Removal of Circulating Neutrophil Extracellular Trap Components With an Immobilized Polymyxin B Filter: A Preliminary Study.

Components of neutrophil extracellular traps (NETs) are released into the circulation by neutrophils and contribute to microcirculatory disturbance in sepsis. Removing NET components (DNA, histones, and proteases) from the circulation could be a new strategy for counteracting NET-dependent tissue damage. We evaluated the effect of hemoperfusion with a polymyxin B (PMX) cartridge, which was originally developed for treating gram-negative infection, on circulating NET components in patients with septic shock, as well as the effect on phorbol myristate acetate (PMA)-stimulated neutrophils obtained from healthy volunteers. Ex vivo closed loop hemoperfusion was performed through PMX filters in a laboratory circuit. Whole blood from healthy volunteers (incubated with or without PMA) or from septic shock patients was perfused through the circuit. For in vivo experiment blood samples were collected before and immediately after hemoperfusion with PMX to measure the plasma levels of cell-free NETs. The level of cell-free NETs was assessed by measuring myeloperoxidase-associated DNA (MPO-DNA), neutrophil elastase-associated DNA (NE-DNA), and cell-free DNA (cf-DNA). Plasma levels of MPO-DNA, NE-DNA, and cf-DNA were significantly increased after 2 h of PMA stimulation. When the circuit was perfused with blood from septic shock patients or PMA-stimulated neutrophils from healthy volunteers, circulating levels of MPO-DNA, NE-DNA, and cf-DNA were significantly reduced after 1 and 2 h of perfusion with a PMX filter compared with perfusion without a PMX filter. In 10 patients with sepsis, direct hemoperfusion through filters with immobilized PMX significantly reduced plasma levels of MPO-DNA and NE-DNA. These ex vivo and in vivo findings demonstrated that hemoperfusion with PMX removes circulating NET components. Selective removal of circulating NET components from the blood could be effective for prevention/treatment of NET-related inappropriate inflammation and thrombogenesis in patients with sepsis.

Increased PD-1 Expression and Altered T Cell Repertoire Diversity Predict Mortality in Patients with Septic Shock: A Preliminary Study.

Sepsis causes impairment of innate and adaptive immunity by multiple mechanisms, including depletion of immune effector cells and T cell exhaustion. Although lymphocyte dysfunction is associated with increased mortality and potential reactivation of latent viral infection in patients with septic shock, the relation between viral reactivation and lymphocyte dysfunction is obscure. The objectives of this study were 1) to determine the relation of lymphocyte dysfunction to viral reactivation and mortality, and 2) to evaluate recovery of lymphocyte function during septic shock, including T cell receptor (TCR) diversity and the expression of programmed death 1 (PD-1). In 18 patients with septic shock and latent cytomegalovirus (CMV) infection, serial blood samples were obtained on days 1, 3, and 7 after the onset of shock, and immune cell subsets and receptor expression were characterized by flow cytometry. TCR diversity of peripheral blood mononuclear cells was analyzed by Multi-N-plex PCR, and CMV DNA was quantified using a real-time PCR kit. A decrease of TCR diversity and monocyte HLA-DR expression were observed in the early stage of septic shock, while CD4+ T cells displayed an increase of PD-1 expression. Significant lymphopenia persisted for at least 7 days following the onset of septic shock. Normalization of TCR diversity and PD-1 expression was observed by day 7, except in patients who died. CMV reactivation was detected in 3 of the 18 patients during the first week of their ICU stay and all 3 patients died. These changes are consistent with the early stage of immune cell exhaustion and indicate the importance of normal lymphocyte function for recovery from septic shock. Ongoing lymphocyte dysfunction is associated with CMV reactivation and dissemination, as well as with unfavorable outcomes.

Clostridium Perfringens Infection in a Febrile Patient with Severe Hemolytic Anemia.

BACKGROUND: Clostridium perfringens (C. perfringens) can cause various infections, including gas gangrene, crepitant cellulitis, and fasciitis. While C. perfringens sepsis is uncommon, it is often rapidly fatal because the alpha toxin of this bacterium induces massive intravascular hemolysis by disrupting red blood cell membranes. CASE REPORT: We present the case of a male patient with diabetes who developed a fatal liver abscess with massive intravascular hemolysis and septic shock caused by toxigenic C. perfringens. The peripheral blood smear showed loss of central pallor, with numerous spherocytes. Multiplex PCR only detected expression of the cpa gene, indicating that the pathogen was C. perfringens type A. CONCLUSIONS: C. perfringens infection should be considered in a febrile patient who has severe hemolytic anemia with a very low MCV, hemolyzed blood sample, and negative Coombs test. The characteristic peripheral blood smear findings may facilitate rapid diagnosis.

Neutrophil extracellular traps in patients with sepsis.

BACKGROUND: Release of neutrophil extracellular traps (NETs) has been identified as an important aspect of innate immunity. We examined whether sepsis had any influence on ex vivo generation of NETs by neutrophils. MATERIALS AND METHODS: We isolated neutrophils from consecutive patients with sepsis (n = 17) and without sepsis (n = 18) admitted to the intensive care unit. Neutrophils were activated by incubation with phorbol-12-myristate-13-acetate (PMA) to induce release of NETs, and NET formation was assessed by measuring the extracellular DNA level. Immunolabeling and fluorescence imaging were also performed. Extracellular killing of bacteria by NETs was studied by co-culture of Escherichia coli and neutrophils in the presence of a phagocytosis inhibitor. To assess in vivo NET formation, plasma levels of cell-free DNA and histones were measured. RESULTS: After stimulation with PMA, neutrophils isolated from septic patients released 4.08 ± 1.02% of their total DNA, whereas neutrophils from nonseptic patients released 29.06 ± 2.94% (P = <0.0001). Immunofluorescent staining of released DNA, elastase, and myeloperoxidase also revealed similar results. Neutrophils from nonseptic patients showed effective extracellular killing of E coli through NETs, whereas neutrophils from septic patients did not (P < 0.001). Plasma levels of cell-free DNA and histones were higher in septic patients than nonseptic patients (P < 0.001). CONCLUSIONS: The ex vivo generation of NETs is downregulated in neutrophils isolated from patients with sepsis. However, it is unclear whether in vivo NET formation is also impaired during sepsis, so further investigation is necessary.

Posterior reversible encephalopathy syndrome occurring after uterine artery embolization for uterine myoma.

This case report describes posterior reversible encephalopathy syndrome (PRES) occurring after uterine artery embolization (UAE) for uterine myoma. This is the first report of PRES occurring after uterine vascular radiologic intervention. The mechanism by which UAE induced PRES is unclear.