Quantifying Myeloperoxidase-DNA and Neutrophil Elastase-DNA Complexes from Neutrophil Extracellular Traps by Using a Modified Sandwich ELISA.

Certain stimuli, such as microorganisms, cause neutrophils to release neutrophil extracellular traps (NETs), which are basically web-like structures composed of DNA with granule proteins, such as myeloperoxidase (MPO) and neutrophil elastase (NE), and cytoplasmic and cytoskeletal proteins. Although interest in NETs has increased recently, no sensitive, reliable assay method is available for measuring NETs in clinical settings. This article describes a modified sandwich enzyme-linked immunosorbent assay to quantitatively measure two components of circulating NETs, MPO-DNA and NE-DNA complexes, which are specific components of NETs and are released into the extracellular space as breakdown products of NETs. The assay uses specific monoclonal antibodies for MPO or NE as the capture antibodies and a DNA-specific detection antibody. MPO or NE binds to one site of the capture antibody during the initial incubation of samples containing MPO-DNA or NE-DNA complexes. This assay shows good linearity and high inter-assay and intra-assay precision. We used it in 16 patients with COVID-19 with accompanying acute respiratory distress syndrome and found that the plasma concentrations of MPO-DNA and NE-DNA were significantly higher than in the plasma obtained from healthy controls. This detection assay is a reliable, highly sensitive, and useful method for investigating the characteristics of NETs in human plasma and culture supernatants.

Acquisition and Analysis of Microcirculation Image in Septic Model Rats.

Background: Microcirculation is a vital sign that supplies oxygen and nutrients to maintain normal life activities. Sepsis typically influences the operation of microcirculation, which is recovered by the administration of medicine injection. Objective: Sepsis-induced variation and recovery of microcirculation are quantitatively detected using microcirculation images acquired by a non-contact imaging setup, which might assist the clinical diagnosis and therapy of sepsis. Methods: In this study, a non-contact imaging setup was first used to record images of microcirculation on the back of model rats. Specifically, the model rats were divided into three groups: (i) the sham group as a control group; (ii) the cecum ligation and puncture (CLP) group with sepsis; and (iii) the CLP+thrombomodulin (TM) group with sepsis and the application of TM alfa therapy. Furthermore, considering the sparsity of red blood cells (RBCs), the blood velocity is estimated by robust principal component analysis (RPCA) and U-net, and the blood vessel diameter is estimated by the contrast difference between the blood vessel and tissue. Results and Effectiveness: In the experiments, the continuous degradation of the estimated blood velocity and blood vessel diameter in the CLP group and the recovery after degradation of those in the CLP+TM group were quantitatively observed. The variation tendencies of the estimated blood velocity and blood vessel diameter in each group suggested the effects of sepsis and its corresponding therapy.

Posterior reversible encephalopathy syndrome (PRES) of the isolated brainstem.

A 71-year-old man had disordered consciousness whose Glasgow Coma Scale was E4V1M5. His blood pressure was high, but there was no abnormality in the cerebrospinal fluid examination. The MRI finding reveals a high-intensity area at the pons without the blood flow interruption. Thus, he has diagnosed with brainstem PRES.

Recombinant thrombomodulin attenuates hyper-inflammation and glycocalyx damage in a murine model of Streptococcus pneumoniae-induced sepsis.

PURPOSE: The anticoagulant agent recombinant thrombomodulin (rTM) activates protein C to prevent excessive coagulation and also possibly regulates hyper-inflammation via neutralization of high-mobility-group B1 (HMG-B1). The glycocalyx layer in endothelial cells also plays a pivotal role in preventing septic shock-associated hyperpermeability. The present study examined the effect of rTM in a murine model of Streptococcus pneumoniae-induced sepsis. METHODS: Male C57BL/6N mice were injected intratracheally via midline cervical incision with 2 × 107 CFU of S. pneumoniae (capsular subtype 19A). Control mice were sham-treated identically but injected with saline. rTM (10 mg/kg) was injected intraperitoneally 3 h after septic insult. Blood concentrations of soluble inflammatory mediators (interleukin [IL]-1ß, IL-6, IL-10, and tumor necrosis factor [TNF]-α) were determined using a microarray immunoassay. Serum concentrations of HMG-B1 and syndecan-1, as a parameter of glycocalyx damage, were determined by enzyme-linked immunosorbent assay. The glycocalyx was also evaluated with electron microscopy. The lungs were removed, and digested to cells, which were then stained with a mixture of fluorophore-conjugated antibodies. Anti-mouse primary antibodies included PE-Cy7-conjugated anti-CD31, AlexaFluor 700-conjugated anti-CD45, PerCP-Cy5.5-conjugated anti-CD326, APC-conjugated anti-TNF-α, PE-conjugated anti-IL-6, and PE-conjugated anti-IL-10. A total of 1 × 106 cells per sample were analyzed, and 2 × 105 events were recorded by flow cytometry, and parameters were compared with/without rTM treatment. RESULTS: The blood concentration of TNF-α was significantly reduced 24 h after intratracheal injection in S. pneumoniae-challenged mice treated with rTM (P = 0.016). Levels of IL-10 in the lung endothelium of rTM-treated S. pneumoniae-challenged mice increased significantly 12 h after intratracheal injection (P = 0.03). Intriguingly, serum HMGB-1 and syndecan-1 levels decreased significantly (P = 0.010 and 0.015, respectively) in rTM-treated mice 24 h after intratracheal injection of S. pneumoniae. Electron microscopy indicated that rTM treatment preserved the morphology of the glycocalyx layer in septic mice. CONCLUSIONS: These data suggest that rTM modulates local inflammation in the lung endothelium, thus diminishing systemic inflammation, i.e., hypercytokinemia. Furthermore, rTM treatment reduced serum syndecan-1 levels, thus preventing glycocalyx damage. The use of rTM to treat sepsis caused by bacterial pneumonia could therefore help prevent both excessive inflammation and glycocalyx injury in the lung endothelium.

Case Report: Lifesaving Hemostasis With Resuscitative Endovascular Balloon Occlusion of the Aorta in a Patient With Cardiac Arrest Caused by Upper Gastrointestinal Hemorrhage.

Resuscitative endovascular balloon occlusion of the aorta (REBOA) is performed to treat hemorrhagic shock, whose cause is located below the diaphragm. However, its use in patients with gastrointestinal hemorrhage is relatively rare. The 45-year-old man with a history of dilated cardiomyopathy had experienced epigastric discomfort and had an episode of presyncope. On his presentation, the patient's blood pressure was 82/64 mmHg, heart rate 140/min, and consciousness level GCS E4V5M6. Hemodynamics stabilized rapidly with a transfusion that was administered on an emergency basis, and a blood sample only showed mild anemia (Hb, 11.5 g/dL). The patient was admitted to investigating the presyncope episode, and the planned endoscopy was scheduled the following day. The patient had an episode of presyncope soon and was found in hemorrhagic shock resulting from a duodenal ulcer rapidly deteriorated to cardiac arrest. Although a spontaneous heartbeat was restored with cardiopulmonary resuscitation, the patient's hemodynamics were unstable despite the emergency blood transfusion administered by pumping. Consequently, a REBOA device was placed, resuscitation was continued, and hemostasis was achieved by vascular embolization for the gastroduodenal artery. The patient was subsequently discharged without complications. However, there is no established evidence regarding the REBOA use in upper gastrointestinal hemorrhage, and the investigations that have been reported have been limited. Further, one recent research suggests that appropriate patient selection and early use may improve survival in these life-threatening cases. As was seen in the present case, REBOA can effectively treat upper gastrointestinal hemorrhage by temporarily stabilizing hemodynamics and enabling a hemostatic procedure to be quickly performed during that time. This report also demonstrated the hemodynamics during the combination of intermittent and partial REBOA to avoid the complications of ischemic or reperfusion injury of the intestines or lower extremities.

A Thrombomodulin Promoter Gene Polymorphism, rs2239562, Influences Both Susceptibility to and Outcome of Sepsis.

Objective: Disseminated intravascular coagulation plays a key role in the pathophysiology of sepsis. Thrombomodulin is essential in the protein C system of coagulation cascade, and functional polymorphisms influence the human thrombomodulin gene (THBD). Therefore, we conducted a multicenter study to evaluate the influence of such polymorphisms on the pathophysiology of sepsis. Methods: A collaborative case-control study in the intensive care unit (ICU) of each of five tertiary emergency centers. The study included 259 patients (of whom 125 displayed severe sepsis), who were admitted to the ICU of Chiba University Hospital, Chiba, Japan between October 2001 and September 2008 (discovery cohort) and 793 patients (of whom 271 patients displayed severe sepsis), who were admitted to the five ICUs between October 2008 and September 2012 (multicenter validation cohort). To assess the susceptibility to severe sepsis, we further selected 222 critically ill patients from the validation cohort matched for age, gender, morbidity, and severity with the patients with severe sepsis, but without any evidence of sepsis. Results: We examined whether the eight THBD single nucleotide polymorphisms (SNPs) were associated with susceptibility to and/or mortality of sepsis. Higher mortality on severe sepsis in the discovery and combined cohorts was significantly associated with the CC genotype in a THBD promoter SNP (-1920*C/G; rs2239562) [odds ratio [OR] 2.709 (1.067-6.877), P = 0.033 and OR 1.768 (1.060-2.949), P = 0.028]. Furthermore, rs2239562 SNP was associated with susceptibility to severe sepsis [OR 1.593 (1.086-2.338), P = 0.017]. Conclusions: The data demonstrate that rs2239562, the THBD promoter SNP influences both the outcome and susceptibility to severe sepsis.

Investigation into Effect of Thrombomodulin Alfa for Septic Model Rats based on Microcirculation Image Analysis.

Sepsis is life threatening organ dysfunction caused by microcirculatory dysfunction. With progression of sepsis, the patients are likely to develop septic shock which is associated with multi organ dysfunction. To treat sepsis and septic shock, Thrombomodulin alfa (TM alfa) was developed. Direct observation of the microcirculation may provide new and rich information in terms of the effect of TM alfa on sepsis. Thus we conducted rodent experiments in which we observed the microcirculation with a non-contact optical imaging setup and measured lactate value from collected blood. From the acquired motion pictures, we estimated the blood velocity. As a result, from experiments, the sham rats showed no significant change in both lactate value and the blood velocity during the observation. On the other hand, lactate value of the septic model rats increased and the blood velocity of them decreased. Lactate value of the septic model rats treated with TM alfa decreased after showing an increase while the blood velocity of them increased after showing a decrease. These findings suggest that microcirculatory alteration may be a sign of sepsis as well as septic shock progression and that the TM alfa may be effective for the treatment of sepsis and septic shock.

Pharmacokinetics, Pharmacodynamics, and Safety of Nivolumab in Patients With Sepsis-Induced Immunosuppression: A Multicenter, Open-Label Phase 1/2 Study.

BACKGROUND: Sepsis often induces an immunosuppressive state, which is associated with high mortality rates. Immunostimulation may be beneficial for sepsis. We investigated the pharmacokinetics, pharmacodynamics, and safety of nivolumab, a human programmed death-1 immune checkpoint inhibitor approved for the treatment of several cancers. METHODS: In this multicenter, open-label phase 1/2 study, a single 480 or 960 mg nivolumab dose was intravenously infused into Japanese patients with immunosuppressive sepsis. Doses were selected to mimic the exposure achieved with the approved dosage for cancer patients (3 mg/kg every 2 weeks [Q2W]). RESULTS: Single 480 and 960 mg nivolumab doses were intravenously infused into five and eight patients, respectively. The maximum concentration after 480 mg (132 µg/mL) was similar to the predicted concentration at the end of infusion with 3 mg/kg Q2W (117 µg/mL). The concentration on Day 28 after 960 mg (33.1 µg/mL) was within the predicted trough concentration range for 3 mg/kg Q2W (90% prediction interval 19.0-163 µg/mL). Absolute lymphocyte counts and monocyte human leukocyte antigen-DR subtype expression levels appeared to increase over time. The incidences of adverse events (AEs) were 80% and 50% in the 480 mg and 960 mg groups, respectively. Drug-related AEs were observed in only one patient in the 480 mg group. No deaths related to nivolumab occurred. CONCLUSIONS: A single dose of 960 mg nivolumab appeared to be well tolerated and sufficient to maintain nivolumab blood concentrations. Both 480 mg and 960 mg nivolumab seemed to improve immune system indices over time. TRIAL REGISTRATION: JAPIC, JapicCTI-173600.

Diagnosis of sepsis-induced disseminated intravascular coagulation and coagulopathy.

Disseminated intravascular coagulation (DIC) is a frequent complication in sepsis. Once patients develop DIC, the mortality rate increases significantly. Moreover, recent studies have suggested that coagulation disorder plays a significant role in the development of organ dysfunction in sepsis. Thus, the early detection of DIC is vital in sepsis care, and the Japanese Association for Acute Medicine established a set of original diagnostic criteria in 2006 (JAAM DIC). Since then, the usefulness of the JAAM DIC has been repeatedly reported, and these criteria have been widely adopted in emergency and critical care settings in Japan. Different criteria have also been released by the International Society on Thrombosis and Haemostasis (ISTH overt-DIC), and the latter criteria are presently considered to be the international standard. Compared with the JAAM DIC, the ISTH overt-DIC criteria are stricter and the timing of diagnosis is later. This discrepancy is because of conceptual differences. As many physicians think sepsis-associated DIC is the target of anticoagulant therapies in Japan, the JAAM DIC criteria were designed to allow the early initiation of treatment. As other countries do not provide DIC-specific treatments, early diagnosis is not necessary, and this situation has led to a significant gap. However, as overt-DIC is a late-phase coagulation disorder, a need for early detection has been advocated, and members of the ISTH have recently proposed the category of sepsis-induced coagulopathy. In this review, we introduce the strengths and weaknesses of the major criteria including JAAM-DIC, ISTH overt-DIC, sepsis-induced coagulopathy, and Japanese Society on Thrombosis and Haemostasis-DIC.

Sepsis-associated disseminated intravascular coagulation and its differential diagnoses.

Disseminated intravascular coagulation (DIC) is a common complication in sepsis. Since DIC not only promotes organ dysfunction but also is a strong prognostic factor, its diagnosis at the earliest possible timing is important. Thrombocytopenia is often present in patients with DIC but can also occur in a number of other critical conditions. Of note, many of the rare thrombocytopenic diseases require prompt diagnoses and specific treatments. To differentiate these diseases correctly, the phenotypic expressions must be considered and the different disease pathophysiologies must be understood. There are three major players in the background characteristics of thrombocytopenia: platelets, the coagulation system, and vascular endothelial cells. For example, the activation of coagulation is at the core of the pathogenesis of sepsis-associated DIC, while platelet aggregation is the essential mechanism in thrombotic thrombocytopenic purpura and endothelial damage is the hallmark of hemolytic uremic syndrome. Though each of the three players is important in all thrombocytopenic diseases, one of the three dominant players typically establishes the individual features of each disease. In this review, we introduce the pathogeneses, symptoms, diagnostic measures, and recent therapeutic advances for the major diseases that should be immediately differentiated from DIC in sepsis.

Atypical Hemolytic Uremic Syndrome With the p.Ile1157Thr C3 Mutation Successfully Treated With Plasma Exchange and Eculizumab: A Case Report.

To describe a case of atypical hemolytic uremic syndrome induced by influenza A infection with the p.Ile1157Thr C3 mutation. DATA SOURCES: Clinical observations of a patient. STUDY SELECTION: Case reports. DATA EXTRACTION: Data extracted from medical records, after patient's consent. DATA SYNTHESIS: Four days prior to presentation to our hospital, a 16-year-old adolescent had a fever and arthralgia with hematuria. He was found to be positive for type A influenza and prescribed oseltamivir and acetaminophen by a primary-care physician. A bleeding tendency and purpura in the extremities and on the trunk developed; therefore, he was transferred to Chiba University Hospital. Hematology revealed severe thrombocytopenia, hyperbilirubinemia, and acute kidney injury. Aspartate aminotransferase, lactate dehydrogenase, and potassium could not be determined because of severe hemolysis. Highly elevated blood urea nitrogen and creatinine levels indicated acute kidney injury. A platelet count of 24,000/µL indicated thrombocytopenia, with low hemoglobin level. Peripheral blood profiling identified schistocytes. Continuous hemodiafiltration and plasma infusion were initiated immediately; however, he became oliguric. Plasma exchange was initiated on ICU day 3, but decreased urine output, hemolysis, and thrombocytopenia persisted. IV eculizumab therapy was initiated on day 7 and resulted in recovery of these symptoms and also successful discontinuation of renal support. The patient showed a stable condition without recurrence of hemolytic findings and acute kidney injury and is currently on maintenance therapy of eculizumab (1,200 mg, every other week) without any relapse of atypical hemolytic uremic syndrome symptoms. A plasma sample collected prior to initiation of plasma exchange showed an disintegrin-like and metalloprotease with thrombospondin type 1 motifs 13 activity level of 104.9%. The absence of both Shiga toxin-producing Escherichia coli in feces led to suspicion of atypical hemolytic uremic syndrome. Subsequent genetic analysis identified a mutation in C3 (p.Ile1157Thr), confirming the diagnosis of atypical hemolytic uremic syndrome. CONCLUSIONS: Although managing thrombocytopenia secondary to infection, inclusion of atypical hemolytic uremic syndrome in the differential diagnosis at an early stage is important in clinical practice.

Immunoadjuvant therapy in sepsis: novel strategies for immunosuppressive sepsis coming down the pike.

Recent efforts have focused on immunoadjuvant therapies for sepsis. The host inflammatory response consequent to initial exposure to pathogens is often followed by anti-inflammatory forces, resulting in increased morbidity and mortality in such critically ill patients. In the subacute stage of sepsis, apoptosis (type I programmed cell death) and subsequently autophagy (type II programmed cell death) have been attracting recent research interest. Although many patients may die during the initial cytokine storm, those who survive this phase might acquire defining characteristics of profound immunosuppression, including failure to clear the primary infection, development of secondary opportunistic infections, and reactivation of latent viruses. Both types of cell death are currently thought to be associated with this subacute immunosuppressive phase of sepsis, and acceleration of autophagy might alleviate immunosuppression through regulation of apoptosis of key immune effector cells. Programmed cell death 1 (PD-1) and its corresponding ligand play a major pathological role in immunosuppression not only in cancer but also in sepsis. Positive costimulatory pathways in T cells, such as CD28 signaling, permit the effector T cell to expand, persist, and effectively clear antigen. However, PD-1 is a negative costimulatory pathway on T cells that broadly enhances immunosuppressive signals across the innate and adaptive immune system. To counter this immunosuppression in sepsis, checkpoint blockade has garnered attention in an area of clinical research. In this review, we introduce some approaches of immunotherapy using anti-PD-1 antibody in infectious diseases and share our future perspectives.

Influence of autophagy on acute kidney injury in a murine cecal ligation and puncture sepsis model.

The role of autophagy in the maintenance of renal homeostasis during sepsis is not well understood. We therefore aimed to determine the influence of autophagy on kidney during sepsis using a murine sepsis model, i.e. cecal ligation and puncture (CLP). In CLP treated animals, the number of autolysosomes observed by electron microscopy increased over time. The number of autophagosomes in CLP animals decreased relative sham operated controls at 24 hrs after CLP, indicating that autophagy flux is already diminishing by that time. Moreover, CLP induced an increase in LC3-II/LC3-I ratio at 6-8 hrs, demonstrated in western blots, as well as an increase in GFP-LC3 dots at 6-8 hrs and 24 hrs, using immunofluorescence and anti-LC3 and LAMP1 antibodies on tissue sections from GFP-LC3 transgenic mice. LC3-II/LC3-I ratio and the number of co-localized GFP-LC3 dots and LAMP1 signals (GFP LC3 + LAMP1 dots) in CLP mice at 24 hrs were significantly reduced compared with data obtained at 6-8 hrs. Notably, acceleration of autophagy by rapamycin resulted in improvement of renal function that was associated with improvement in the histologic severity of tubular epithelial injury in CLP treated animals. Autophagy in the kidney was significantly slowed in the kidney during the acute phase of sepsis; nonetheless, autophagy in kidney appears to play a protective role against sepsis.

Blocking Liver Autophagy Accelerates Apoptosis and Mitochondrial Injury in Hepatocytes and Reduces Time to Mortality in a Murine Sepsis Model.

Autophagy plays an important role in cell survival, sequestering, and degrading a wide variety of substrates. Although an increase of autophagosomes in liver has been reported in sepsis patients as well as in septic mice, the influence of autophagy on liver injury, the interaction between autophagy, and other types of cell death in sepsis remain unclear. The aim of this study was to elucidate the contribution of liver autophagy to the pathophysiology of sepsis. We performed a cecal ligation and puncture on liver-specific autophagy-deficient (Alb-Cre/Atg5) mice (6-8-week-old male). When compared with controls (C57BL/6), we found a significant accumulation of p62 in the liver and demonstrated a greater number of cleaved caspase-3 immunoreactive hepatocytes in these knockout (KO) mice. Additionally, we confirmed a significant increase in autophagic vacuoles in the control mice relative to KO mice; in contrast, cell shrinkage and nuclear fragmentation (morphological characteristics of apoptosis) were preferentially seen in the KO mice by transmission electron microscopy. Severe mitochondrial damage was also prominent in KO mice, relative to controls, associated with an increase of reactive oxygen species in hepatocytes. Serum aspartate transaminase levels (P = 0.005) and serum interleukin-6 levels (P = 0.020) were significantly increased in the KO mice compared with controls. Deficiency of autophagy in liver significantly decreased survival in the murine sepsis model (P = 0.025). In conclusion, blocking liver autophagy accelerates time to mortality in the murine sepsis model, suggesting that liver autophagy plays a protective role for organ failure through degradation of damaged mitochondria, as well as prevention of apoptosis.

The Effects of Fasting and Massive Diarrhea on Absorption of Enteral Vancomycin in Critically Ill Patients: A Retrospective Observational Study.

PURPOSE: Although vancomycin (VCM) is not absorbed from healthy intestinal mucosa, elevations in the serum VCM concentrations have been reported in some cases. The aims of this study are to evaluate the necessity of therapeutic drug monitoring (TDM) during enteral VCM administration in critically ill patients. MATERIALS AND METHODS: In this retrospective study, we enrolled 19 patients admitted to our intensive care unit who were treated with enteral VCM from December 2006 to January 2014. Clinical factors were compared between two groups: Group E whose serum concentrations were detectable, and Group N whose concentrations were below the detection limit of the VCM assay. RESULTS: Group E comprises 7 patients, and Group N comprises 12 patients. The fasting duration in Group E was significantly longer compared with that in Group N (17 vs. 8 days, p = 0.023). Furthermore, there was a significant correlation between the serum VCM concentrations and the fasting duration (r = 0.79, p < 0.0001), and the amount of diarrhea (r = 0.46, p = 0.046). No difference was observed in the amount of diarrhea at the time of TDM (Group E; 1,850 mL vs. Group N; 210 mL, p = 0.055) and in the Sequential Organ Failure Assessment subscore for the renal system at the time of TDM (Group E; 4.0 vs. Group N; 1.5, p = 0.068). CONCLUSION: Long durations of fasting and massive diarrhea were associated with elevations in the serum VCM concentrations, which suggested that TDM might be necessary during enteral VCM administration in critically ill patients. TRIAL REGISTRATION: UMIN Clinical Trials Registry identifier UMIN000016955.

Pharmacokinetics of Standard- and Reduced-Dose Recombinant Human Soluble Thrombomodulin in Patients with Septic Disseminated Intravascular Coagulation during Continuous Hemodiafiltration.

INTRODUCTION: Recombinant human soluble thrombomodulin (rTM) is reportedly excreted by the kidneys; therefore, the recommended dose for patients with renal impairment is one-third of the standard dose. The aim of this study was to evaluate whether this reduced dose of rTM achieves effective drug concentrations that are comparable to those of the standard dose in treating sepsis-induced disseminated intravascular coagulation (DIC) during continuous hemodiafiltration (CHDF). METHODS: Eight patients in an intensive care unit were randomized to receive either reduced-dose (0.02 mg/kg, n = 4) or standard-dose (0.06 mg/kg, n = 4) rTM. We evaluated the effect of standard dose in comparison to that of reduced dose on the pharmacokinetics (PKs) of rTM for the sepsis-induced DIC patients receiving CHDF. Patients received rTM during a 30-min infusion for six consecutive days. PK parameters of rTM were analyzed using the one-compartment model. RESULTS: The elimination half-life, clearance (T1/2), and distribution volume of sTM were similar between the reduced and standard doses. The maximum concentration (Cmax) and area under the concentration-time curve (AUC) of sTM were approximately 2.5 times higher with standard-dose daily infusions than that with reduced-dose drip infusions (p = 0.041 and 0.062, respectively). The time when the blood concentration of sTM was >500 ng/mL, i.e., the holding time, was significantly longer with standard-dose infusions than those with reduced dose (p = 0.039). CONCLUSION: rTM displayed dose-dependent PK behavior at clinically relevant doses. During CHDF, effective blood concentration of rTM was not achieved with the reduced dose, and rTM was found to not bioaccumulate. Therefore, this pilot study suggests that reducing the rTM dose is unnecessary, even in sepsis-induced DIC patients who require CHDF. However, we need to perform a definitive study to determine the dosage of rTM for the case.

Pharmacokinetics of recombinant human soluble thrombomodulin in disseminated intravascular coagulation patients with acute renal dysfunction.

Recombinant human soluble thrombomodulin (ART-123) is a novel anticoagulant for patients with disseminated intravascular coagulation (DIC). It is widely used in clinical settings throughout Japan. Furthermore, a global Phase 3 study is currently being conducted. In healthy subjects, ART-123 is excreted mainly via the kidneys. Therefore, ART-123 dose decrease was recommended in DIC patients with severe renal dysfunction. However, the pharmacokinetics of ART-123 in DIC patients with severe acute renal dysfunction has not been elucidated. In an open-label, multicentre, prospective, clinical pharmacological study, we investigated the pharmacokinetics and safety of ART-123 upon repeated administration to DIC patients. ART-123 was administered to patients at a dose of 130 or 380 U/kg/day for six consecutive days. Plasma concentrations of ART-123 were measured at 21 time points until eight days after the final administration. Urinary excretion rates during the first 24 hours (h) were calculated. Patient renal functions were evaluated by measuring 24-h creatinine clearance (Ccr). Forty-three patients were enrolled in the present study. The urinary excretion rates of ART-123 correlated closely with 24-h Ccr. Total body clearance of ART-123 was also weakly related with 24-h Ccr. However, the plasma concentrations of ART-123 were not considerably different among patients with different renal function. Two patients had subcutaneous haemorrhage as an adverse event related to ART-123. In conclusion, plasma concentrations of ART-123 may not be different among patients with different renal functions. ART-123 was well tolerated in these patients.

Suppression of T Cell Autophagy Results in Decreased Viability and Function of T Cells Through Accelerated Apoptosis in a Murine Sepsis Model.

OBJECTIVE: While type 1 programmed cell death (apoptosis) of T cells leads to immunosuppression in sepsis, a crosstalk between apoptosis and autophagy (type 2 programmed cell death) has not been shown. The aim of this study is to elucidate the details of the interaction between autophagy and immunosuppression. DESIGN: Laboratory investigation in the murine sepsis model. SETTING: University laboratory. SUBJECTS: Six- to 8-week-old male mice. INTERVENTIONS: We investigated the kinetics of autophagy in T cells from spleen in a cecal ligation and puncture model with green fluorescent protein-microtubule-associated protein light chain 3 transgenic mice. We analyzed apoptosis, mitochondrial homeostasis and cytokine production in T cells, and survival rate after cecal ligation and puncture using T cell-specific autophagy-deficient mice. MEASUREMENTS AND MAIN RESULTS: We observed an increase of autophagosomes, which was assessed by flow cytometry. However, an autophagy process in CD4 T cells during sepsis was insufficient including the accumulation of p62. On the other hand, a blockade of autophagy accelerated T cell apoptosis compared with the control mice, augmenting the gene expression of Bcl-2-like 11 and programmed cell death 1. Furthermore, mitochondrial accumulation in T cells occurred via a blockade of autophagy during sepsis. In addition, interleukin-10 production in CD4 T cells from the cecal ligation and puncture-operated knockout mice was markedly increased. Consequently, deficiency of autophagy in T cells significantly decreased the survival rate in the murine sepsis model. CONCLUSIONS: We demonstrated that blocking autophagy accelerated apoptosis and increased mortality in concordance with the insufficient autophagy process in CD4 T cells in the murine sepsis model, suggesting that T cell autophagy plays a protective role against apoptosis and immunosuppression in sepsis.

Complete remission of thrombotic microangiopathy after treatment with eculizumab in a patient with non-Shiga toxin-associated bacterial enteritis: A case report.

UNLABELLED: To describe a case of complete remission of thrombotic microangiopathy after treatment with eculizumab in a patient with non-Shiga toxin-associated bacterial enteritis. CASE REPORT: Medical/surgical intensive care unit (ICU) of a university teaching hospital.A 62-year-old man presented to a local hospital with mucous and bloody stool persisting for 1 month and worsening abdominal pain for 2 weeks. He had thrombocytopenia and renal dysfunction and was admitted with a diagnosis of sepsis due to intraabdominal infection. However, he did not respond to antimicrobial therapy, and after 7 days he was transferred to the Chiba University Hospital ICU.Antimicrobial therapy was continued, and continuous hemodiafiltration was initiated on ICU day 3, but the patient's condition deteriorated and he became anuric. Plasma exchange (PE) was initiated on ICU day 11, but anuria and thrombocytopenia persisted. Intravenous eculizumab therapy was initiated on day 26 and resulted in quick recovery of urine output and platelet count and successful discontinuation of renal support.The diagnosis of thrombotic microangiopathy was established by the presence of schistocytes on the peripheral blood smear on ICU day 9. A plasma sample collected prior to initiation of PE showed a disintegrin-like and metalloproteinase with thrombospondin type 1 motifs member 13 (ADAMTS13) activity level of >10% (25.1%). The absence of both Shiga-toxin producing E coli in feces and anti-Shiga-toxin antibody in blood led to suspicion of atypical hemolytic uremic syndrome (aHUS). Genetic test identified a nonsynonymous mutation (p.Ala311Val) in the membrane cofactor protein gene (MCP).Although the pathological significance is currently unknown, this mutation may have been the cause of adult-onset aHUS in our patient. In this case, eculizumab was successfully introduced and discontinued, and the patient remained relapse-free after 1 year of follow-up. The duration of eculizumab therapy for patients with aHUS should be determined on a case-by-case basis and possibly according to the causative genetic mutation, even though discontinuation of eculizumab therapy once initiated is not generally recommended.