The (I/Y)XGG motif of adenovirus DNA polymerase affects template DNA binding and the transition from initiation to elongation.

Adenovirus DNA polymerase (Ad pol) is a eukaryotic-type DNA polymerase involved in the catalysis of protein-primed initiation as well as DNA polymerization. The functional significance of the (I/Y)XGG motif, highly conserved among eukaryotic-type DNA polymerases, was analyzed in Ad pol by site-directed mutagenesis of four conserved amino acids. All mutant polymerases could bind primer-template DNA efficiently but were impaired in binding duplex DNA. Three mutant polymerases required higher nucleotide concentrations for effective polymerization and showed higher exonuclease activity on double-stranded DNA. These observations suggest a local destabilization of DNA substrate at the polymerase active site. In agreement with this, the mutant polymerases showed reduced initiation activity and increased K(m)(app) for the initiating nucleotide, dCMP. Interestingly, one mutant polymerase, while capable of elongating on the primer-template DNA, failed to elongate after protein priming. Further investigation of this mutant polymerase showed that polymerization activity decreased after each polymerization step and ceased completely after formation of the precursor terminal protein-trinucleotide (pTP-CAT) initiation intermediate. Our results suggest that residues in the conserved motif (I/Y)XGG in Ad pol are involved in binding the template strand in the polymerase active site and play an important role in the transition from initiation to elongation.

Complement anaphylatoxin C3a and C5a formation in premature children with respiratory distress.

UNLABELLED: Premature children (n = 25) with respiratory distress (RD) were studied regarding complement activation and formation of the anaphylatoxins C3a and C5a. Blood samples were drawn on admission to the paediatric intensive care unit. In 18 of the patients RD was accompanied by other perinatal complications like pneumothorax or intracerebral haemorrhages. Seven of the premature children had RD without such complications. Preterm children with RD and with peri- and postnatal complications such as pneumothorax or intracerebral haemorrhage had increased concentrations in plasma of the anaphylatoxins C3a and C5a compared with preterm children with RD without these complications. There was a positive correlation between the plasma C3a and C5a concentrations in the preterm children. CONCLUSION: The present study indicates that isolated RD will appear without signs of complement activation and that complications like pneumothorax or intracerebral haemorrhages are associated with release of the anaphylatoxins C3a and C5a.

Anti-inflammatory agents inhibit leukocyte accumulation and vascular leakage induced by trypsin and trypsin-digested serum in hamster cheek pouch.

In the present study we investigated the effect of nordihydroguaiaretic acid (NDGA), indomethacin, and cortisone on trypsin-induced acute inflammation in the hamster cheek pouch. Permeability changes, evaluated by fluorescence microscopy after injection of FITC-dextran (MW 150,000), induced by trypsin (2.5 microM) and trypsinated serum (2.5 microM) were significantly suppressed by pretreatment with NDGA (20 mg/kg) and indomethacin (20 mg/kg). Pretreatment with cortisone (40 mg/kg) reduced the permeability changes induced by trypsinated serum but had no significant effect on trypsin-induced leakages. Accumulation of polymorphonuclear leukocytes, as calculated by a whole tissue histological technique, induced by trypsin or trypsinated serum, was significantly reduced by pretreatment with cortisone, NDGA, or indomethacin. These results indicate a role of both cyclooxygenase and lipoxygenase products in trypsin-induced acute inflammation in the hamster cheek pouch.

The immunologic response to thermal injury.

Thermal injury is associated with altered immune defense. Extensive and deep thermal injuries lead to depressed immune defense function with both cellular and humoral defense affected. There is an intricate interaction between various components of the immune system. The altered specific immune response is seen as a depressed ability to produce active rosette-forming cells. Depressed stimulation of lymphocyte proliferation as well as the mixed lymphocyte response have also been recorded following burns. These effects are modulated by the release of kinins, prostaglandins, anaphylatoxins, superoxides, and leukotrienes, all of which can influence the inflammatory response following thermal injury. The humoral immunity is altered as seen by decreased levels of immunoglobulins, activation of complement with release of anaphylatoxins, and formation of membrane attacking complexes leading to inflammation and cytolysis. The immune response to burns is also affected by factors other than this injury, such as nutrition or diseases such as diabetes mellitus or disorders of the lymphoproliferative type. The immune response is also influenced by some drugs used for other reasons such as steroids, chemotherapeutic agents, and topical agents used for burn wound care. The immune reaction to a burn is also influenced by the additive effect of superimposed infections. Removal of injured tissue without the need for extensive transfusion will improve the ability of the burned patients to use their immune defense system in a fruitful way.

Activation of the complement cascade by trypsin.

In twenty-three patients with acute pancreatitis, the plasma levels of immunoreactive trypsin were determined with a RIA method. The patients were divided into groups according to the severity of the disease. Ranson's criteria and the development of multisystem organ failure were used for the classification. Elevated plasma levels of immunoreactive trypsin were found in all groups after admittance. Incubation of fresh human serum and plasma with bovine trypsin in concentrations between 10(-6) and 10(-4) M at 20 degrees C activated the complement cascade. The anaphylatoxins C3a and C5a were determined with a RIA and the terminal complement complex (TCC) with an ELISA method. C3a and C5a were released and TCC was formed. The effect of trypsin on leukocyte activation was determined with a chemiluminescence technique. Trypsin dissolved in saline did not activate the leukocytes. However, serum digested by trypsin-activated leukocytes in a dose-dependent manner. The present study supports the theory that trypsin can activate complement components and results in formation of split products which have potent vascular, and leukocyte activating effects.

Trypsin-induced vascular permeability and leukocyte accumulation in hamster cheek pouch: the role of complement activation.

Trypsin-induced acute inflammation was studied in hamster cheek pouch using intravital microscopy, correlative histology, and electron microscopy. Vascular permeability changes were monitored with intravital fluoroscopy, after intravenous injection of FITC-dextran (Mw 150,000), by counting the number of FITC-dextran leakages around the vessels. The number of extravasated polymorphonuclear leukocytes (PMNLs) was calculated by a histological technique. A dose-dependent increase in the number of FITC-dextran leakages, as well as the number of accumulated PMNLs, was found when trypsin was locally deposited in concentrations of 0.25-2.5 microM (15 microliters during 5 min). Local deposition of autologous serum treated with trypsin at final concentrations of 0.25-2.5 microM caused an increase in vascular permeability as equally pronounced as that of pure trypsin, but only a moderate PMNL accumulation which was not dose dependent. Trypsin at a 25 microM concentration resulted in numerous microbleedings and cessation of flow. The electron microscopy demonstrated inflammatory events (PMNL adhesion, diapedesis, and interstitial infiltration) in all treatment groups but they were more pronounced after trypsin exposure. Trypsin did not cause disintegration, cellular lysis, or increased mast cell degranulation. The permeability changes induced by trypsin (2.5 microM) and trypsinated serum (2.5 microM) were significantly suppressed by the addition of the chelating agent potassium-EDTA to the reaction mixture, indicating a calcium- or magnesium-dependent mechanism. Pretreatment of the animals with cobra venom factor (CVF), by which the plasma C3 concentration was reduced to less than 10%, inhibited the vascular leakages almost completely. The trypsin-induced accumulation of PMNLs was significantly reduced by potassium-EDTA as well as by pretreatment with CVF (P less than 0.01). These findings indicate a central role of complement activation in trypsin-induced acute inflammation in the hamster cheek pouch.

Complement activation and reinfusion of wound drainage blood.

Eighteen patients undergoing total hip replacement (n = 13) or knee arthroplasty (n = 5) due to osteoarthritis or osteoarthrosis were prospectively studied in an investigation of complement activation and anaphylatoxin release in association with reinfusion of aspirated wound blood. Twelve of the patients needed blood transfusions and received an average of 390 +/- 75 ml (+/- SD) of autologous blood within 45 min. Plasma complement components, anaphylatoxins, and inhibitors were studied 1 min before and 15 min after the start of and 15 min after the completion of autologous transfusion. Samples also were taken from the collected blood, before and after passing it through a microporous filter. Blood gases and systemic complement samples were drawn simultaneously. There were no significant changes in systemic complement variables before, during, or after transfusion of autologous blood. However, in the aspirated blood, increased concentration of anaphylatoxins (C3a and C5a) and terminal complement complexes (TCC) were present (P less than 0.001). There were no differences observed between samples drawn before and after filtration of the blood. The concentration of C5 was less in the collected blood than in the systemic blood (P less than 0.05). No changes in blood gases were observed. This study demonstrated that postoperatively salvaged whole blood underwent anaphylatoxin formation and complement activation. However, after reinfusion of this blood, neither systemic complement activation nor clinical complications were observed.

Accumulation of anaphylatoxins and terminal complement complexes in inflammatory fluids.

The presence of anaphylatoxins (C3a and C5a) and terminal complement complexes (TCC) in different inflammatory fluids and plasma was studied in 33 patients. Anaphylatoxins were assayed using a radioimmunoassay technique, and the terminal complement complexes were determined by an ELISA method. Patients with peritoneal (n = 14), pleural (n = 7), pericardial (n = 6) or burn bullae fluid (n = 6) were studied. High C3a and TCC concentrations were found in all these fluids. Elevated C3a and TCC concentrations in inflammatory fluids were found not only in patients with elevated plasma C3a and TCC concentrations, but also in patients with normal plasma levels. No increases in C5a concentration were observed in pleural or burn bullae fluid. In one patient with pericarditis, and in subjects with acute pancreatitis with ascites, high C5a levels were found in the fluid. However, the high TCC concentration in the fluids suggests that C5a had been formed but was probably removed by leucocytes present in the fluid.

Anaphylatoxin generation in acute pancreatitis.

Fifty-one patients with elevated serum amylase and clinical signs of acute pancreatitis were studied prospectively. The concentrations of anaphylatoxins (C3a and C5a) were measured with a radioimmunoassay and the activity of their inactivator was determined. The pancreatitis was classified as mild, moderate, or severe according to Ranson's 11 signs, appearance of peritoneal fluid, and development of multisystem organ failure (MSOF). Plasma C3a and C5a concentrations were elevated during attacks of acute pancreatitis. Anaphylatoxin levels correlated with the severity of the disease (C3a, P less than 0.001; C5a, P less than 0.05). The highest and most persistent levels were found in the group with MSOF. C3a levels decreased rapidly during recovery. In patients with complications like abscess or pseudocyst, the C3a elevation persisted until adequate treatment was instituted. In this study, no significant changes of the inactivator levels were found, except at discharge when the inactivator level of the severe group was elevated compared to that of the moderate and mild groups (P less than 0.05).

Altered concentrations of terminal complement complexes, anaphylatoxins, and leukotrienes in the coronary sinus during cardiopulmonary bypass.

The aim of this study was to determine if the hypoperfused heart activates complement with formation of anaphylatoxins, terminal complement complexes (TCC), or leukotrienes during cardiopulmonary bypass. Fifteen patients undergoing elective cardiopulmonary bypass surgery were studied regarding complement and leukotriene activation. Blood samples were drawn serially from the radial artery and coronary sinus. The plasma concentrations of the complement components C1INH, C3, C4, and C5 decreased during the procedure, whereas C3a and TCC increased. Protamine reversal of heparin further increased the plasma levels of C3a and TCC. No significant changes in plasma levels of C5a and leukotriene C4 were observed during cardiopulmonary bypass. The activity of the anaphylatoxin inactivator (AI) decreased in both the radial artery and the coronary sinus. There were no significant differences between the concentrations of complement components and leukotriene C4 in blood from the radial artery and coronary sinus. The levels of C3a and TCC increased and C1INH, C3, C4, C5, and the anaphylatoxin inactivator activity decreased to the same extent in the coronary sinus and the radial artery. Thus, the heart does not appear to be the primary site for the altered concentrations of these endogenous vasoactive substances.

Complement activation and anaphylatoxin (C3a and C5a) formation in preeclampsia and by amniotic fluid.

The aim of this study was to determine whether the biologically active complement peptides C3a and C5a are formed in pregnancy and whether amniotic fluid can activate complement. C3a and C5a are formed when complement is activated. They increase smooth-muscle contraction, vascular permeability, and histamine release from mast cells and basophils. Thirty pregnant women were studied, 16 with uncomplicated and 14 with preeclamptic pregnancies. The plasma C3a and C5a concentrations before delivery were significantly higher in the preeclamptic than in the normal group. The concentrations returned to normal within 1 week. Plasma, serum, and amniotic fluid from 12 pregnant women (eight uncomplicated and four preeclamptic pregnancies) were drawn in connection with delivery. Amniotic fluid was incubated in fresh autologous serum at 37C for 15 minutes. A dose-dependent formation of C3a and C5a was registered with increasing amounts of amniotic fluid.

Inhibition of complement activation by high-dose corticosteroids in total hip arthroplasty.

Anaphylatoxins are released during total hip arthroplasties (THA) when methylmethacrylate is used. These toxins may be responsible for hemodynamic and pulmonary instability during surgery. Recent studies have shown that the release of anaphylatoxins may be inhibited by high-dose corticosteroids (HDC). In a double-blind study 30 consecutive patients with osteoarthritis or failed hip fractures were randomized into two groups; 15 patients received HDC at the beginning of the operation and 15 patients, designated the control group, received infused saline. Anaphylatoxin formation, arterial oxygen tension, and blood pressure were determined preoperatively, during the operation, and one day postoperatively. The patients who received HDC had no significant alteration regarding the anaphylatoxins or arterial oxygen tension during surgery. However, in the control group elevated C3a levels and decreased PaO2 levels were found. Corticosteroids therefore inhibit complement activation and anaphylatoxin release in hip arthroplasty surgery.

Anaphylatoxin formation in sepsis.

Complement activation and anaphylatoxin formation were studied in 27 septic patients. The patients were treated with antibiotics and high-dose corticosteroids. Blood samples were drawn on admission and every week thereafter. Plasma levels of complement components C1INH, C3, C4, and C5 were low before the start of treatment but were above normal one week later in both successfully and unsuccessfully treated patients. In contrast, plasma levels of anaphylatoxins C3a/C3adesArg and C5a/C5adesArg were elevated on admission. After successful treatment, plasma levels of C3a/C3adesArg and C5a/C5adesArg returned to normal within one week. Nine patients had ongoing sepsis one week after the start of treatment and a persistent rise in anaphylatoxin concentration. They developed multisystem organ failure with respiratory, hepatic, and renal insufficiency. In vitro studies of Escherichia coli incubation in fresh serum indicated a dose-related formation of C3a/C3adesArg and C5a/C5adesArg. High concentrations of methylprednisolone inhibited the anaphylatoxin formation in vitro.

Terminal complement complexes and anaphylatoxins in septic and ischemic patients.

Terminal complement complex (TCC) and anaphylatoxin formation in 18 patients with sepsis and 20 patients with acute limb ischemia were studied before the start of treatment and seven days later. The septic or ischemic patients had elevated levels of plasma TCC before start of therapy. In successfully treated patients these concentrations were within the normal range one week later. Similarly, the plasma anaphylatoxin level was increased before therapy and returned to the normal range within seven days. Escherichia coli incubated in vitro in fresh human serum at body temperature started formation of TCC in a dose-related manner. As complement will induce cellular lysis via TCC and edema via anaphylatoxins, anemia and impaired respiration in these patients may be influenced by increased concentrations of terminal complement complexes and of C3a and C5a.

Complement and anaphylatoxin responses to cross-clamping of the aorta. Studies during general anaesthesia with or without extradural blockade.

Complement activation and anaphylatoxin formation were studied in 28 patients undergoing aortic reconstructive surgery under general anaesthesia. In 12 of the patients an extradural catheter was placed in the L1-2 space and 2% mepivacaine was injected until blockade reached the T4 level. The remaining 16 patients were operated upon under general anaesthesia alone. Complement activation was not obvious until aortic cross-clamping. The release of anaphylatoxin was less pronounced during cross-clamping in the extradural group.

The ischaemic leg as a source of complement activation.

Complement activation before and after surgical resection was studied in 40 patients with ischaemic legs (17 with acute and 23 with chronic ischaemia). Plasma anaphylatoxin concentrations (C3a and C5a) and anaphylatoxin inhibitor (AI) activity were studied pre-operatively, and 1 h, 24 h and 1 week postoperatively. Increased plasma anaphylatoxin concentrations were found pre-operatively. Anaphylatoxin levels in plasma had returned to normal one week postoperatively. Patients with acute ischaemia had higher plasma C3a and C5a pre-operatively and during the first 24 h postoperatively than patients with chronic ischaemia. Anaphylatoxins increase vascular permeability, smooth muscle contraction and histamine release from mast cells. Their enhanced activity might be one of the explanations for respiratory, hepatic and renal insufficiency before surgical resection in patients with extensive ischaemia.

In vivo determination of modulated migration of radiolabeled leukocytes.

A method to determine migration of phagocytic cells in vivo has been evaluated. It was used to analyze leukocyte migration during ischemia, peritonitis, and after pretreatment with methylprednisolone. Phagocytic cells were labeled in vitro with 150 megabecquerel (4.05 mCi) of technetium Tc 99m colloid and reinfused. Saline 0.03 mg and 3.0 mg of endotoxin per kilogram per rat were injected subcutaneously in duplicate. Four hours later, a standardized amount of skin and subcutaneous tissue from the area of injection was removed and weighed. A dose-related accumulation of radiolabeled leukocytes to endotoxin was found in healthy controls but not in animals with hypoperfusion or peritonitis or those pretreated with methylprednisolone. This in vivo method to calculate leukocyte migration will give reproducible results with a mean difference between duplicate samples of less than 6%.

Increased prolactin binding and morphological changes in the wound epithelium of regenerating limbs of Notophthalmus viridescens.

Distribution of prolactin has been examined in regenerating forelimbs from the newt Notophthalmus viridescens. Specific prolactin binding was demonstrated in homogenates of unamputated tissue, and of regenerating limbs at from 3 to 21 days postamputation. Labeled prolactin that was injected intraperitoneally into animals with one regenerating limb accumulated in the most distal portion of the regenerate at 7 and 14 days postamputation. Light-microscopic autoradiography demonstrated that labeled prolactin was localized most heavily in the apical, outer layer of the wound epithelium. Scanning electron microscopy demonstrated that, in addition to changes in prolactin affinity following amputation, morphological changes occurred in the apical wound epithelium as well. Cell surfaces of the stump epidermis were characterized by periodic dispersion of papillae among a network of interconnecting structures 1-2 microns across. By contrast, the surfaces of cells from the area in which labeled prolactin was found to localize most intensely were characterized by lack of papillae and, depending on the stage of regeneration, a pattern of microvilli and microplicae. These morphological alterations appear to reflect functional and biochemical differences between stump epidermis and wound epithelium.