Human autoantibodies to poly(adenosine diphosphate-ribose) polymerase.

The chromatin-bound enzyme poly(ADP-ribose) polymerase (ADPRP) is strongly stimulated by DNA with single- or double-stranded breaks, and transfers the ADP-ribose moiety of NAD to nuclear proteins. The activation of ADPRP is important for DNA repair and replication, and also has been postulated to play a role in the pathogenesis of lymphocyte dysfunction associated with chronic inflammatory diseases, and inborn errors of nucleoside metabolism. We have detected high titers of IgG autoantibodies to the ADPRP protein in six patients with rheumatic complaints. No other autoantibodies were detected in any of the six sera. The specificity of the anti-enzyme antibodies was established by (a) immunoprecipitation of ADPRP activity, (b) immunoprecipitation and immunoblotting of both the native 116-kD enzyme and its proteolytic digestion products. ADPRP was purified from human thymus and calf thymus. The autoantibodies reacted equivalently with both enzymes. The anti-ADPRP antibodies had a distinctive immunofluorescent pattern with HEp-2 cells, reacting intensely with nucleoli and metaphase chromosomes, and diffusely with the nucleus. Autoantibodies to ADPRP have not been described previously. The presence of a specific immune response against an enzyme that has been associated with various immunodeficiency syndromes raises intriguing possibilities concerning the relationship between DNA damage, immunodeficiency, and autoimmunity.     

Resistance to acute septic peritonitis in poly(ADP-ribose) polymerase-1-deficient mice

Sepsis is associated with a widespread production of proinflammatory cytokines and various oxidant species. Activation of the enzyme poly(ADP-ribose) polymerase (PARP) has been shown to contribute to cell necrosis and organ failure in various diseases associated with inflammation and reperfusion injury. The aim of the current study was to elucidate the role of PARP activation in the multiple organ dysfunction complicating sepsis in a murine model of polymicrobial sepsis induced by cecal ligation and puncture (CLP). Mice genetically deficient in PARP (PARP-/-) and their wild-type littermates (PARP+/+) were subjected to CLP. After 12 and 24 h, the proinflammatory cytokines TNF-alpha and IL-6, as well as the anti-inflammatory cytokine IL-10, and nitrite/nitrate were measured in plasma samples. Organs were harvested for the measurement of myeloperoxidase (MPO) and malondialdehyde (MDA) levels, and immunohistochemical staining for nitrotyrosine and poly(ADP ribose) was performed in gut sections. PARP-/- mice, and their wild-type littermate showed a similar time-dependent increase in plasma nitrite/nitrate and in gut and lung MDA content, as well as the presence of nitrotyrosine in the gut. In contrast to wild-type mice showing a PARP activation in the gut, PARP-/- mice had no staining for poly(ADP ribose). PARP-/- mice had significantly lower plasma levels of TNF-alpha, IL-6, and IL-10, and they exhibited a reduced degree of organ inflammation, indicated by decreased MPO activity in the gut and lung. These effects were associated with a significant improvement in the survival of CLP in PARP-/- mice. Thus, PARP activation has an important role in systemic inflammation and organ damage in the present model of polymicrobial septic shock.     

Human autoantibodies to poly(adenosine diphosphate-ribose) polymerase recognize cross-reactive epitopes associated with the catalytic site of the enzyme.

The factors responsible for the production of autoantibodies against self-components are not well understood. We have identified monospecific human autoantibodies to poly(ADP-ribose) polymerase (ADPRP) in the sera of rheumatic patients. Since this nuclear enzyme has been extensively characterized, and its entire structure is known, we could investigate in detail the epitope specificity of the human autoantibodies, and their effects on the biological functions of the enzyme. All sera with autoantibodies to ADPRP recognized the NAD-binding domain of the enzyme, as demonstrated by either immunoblotting or immunoprecipitation of partially proteolyzed ADPRP. The autoantibodies also inhibited the catalytic activity of the purified enzyme, as measured by the transfer of ADP-ribose from [32P]NAD to either histones or to ADPRP itself. Because comparative structural analyses have shown that the active sites of enzymes are often conserved during evolution, we tested the ability of the autoantibodies to react with ADPRP from lower eukaryotes. The human autoantibodies reacted with ADPRP in cellular extracts from mammalian, avian, amphibian, arthropod, and protozoan cells, and also inhibited the catalytic activity of the various enzymes. Collectively, these experiments indicate that the human autoantibodies to ADPRP recognize a distinct group of evolutionarily conserved antigenic determinants that are closely related to the catalytic site of the enzyme. The results are consistent with the hypothesis that the epitope selectivity of human autoantibodies to ADPRP is influenced by cross-reactive antigens in the external environment.     

Potential role of poly(adenosine 5'-diphosphate-ribose) polymerase activation in the pathogenesis of myocardial contractile dysfunction associated with human septic shock

OBJECTIVE: Sepsis is associated with increased production of superoxide and nitric oxide, with consequent peroxynitrite generation. Cardiodepression is induced in the heart during oxidative stress associated with septic shock. Oxidative and nitrosative stress can lead to activation of the nuclear enzyme poly(adenosine 5'-diphosphate [ADP]-ribose) polymerase (PARP), with subsequent loss of myocardial contractile function. The aim of the study was to investigate whether cardiodepression found in septic patients is associated with plasma markers of myocardial necrosis and with myocardial PARP activation. DESIGN: Prospective and observational study. SETTING: University hospital intensive care unit for clinical and surgical patients. PATIENTS: Twenty-five patients older than 18 yrs presenting with severe sepsis or septic shock. Patients with history of chronic heart failure, cancer, coronary artery disease, diabetes, or acquired immune deficiency syndrome were excluded. INTERVENTIONS: Patients were followed for 28 days, and biochemical and hemodynamic data were collected on days 1, 3, and 6 of sepsis. The groups were survivors and nonsurvivors, defined only after the end of clinical patient evolution. Heart sections from patients who died were analyzed with hematoxylin-eosin and Picro Sirius-Red immunostaining and with electron microscopy. MEASUREMENTS AND MAIN RESULTS: The study population included 25 individuals, of whom 12 (48%) died during the 6 days of follow-up. The initial data of the inflammation marker C-reactive protein and Acute Physiologic and Chronic Health.Evaluation severity were similar in both groups (nonsurvivors, 26 +/- 2; survivors, 24 +/- 5; NS). Overall, an increase in plasma troponin level was related to increased mortality risk. In patients who died, significant myocardial damage was detected, and histologic analysis of heart sections showed inflammatory infiltration, increased collagen deposition, and derangement of mitochondrial cristae. Immunohistochemical staining for poly(ADP-ribose) (PAR), the product of activated PARP, was demonstrated in septic hearts. There was a positive correlation between PAR staining densitometry and troponin I (r(2) = 0.73; p < .05), and the correlation of PAR staining densitometry and left ventricular systolic stroke work index was r(2) = 0.33 (p = .0509). CONCLUSION: There is significant PARP activation in the hearts of septic patients with impaired cardiac function. We hypothesize that PARP activation may be partly responsible for the cardiac depression seen in humans with severe sepsis.     

Role of adenosine A(1) receptors in modulating extracellular adenosine levels.

The purpose of this investigation was to test the hypothesis that A(1) receptors modulate extracellular levels of adenosine in cardiovascular tissues. Rat cardiac fibroblasts and human aortic vascular smooth muscle cells were cultured to confluence and various pharmacological agents were applied to the cultures. The extracellular fluid was extracted and adenosine concentrations were measured by HPLC. Three selective A(1) receptor antagonists, namely 8-cyclopentyl-1,3-dipropylxanthine, xanthine amine congener, and N-0840, at a concentration of 10 nM significantly increased extracellular levels of adenosine in both rat cardiac fibroblasts and human aortic vascular smooth muscle cells. Further studies in rat cardiac fibroblasts revealed that the effects of A(1) receptor blockade on extracellular adenosine levels were concentration dependent and prevented by inhibition of G(i) proteins with pertussis toxin or blockade of ecto-5'-nucleotidase with alpha, beta-methyleneadenosine-5'-diphosphate. In cardiac fibroblasts in which the extracellular levels of endogenous adenosine were increased, the ability of A(1) receptor blockade to augment extracellular adenosine was attenuated. A time-course study revealed a time lag of several hours between blockade of A(1) receptors and increases in extracellular adenosine levels. These data suggest that A(1) receptors function to detect the long-term levels of extracellular adenosine, and appropriately adjust extracellular adenosine levels by a slow-onset mechanism involving G(i) proteins and ecto-5'nucleotidase.     

Mesenteric injury after cardiopulmonary bypass: role of poly(adenosine 5'-diphosphate-ribose) polymerase

OBJECTIVES: To investigate the effects of the ultrapotent poly(adenosine 5'-diphosphate-ribose) polymerase (PARP) inhibitor INO-1001 on cardiac and mesenteric function during reperfusion in an experimental model of cardiopulmonary bypass with cardioplegic arrest. DESIGN: Prospective, randomized, and blinded experimental study. SETTING: Research laboratory. SUBJECTS:: Twelve anesthetized dogs underwent cardiopulmonary bypass with hypothermic cardioplegic cardiac arrest. INTERVENTIONS: After 60 mins of hypothermic cardiac arrest, either PARP inhibitor INO-1001 (1 mg/kg, n = 6) or vehicle (control, n = 6) was administered during reperfusion. MEASUREMENTS AND MAIN RESULTS: Left ventricular hemodynamic variables were measured by combined pressure-volume-conductance catheters. Coronary and mesenteric blood flow and vasodilatory responses to acetylcholine and sodium nitroprusside as well as mesenteric lactate and creatinine phosphokinase release were also determined. The administration of INO-1001 led to a significantly improved recovery of left ventricular systolic function (p < .05) after 60 mins of reperfusion. Coronary and mesenteric blood flow were also significantly higher in the INO-1001 group (p < .05). Although the vasodilatory response to sodium nitroprusside was similar in both groups before and after cardiopulmonary bypass and similar in response to acetylcholine before cardiopulmonary bypass, PARP-inhibited dogs had lower mesenteric vascular resistance after cardiopulmonary bypass (p < .05). Mesenteric lactate and creatinine phosphokinase release was significantly lower in the PARP inhibitor treated group (p < .05). CONCLUSION: PARP inhibition with INO-1001 improves the recovery of myocardial function and prevents mesenteric vascular dysfunction and tissue injury after cardiopulmonary bypass with hypothermic cardiac arrest.     

Tumor treating fields combined with a poly (adenosine diphosphate-ribose) polymerase inhibitor during radiotherapy for rapidly progressing IDH-wildtype diffuse astrocytoma: a case report

A 57-year-old woman was diagnosed with IDH-wildtype (IDHwt) astrocytoma (World Health Organization grade II) with the molecular characteristics of glioblastoma. She underwent concurrent radiotherapy and chemotherapy according to the Stupp protocol in combination with a multi-target antiangiogenic drug and additional intrathecal chemotherapy using methotrexate. During treatment, the patient’s tumor showed rapid progression. The chemotherapy with temozolomide was stopped and replaced with radiotherapy combined with tumor treating fields (TTF), the poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitor niraparib, and anlotinib. After the radiotherapy was completed, the symptoms of increased intracranial pressure and epilepsy were well controlled. Considering the patient’s tolerance to the treatment, the combined therapy of TTF and anlotinib was continued, and osimertinib, a third-generation epidermal growth factor receptor–tyrosine kinase inhibitor with good permeability of the blood–brain barrier, was added. The patient was regularly followed up and had no obvious adverse drug reactions. Head magnetic resonance imaging (plain scan + enhanced scan) suggested that the lesions were stable. For rapidly progressing glioblastomas or histological grade II/III IDHwt astrocytomas, the combination of TTF and a PARP inhibitor during radiotherapy may have a synergistic effect on tumor control and is well tolerated by patients.     

Role of coagulation FVIII in septic peritonitis assessed in hemophilic mice

BACKGROUND: Inhibition of blood coagulation appears to be an important therapeutic strategy to improve the outcome in sepsis. However, the beneficial effect of anticoagulant treatment in sepsis is solely based on experimental data using inhibitors of the extrinsic coagulant pathway. The role of the intrinsic pathway of coagulation in the pathogenesis of sepsis has not been explored yet. OBJECTIVE: In the current study, we contribute to determine the role of factor (F)VIII, the key player of the intrinsic coagulant pathway, on host defense against peritonitis. METHOD: Hemizygous FVIII-deficient mice and their wild-type littermates were challenged with 1 x 10(4) bacteria in a septic peritonitis model. RESULTS: The intraperitoneal injection of Escherichia coli led to growth and dissemination of bacteria and provoked an inflammatory response as evident from elevated cytokine levels, increased cell influx into tissues, liver necrosis, and endothelialitis resulting in mortality. The FVIII-deficient genotype slightly reduced bacterial outgrowth but had no effect on markers of inflammation and/or survival. In addition, FVIII-deficient mice showed profound activation of coagulation, thereby improving the hemophilic phenotype of FVIII-deficient mice. CONCLUSION: FVIII deficiency slightly modifies host defense in septic peritonitis in mice, but does not influence the final outcome of peritonitis. Therefore, we question the importance of the intrinsic coagulant pathway during sepsis.     

Role of adenosine A(1) receptor in angiotensin II- and norepinephrine-induced renal vasoconstriction

We investigated the contributions of adenosine A(1) receptors to angiotensin II- and norepinephrine-induced renal vasoconstriction. Intrarenal administrations of angiotensin II (3, 10, and 30 ng) or norepinephrine (100 and 500 ng) produced dose-dependent renal vasoconstriction in anesthetized dogs. Under resting conditions, angiotensin II (30 ng) and norepinephrine (500 ng) significantly decreased renal blood flow by -43 +/- 3 and -19 +/- 2%, respectively (n = 21). Intra-arterial infusion of adenosine (5 microg/kg/min) significantly augmented renal blood flow responses to both angiotensin II and norepinephrine (-64 +/- 4 and -45 +/- 14%, n = 7). Renal blood flow responses to angiotensin II and norepinephrine were also augmented by inhibition of cellular uptake of adenosine with dipyridamole (10 microg/kg/min, n = 6). Blockade of adenosine A(1) receptors with 8-(noradamantan-3-yl)-1,3-dipropylxanthine (KW-3902; 10 microg/kg/min) did not alter basal renal blood flow but significantly attenuated angiotensin II- and norepinephrine-induced renal vasoconstriction (-34 +/- 6 and -9 +/- 3%, n = 7). Furthermore, KW-3902 completely prevented augmentation of renal blood flow responses to angiotensin II and norepinephrine produced by adenosine or dipyridamole (n = 7 and 6, respectively). Administrations of angiotensin II (30 ng) or norepinephrine (500 ng) into the common carotid artery significantly decreased carotid blood flow by -20 +/- 5 and -41 +/- 10%, respectively; however, neither adenosine (5 microg/kg/min) nor KW-3902 (10 microg/kg/min) affected the carotid blood flow responses to angiotensin II and norepinephrine (n = 5, respectively). Adenosine concentrations in dialysates were not significantly changed by administrations of angiotensin II (from 19 +/- 3 to 24 +/- 4 nM, n = 6) or norepinephrine (from 16 +/- 3 to 19 +/- 3 nM, n = 6). These results suggest that basal interstitial adenosine levels influence both angiotensin II and norepinephrine-induced vasoconstriction via A(1) receptors in the kidney but not in the area drained by the common carotid artery. The responses of adenosine to angiotensin II- and norepinephrine-induced renal vasoconstriction may not be mediated through de novo intrarenal adenosine accumulation due to angiotensin II- and norepinephrine-induced renal vasoconstriction.     

Role of T cells for cytokine production and outcome in a model of acute septic peritonitis

Although it is generally accepted that early defense mechanisms are controlled by cells of the innate immune system, T cells were found to be crucial for host resistance against acute septic peritonitis. However, the mechanisms by which T cells mediate protection are not fully understood. Here, we demonstrate mice deficient for recombinase-activating gene (RAG) 1, which lack mature B and T cells, showed enhanced susceptibility and impaired bacterial clearance in a model of acute septic peritonitis. Whereas B-cell-deficient muMT mice showed no significant difference in the survival rate after peritonitis induction, T-cell-deficient Balb/c nude mice exhibited reduced survival. Importantly, analysis of cytokine production in both RAG-1-deficient and T-cell-deficient nude mice indicated strongly attenuated production of IL-12, interferon (IFN) gamma, and IL-10 during sepsis. Reduced cytokine levels were detected both in serum and in organ extracts of septic mice. Direct analysis of T cells isolated from septic mice demonstrated that T cells respond to an acute septic challenge by increased production of IFN-gamma and IL-10. Moreover, bacterial numbers in spleens of septic RAG-1-deficient mice were significantly increased as compared with controls, suggesting that T cells are engaged in the early antibacterial immune defense during sepsis, possibly via the production of IFN-gamma. In summary, these results imply that T cells contribute to protective immune responses against acute systemic infections via their ability to produce crucial immune mediators.     

DAP12 (KARAP) amplifies inflammation and increases mortality from endotoxemia and septic peritonitis

DAP12 (KARAP) is a transmembrane signaling adaptor for a family of innate immunoreceptors that have been shown to activate granulocytes and monocytes/macrophages, amplifying production of inflammatory cytokines. Contrasting with these data, recent studies suggest that DAP12 signaling has an inhibitory role in the macrophage response to microbial products (Hamerman, J.A., N.K. Tchao, C.A. Lowell, and L.L. Lanier. 2005. Nat. Immunol. 6:579-586). To determine the in vivo role for DAP12 signaling in inflammation, we measured the response of wild-type (WT) and DAP12-/- mice to septic shock. We show that DAP12-/- mice have improved survival from both endotoxemia and cecal ligation and puncture-induced septic shock. As compared with WT mice, DAP12-/- mice have decreased plasma cytokine levels and a decreased acute phase response during sepsis, but no defect in the recruitment of cells or bacterial control. In cells isolated after sepsis and stimulated ex vivo, DAP12 signaling augments lipopolysaccharide-mediated cytokine production. These data demonstrate that, during sepsis, DAP12 signaling augments the response to microbial products, amplifying inflammation and contributing to mortality.     

Role of the factor V Leiden mutation in septic peritonitis assessed in factor V Leiden transgenic mice

OBJECTIVE: The factor V Leiden (FVL) mutation (Arg506Glu) results in the production of an FV protein that when activated is relatively resistant to inactivation by activated protein C and thereby leads to predisposition to thrombosis. The rather high prevalence of the FVL mutation in the general population prompted speculation about a potential survival benefit for individuals carrying the FVL allele. Indeed, both clinical and experimental animal data suggest that a heterozygous FVL genotype might protect against the lethal consequences of sepsis. We sought to confirm the survival advantage of heterozygous FVL mice in septic disease. DESIGN: Controlled animal experiment. SETTING: Academic research laboratory. SUBJECTS: Wild-type, heterozygous, and homozygous FVL mice subjected to 1 x 10 live bacteria as model for septic peritonitis. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The intraperitoneal injection of E. coli led to growth and dissemination of bacteria and provoked an inflammatory response as evident from elevated cytokine levels (interleukin-6, interleukin-10, and tumor necrosis factor-alpha), induced thrombin-antithrombin complex levels, increased granulocyte influx into the peritoneal cavity, liver necrosis, and adhesion of leukocytes to the vessel wall, resulting in approximately 50% mortality after 72 hrs. The FVL genotype had no significant effect on bacterial outgrowth, markers of inflammation (i.e., tumor necrosis factor-alpha levels of 152 [96.2-200], 152 [99.7-1745], and 110 [99.7-177] pg/mL in peritoneal lavage fluid at t = 20 hrs for wild-type, heterozygous, and homozygous FVL mice, respectively), thrombin generation (i.e., thrombin-antithrombin complex levels of 19.9 [9.31-37.4], 10.4 [6.55-15.8], and 12.6 [8.24-29.0] ng/mL in peritoneal lavage fluid at t = 6 hrs for wild-type, heterozygous, and homozygous FVL mice, respectively), and/or survival (50%, 36%, and 50% for wild-type, heterozygous, and homozygous FVL mice, respectively). CONCLUSIONS: The FVL allele has no beneficial effect in mouse septic peritonitis, and the general protective effect of FVL in sepsis needs further investigation.     

Activation of poly(ADP-ribose) polymerase in circulating leukocytes during myocardial infarction

Myocardial ischemia-reperfusion can lead to increased oxidative stress both locally and in circulating leukocytes. Oxidant-mediated DNA single strand breaks are known to activate the nuclear enzyme poly(ADP-ribose) polymerase (PARP) in various forms of shock, inflammation, and ischemia-reperfusion injury. The aim of the current study was to investigate whether a local insult such as myocardial ischemia-reperfusion is sufficient to lead to activation of PARP in circulating leukocytes. In anesthetized rats myocardial ischemia-reperfusion was induced by transient ligation of the left anterior descending coronary artery. There was a marked increase in poly(ADP-ribosyl)ation of proteins in homogenates of leukocytes isolated from rats at the end of the reperfusion period. Poly(ADP-ribosyl)ation was inhibited by administration of the pharmacologic PARP inhibitor INO-1001 (30 mg/kg) to the rats. We conclude that local insults, such as myocardial reperfusion injury, are sufficient to activate PARP in circulating leukocytes. PARP activation in circulating cells may mediate certain systemic effects of local ischemia-reperfusion injury such as inflammatory mediator production and remote organ injury.     

Drug-induced hypothermia begun 5 minutes after injury with a poly(adenosine 5'-diphosphate-ribose) polymerase inhibitor reduces hypoxic brain injury in rat pups

OBJECTIVE: Poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors have shown promise in hypoxic ischemic brain damage. We wished to see if GPI-6150 (1,11b-dihydro-[2H]benzopyrano[4,3,2-de]isoquinolin-3-one), a specific PARP inhibitor, would reduce brain injury in a newborn animal model. DESIGN: Randomized controlled trial. SETTING: University laboratory. SUBJECTS: Seven-day-old rat pups. INTERVENTION: Subjects had the right carotid artery ligated and then received either vehicle or 5, 15, or 30 mg/kg GPI-6150 intraperitoneally 5 mins after the hypoxia. Hypoxia was produced by exposing the pups to 8% oxygen for 2.5 hrs. MEASUREMENTS AND MAIN RESULTS: Twenty-two days later, the brains were scored from normal to severely damaged and were weighed by a blinded observer. Twenty-four of 53 (45%) vehicle-treated pups, 11 of 22 (50%) 5 mg/kg treated pups, 22 of 23 (96%) 15 mg/kg treated pups (p <.01 vs. vehicle), and 16 of 31 (52%) 30 mg/kg treated pups were scored as normal. Right hemisphere weight was reduced by 15 +/- 2.6% in the vehicle group, 5.9 +/- 2.8% in the 5 mg/kg group, -0.4 +/- 1.7% in the 15 mg/kg group (p <.01 vs. vehicle), and 13.3 +/- 3.1% in the 30 mg/kg group. GPI-6150 decreased rectal temperature from 33 +/- 0.4 to 29 +/- 0.7 degrees C for 3 hrs after dosing, but temperatures returned to normal by 6 hrs. We maintained the body temperature at 35 degrees C for 6 hrs after injury in a group of pups treated with 15 mg/kg. Nine of 25 (41%) vehicle-treated and 15 of 26 (58%) GPI-6150-treated pups were scored as normal (p = nonsignificant). Right hemisphere weight was reduced by 25 +/- 4% in the vehicle group and 20 +/- 5% in the GPI-6150 group (p = nonsignificant). CONCLUSIONS: GPI-6150 at a dose of 15 mg/kg dramatically decreased the number of pups sustaining brain injury, relative to vehicle, but is dependent on an induced decrease in core temperature to produce the effect.     

Burn and smoke injury activates poly(ADP-ribose)polymerase in circulating leukocytes

The nuclear enzyme poly(ADP-ribose)polymerase (PARP) plays a significant role in the pathogenesis of various forms of critical illness. DNA strand breaks induced by oxidative and nitrative stress trigger the activation of PARP, and PARP, in turn, mediates cell death and promotes proinflammatory responses. Until recently, most studies focused on the role of PARP in solid organs such as heart, liver, and kidney. We investigated the effect of burn and smoke inhalation on the levels of poly(ADP-ribosylated) proteins in circulating sheep leukocytes ex vivo. Adult female merino sheep were subjected to burn injury (2× 20% each flank, 3 degrees) and smoke inhalation injury (insufflated with a total of 48 breaths of cotton smoke) under deep anesthesia. Arterial and venous blood was collected at baseline, immediately after the injury and 1 to 24 h after the injury. Leukocytes were isolated with the Histopaque method. The levels of poly(ADP-ribosyl)ated proteins were determined by Western blotting. The amount of reactive oxygen species was quantified by the OxyBlot method. To examine whether PARP activation continues to increase ex vivo in the leukocytes, blood samples were incubated at room temperature or at 37°C for 3 h with or without the PARP inhibitor PJ34. To investigate whether the plasma of burn/smoke animals may trigger PARP activation, burn/smoke plasma was incubated with control leukocytes in vitro. The results show that burn and smoke injury induced a marked PARP activation in circulating leukocytes. The activity was the highest immediately after injury and at 1 h and decreased gradually over time. Incubation of whole blood at 37°C for 3 h significantly increased poly(ADP-ribose) levels, indicative of the presence of an ongoing cell activation process. In conclusion, PARP activity is elevated in leukocytes after burn and smoke inhalation injury, and the response parallels the time course of reactive oxygen species generation in these cells.