Mechanism of alternative complement pathway dysregulation by a phosphorothioate oligonucleotide in monkey and human serum.

The species sensitivity and mechanism of complement pathway activation by a phosphorothioate oligonucleotide were investigated in monkey and human serum. Increasing concentrations of a phosphorothioate oligonucleotide, ISIS 2302, were incubated in either monkey or human serum. Complement activation in monkey serum was selective for the alternative pathway and occurred at concentrations ≥ 50 µg/mL ISIS 2302. By comparison, complement activation in human serum was absent. A similar difference in sensitivity for activation was also observed for a representative 2'-methoxyethyl (MOE)-modified oligonucleotide. The absence of oligonucleotide-induced complement activation was also observed in dogs. Protein binding with ISIS 2302 and enzyme competition studies suggested that factor H was important in oligonucleotide-mediated complement activation process, and addition of factor H to serum effectively prevented the activation in monkey serum. Furthermore, based on the immunoassay for factor H, there was an apparent decrease in factor H concentration as the ISIS 2302 concentration increased. This result suggests that ISIS 2302 binds to factor H and interferes with the factor H antibody from the immunoassay. Factor H is a regulatory protein that limits alternative pathway activation. Disruption of factor H interaction with C3 convertase by oligonucleotide could promote activation in this pathway.

Dietary aspartyl-phenylalanine-1-methyl ester delays osteoarthritis and prevents associated bone loss in STR/ORT mice.

OBJECTIVE: STR/ORT mice provide a well-known model for murine idiopathic OA, with histological joint lesions resembling those of human OA. This model was used to investigate protective effects of the dipeptide aspartyl-phenylalanine-1-methyl ester (Asp-Phe-OMe or aspartame) via the oral route vs a regular diet. METHODS: STR/ORT mice were housed individually and fed diets with or without Asp-Phe-OMe (4 mg/kg), after weaning at the age of 3 weeks, until 15 months of age (average of 20 animals per group). The study groups were kept blinded to the investigators, who measured food consumption and body weight and performed gait mobility tests. Radiographic scans were also performed at regular time intervals to evaluate differential radiographic anomalies associated with progress of OA in response to oral Asp-Phe-OMe therapy. RESULTS: The Asp-Phe-OMe-fed animals presented a pattern of significantly delayed disease onset. In addition, their muscle and bone mass were highly preserved, even at later time points after OA was established. Moreover, control animals presented a higher variability in gait motility in comparison with the Asp-Phe-OMe-fed animals, suggesting a protective effect from movement limitations associated with advanced OA. CONCLUSION: Asp-Phe-OMe, given orally, delays OA in the spontaneous STR/ORT model, improves bone cortical density and muscle mass, and may contribute to a better quality of life for these diseased animals.

Pancreatic enzymes generate cytotoxic mediators in the intestine.

Recent evidence indicates that shock is accompanied by a failure of the mucosal barrier in the intestine and entry of pancreatic digestive enzymes into the wall of the intestine. To investigate the formation of cytotoxic mediators produced by enzymatic digestion of the intestine, we applied homogenates of rat small intestinal wall to human neutrophils and used flow cytometry measurements of propidium iodide uptake to determine cytotoxicity. We show that homogenates of the small intestine after ischemia by occlusion of the superior mesenteric and celiac arteries for 3 h, but not without ischemia, are cytotoxic. Digestion of homogenates of nonischemic intestinal wall with purified trypsin, chymotrypsin, or elastase, proteases normally present in the intestinal lumen, yielded cytotoxic mediators. Before cell death, we saw cell damage in the form of bleb formation and flow cytometry measurements of cell size changes due to blebbing. Cytotoxicity was prevented by serine protease inhibition with phenylmethylsulfonyl fluoride (PMSF) before, but not after proteolytic digestion of the wall homogenates, indicating that enzymatic action of proteases on the homogenate is necessary for cytotoxicity. Cytotoxicity of wall homogenates digested by enzymes in the fluid collected from the lumen of the intestine was greater than digests by the individual purified proteases. Cytotoxicity is undetectable if digestive enzymes in the luminal fluid are inhibited with a combination of enzyme inhibitors PMSF and 6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfonate before addition of wall homogenates. Passage of digested intestinal wall homogenates across a hydrophobic glass-fiber filter reduced cytotoxicity. Furthermore, we found that luminal fluid itself may be cytotoxic, possibly because of digestion of ingested food. To test whether digested food can be cytotoxic, we homogenized rat food and digested it in vitro with chymotrypsin or endogenous enzymes in luminal fluid. Cytotoxicity was significantly increased after digestion of food by luminal fluid compared with luminal fluid or undigested food. These results indicate the presence of a previously unknown mechanism for hemorrhagic necrosis in shock.

Intraluminal pancreatic serine protease activity, mucosal permeability, and shock: a review.

Shock states are characterized by a pronounced activation of numerous cell types that lead to an acute inflammatory reaction. The exact mechanism by which these inflammatory cells are activated is not known. Numerous studies have implicated the gastrointestinal tract as one of the main sites for the generation of inflammatory mediators and initiation of an acute systemic response. The pancreas is known to secrete powerful digestive enzymes, and we hypothesize that they may play a leading role in the pathogenesis of multiorgan failure after the onset of shock. We carried out a search in PubMed for all relevant studies related to the role of the pancreas in shock. Studies that included information concerning the role of pancreatic enzymes in shock were then summarized. Our article serves to review the current hypotheses on how digestive enzymes produced by the pancreas may play a pivotal role in initiating the systemic inflammatory response. We further hypothesize how these enzymes and/or their products may ultimately contribute to multiorgan failure and death.

Contribution of anaphylatoxins to allergic inflammation in human lungs.

The contribution of complement activation to allergic asthma remains controversial. In order to elucidate the role played by the complement split products, anaphylatoxins C3a and C5a, we evaluated their effects on production of cysteinyl-leukotrienes (cysLTs) by human lung fragments following an anaphylactic reaction. The lung tissues obtained from two patients with lung cancer showed C5aR-, C5L2R-, and C3aR-mRNA expression. When the chopped lung fragments passively sensitized with human IgE were incubated with anti-human IgE antibody, a significant amount of cysLTs was generated in comparison with the control (without anti-IgE antibody). The co-addition of human C5a at doses of 0.1 to 10 ng/ml to the anti-IgE antibody potentiated cysLT production. The response was bell-shaped in distribution, significant, and peaked at a C5a concentration of 1 ng/ml. The co-addition of human C3a up to 1,000 ng/ml seemed to increase cysLT production, but not to any significant extent. A novel C5a receptor complementary peptide, acetylated peptide A, dose-dependently inhibited cysLT production by the human lung fragments following the anaphylactic reaction in the presence of 1 ng/ml C5a. However, this peptide did not inhibit cysLT production in the presence of 100 ng/ml C3a. It is suggested that the anaphylatoxin C5a potentiates cysLT production in human lung tissues and contributes to allergic inflammation in disorders such as asthma, thus acetylated peptide A may be useful for suppressing allergic inflammation in the lungs.

A new hypothesis for microvascular inflammation in shock and multiorgan failure: self-digestion by pancreatic enzymes.

Shock is accompanied by a severe inflammatory cascade in the microcirculation, the origin of which has been hypothesized in the past to be associated with specific mediators such as endotoxin, oxygen free radicals, nitric oxide, cytokines, and lipid products. But no intervention with clinical effectiveness has been derived from these ideas to date. The authors propose here a new hypothesis suggesting that degradative enzymes, synthesized in the pancreas as part of normal digestion, may play a central role in shock and multiorgan failure. These powerful enzymes have the ability to digest almost every biological material. Self-digestion (i.e. autodegradation) is prevented by compartmentalizing the fully activated degradative enzymes in the intestinal lumen by the mucosal barrier. In shock, maintenance of the mucosal barrier is impaired and it becomes permeable to pancreatic enzymes. Digestive enzymes thereby gain access to the wall of the intestine and initiate self-digestion of submucosal extracellular matrix proteins and interstitial cells. The process leads to generation and release of a host of strong inflammatory mediators. The authors hypothesize that inhibition of pancreatic enzymes in the lumen of tile intestine can serve to attenuate formation of these inflammatory mediators in ischemic tissues following hemorrhagic shock, and consequently prevent cell and tissue injury as well as multiorgan failure.

Characterization of two classes of pancreatic shock factors: functional differences exhibited by hydrophilic and hydrophobic shock factors.

Pancreatic tissue homogenate induces a powerful pathophysiologic response sufficient to produce lethal shock in a rat. However, limited progress has been made in the biochemical characterization of these pancreas-derived active factors or their mechanisms of action. It has been shown that the pancreas is a major source of these shock-inducing factors and that they are generated by pancreatic proteinases. Porcine pancreas was homogenized and the filtered homogenate was subjected to organic extraction both before and after incubation for 2.5 h at 37 degrees C. The aqueous and lipid extracts of pancreatic homogenates were collected and analyzed for their ability to activate human neutrophils and to induce lethal shock in the rat. Neutrophil activation, a presumed hallmark of shock, was determined by chemiluminescence and myeloperoxidase (MPO) release. Only the intact homogenate and lipid extracts stimulated the neutrophils, and the aqueous extracts proved to be inactive. Neutrophils exhibited enhanced cellular activation when exposed to substimulatory levels of either formyl-methionyl-leucyl-phenylalanine (FMLP) or platelet-activating factor (PAF) followed by substimulatory levels of the lipid extracts, but not by the aqueous extracts. Both the lipid and aqueous extracts induced dramatic decreases in heart rate and blood pressure when injected in the rat, often resulting in lethal shock. In all cases, incubation of the homogenates at 37 degrees C enhanced the potency of the extracts. Our results demonstrated that the pancreas-derived homogenate and lipid factors were capable of inducing both neutrophil activation and shock. These results support the hypothesis that shock is produced via neutrophils that have been activated by inflammatory components. However, the shock-inducing factors in the aqueous extracts (i.e., hydrophilic fraction of the homogenate) apparently function via a pathway independent of neutrophil activation. This is the first evidence that there are both hydrophobic and hydrophilic factors generated in tissue homogenates capable of inducing shock, and that these different chemical classes of factors appear to function via separate mechanisms.

Elevated kallikrein activity in plasma from stable liver transplant recipients.

Extensive in vitro conversion of complement components C3 and C4 has been observed in EDTA plasma obtained from a number of stable orthotopic liver transplant recipients (LTR) [Clin. Chem. 45 (1999) 1190]. Consequently, we designed a chromogenic substrate (Ac-Ala-Gly-Leu-Thr-Arg-p-nitroanilide, AGLTR-pNA), based on the C1s cleavage site in complement component C4, in an attempt to identify the plasma proteinase(s) that cleaves C4 in vitro. Average peptidase activity in EDTA plasma obtained from stable LTR (n = 16) was significantly higher (P<0.01) than that in plasma from healthy non-transplant donors (n = 16). This peptidase activity was also detected using commercial substrates designed for specific coagulation proteinases. The plasma proteinase was not inhibited by hirudin, a thrombin inhibitor, but was inhibited by the plasma kallikrein inhibitor D-Phe-Phe-Arg-chloromethylketone, which fails to inhibit C1s. We concluded that the peptidase detected inLTR plasma, using chromogenic substrates including AGLTR-pNA, was plasma kallikrein. Western blot analysis confirmed the presence of kallikrein-alpha-2-macroglobulin complexes (alpha2M) in LTR plasmas. We also demonstrated that kallikrein was not the proteinase responsible for the in vitro cleavage of C4. Elevation of the plasma peptidase activity correlated significantly with recurrent hepatitis C virus (HCV) infection in these liver recipients with a P value <0.02. Significant correlation was not observed between complement activation (i.e. the C4a levels) and recurrent HCV infection (P>0.15); however, C4a levels did correlate with rejection (P<0.02). These results suggest that elevation in plasma peptidase activity and activation of complement do signal different pathological events in LTR, events that appear related to HCV-induced infection and immune tissue injury, respectively.