receptor Adapter Protein) interacts with Toll-like receptor-4 (TLR-4) and MyD88 (Myeloid Differentiation protein 88). Since Toll-like receptors produce varying transcriptional responses via MyD88, TIRAP may provide specificity to TLR-4 signaling. The carboxyl portion of TIRAP has been implicated as the region which may modulate TLR-4 signaling. The precise molecular mechanism has not been delineated. We sought to identify the domain critical to TLR-4 signaling using a series of TIRAP deletion mutants. Methods: Multiple mutant DNA sequences were created by progressively shortening the carboxyl terminus of TIRAP. Mutants not containing the critical signaling region were expected to abrogate TLR-4 signaling by competitively inhibiting endogenous TIRAP. Mutant sequences were ligated into the pIRES2-EGFP vector and transfected into RAW 264.7 macrophages. The cells were co-transfected with a luciferase reporter vector containing the tumor necrosis factor-alpha promoter region and a Rinella luciferase vector to normalize for transfection variability. Cells were stimulated with endotoxin and assayed for luciferase production. Results: Macrophages transfected with the TIRAP sequence displayed a potent response to endotoxin stimulation (10.78 normalized luciferase units). All mutants of TIRAP demonstrated an abrogated response to endotoxin stimulation (5.55 − 6.82 NLU; p Conclusions: The terminal region of TIRAP appears critical to TLR-4 signaling. Mutants missing the carboxyl terminus cannot induce a full response to endotoxin. These results help to identify the domain of TIRAP which may provide specificity to MyD88-dependent TLR-4 signaling.