Toraymyxin® (Toray Medical Co., Ltd, Tokyo, Japan) has been developed as a direct hemoperfusion column that contains polymyxin B-immobilized fiber to bind endotoxins in the patients’ blood. Toraymyxin was approved by the Japanese National Health Insurance system for the treatment of endotoxemia and septic shock in 1994. We reviewed and analyzed clinical history and evidence of Toraymyxin, and assessed the current status of Toraymyxin use for the treatment of severe sepsis and septic shock.
Our review shows that Toraymyxin appeared to be effective in improving hemodynamics and respiratory function in septic shock requiring emergency abdominal surgery. The recent large-scale RCTs could not demonstrate whether prognosis is improved by Toraymyxin. The clinical studies based on large-scale data-base from Japan revealed that Toraymyxin appeared to have a survival benefit in patients with severe condition of septic shock. We also commented on the revised version of health insurance adaptation of Toraymyxin in April, 2020.
Periodontal disease is one of the major diseases afflicting human beings and is caused by a bacterial infection leading to gingival inflammation, periodontal tissue destruction, alveolar bone loss, and eventually to tooth loss. Chronic periodontitis is one of the major periodontal diseases, and the periodontal pathogen Porphyromonas gingivalis is frequently isolated from diseased sites in the patients with this disease. The major virulence factors produced by P. gingivalis induce cysteine proteinases, gingipains. We found that gingipains cleave CD14 on human monocytes and human gingival fibroblasts and ICAM-1 on human gingival epithelial cells, resulting in immune evasion by the bacterium in periodontal tissues. Furthermore, gingipains are capable of inducing the production of proinflammatory molecules, such as IL-33 by human gingival epithelial cells, and IL-31 by human mast cells. Porphyromonas gingivalis-induced IL-33 and IL-31 attenuated the production of LL-37, an antimicrobial peptide, and the expression of claudin-1, a tight junction molecule, respectively, in gingival epithelial cells. These findings suggest that IL-33 and IL-31 induced by gingipains resulted in the induction of chronic inflammation and the dysfunction of epithelial barrier. On the basis of our findings, the pathogenicity of gingipains is divided into two phases : (a) the acute inflammation during severe infection, where P. gingivalis produces a large amount of gingipains resulting in the proteolytic degradation of the host molecules, and (b) chronic inflammation during mild infection, where P. gingivalis produce a small amount of gingipains, resulting in the induction of chronic inflammatory conditions and the consequent impairment of innate immunity.
Emerging data suggest that sepsis survivors frequently experience long-term mental impairment after discharge. This is assumed to be caused by sepsis-associated encephalopathy (SAE) ; however, the relationship between the onset of long-term mental impairments and SAE is still unclear. In addition, several studies have revealed that the development of mental impairment results in neuroinflammation, which is induced by aberrant activated immune cells, including glial cells and T cells. However, it is still unclear how these immune cells contribute to the pathogenesis and attenuation of SAE. In this study, we performed animal behavioral tests and found that anxiety-like behavior was exacerbated in mice with sepsis. Furthermore, these mice had elevated levels of pro-inflammatory cytokines such as interleukin (IL)-1β in the brain, depending on the severity of sepsis. Interestingly, sepsis induced an increase in microglia and a decrease in astrocytes in mice during the acute/subacute phase ; T cell numbers also increased in the brains of mice with sepsis. To investigate how brain-accumulated T cells influence the development of SAE and mental impairments, we treated mice with sepsis with FTY720 to inhibit recruitment. This immunomodulator delayed the recovery of anxiety-like behavior, and the number of astrocytes in the brain remained reduced during the chronic phase of sepsis in mice. Additionally, the expression of Il1b in the brains of FTY720-treated mice with sepsis had increased during the chronic phase. Our findings suggest that infiltrated T cells contribute to the attenuation of SAE and alleviate SAE-induced mental disorders by alleviating neuroinflammation in the chronic phase of sepsis.
Recently, a cecal slurry (CS) method was established by Wynn JL, in which a fecal suspension (CS) was prepared from contents of the appendix of adult mice and intraperitoneally administered to the subject mice. In addition, by creating and storing a large amount of CS preservation solution at once following the method reported by Starr ME, the experimental reproducibility of CS model has been improved.
We have applied these techniques to 4-day old newborn mice and created preterm sepsis mouse model. We believe that CS model is the best solution for modeling neonatal sepsis in the condition comparing between same-age newborn pups. In this paper, the current status of our application of this model to elucidate clinical questions will be outlined.
Rates of mortality due to sepsis remains high. Endotoxin is the cell wall of gram negative bacteria and it is one of the most major mediators of inflammation in sepsis. Therefore, the measurement of endotoxin in blood is important to select clinical treatment for sepsis/septic shock patients. The standard assay for determination of endotoxin has been the limulus amebocyte lysate assay. However, some reports founded that the usefulness of this assay was limited. Endotoxin activity assay (EAA) is another method for measurement of endotoxin levels in blood. Some studies reported that EAA had a significant correlation with mortality, severity and other biomarkers. Moreover, the EUPHRATES trial showed the possibility that EAA had an usefulness for selecting responsive patient population to PMX-DHP. However, it remains unclear whether EAA is useful for septic patients and we need more research to evaluate its potential. This report serves to present the available knowledge concerning EAA while referencing to previous reports and data from our intensive care units.
Toll-like receptors (TLRs) are transmembrane proteins classified into pattern recognition receptors (PRRs). TLRs are found in cell surface and/or endocytic vehicles in immune cells. TLRs activate innate immunity by recognizing pathogen-associated molecular patterns (PAMPs) derived from bacteria and viruses and damage-associated molecular patterns (DAMPs) produced by damaged tissue. The activation of innate immunity induces adaptive immunity that plays an important role in the host defense against pathogenic infection. On the other hand, abnormal activation of innate immunity causes immune disorders such as autoimmune disease. Therefore, TLRs are important molecules in the immune system. So far, 10 types of TLRs (TLR1 to TLR10) in humans and 12 types of TLRs (TLR1 to TLR9, TLR11 to TLR13) in mice have been found. TLR1, TLR2, TLR4, and TLR6 are found on the cell surface, and TLR3, TLR7, TLR8, and TLR9 are found in the endosomal compartment. Each TLR recognizes specific PAMPs and DAMPs ; lipopeptides for TLR1/2 and TLR2/6 ; nucleic acid structures for TLR3, TLR7, TLR8 and TLR9 ; lipopolysaccharide (LPS) structures and heat-shock proteins for TLR4. In recent years, various ligands activating TLRs signal have also been chemically synthesized and have been utilized as effective adjuvants in antiviral and anticancer therapy. Regarding TLR7 ligand (TLR7L), conjugates with peptides, polymers, proteins, glycans, and nanoparticles have attracted much attention because they have great potencies on the humoral and cellular immune response. In this paper, we describe recent progress in the synthesis and biological property of TLR7 ligand conjugate containing our recent study.
Bee venom (BV) induces skin inflammation, characterized by erythema, blisters, edemas, pain, and itching. Although BV has been found to have an inhibitory effect on toll-like receptors (TLRs), we here show that BV enhances keratinocyte responses to polyinosinic-polycytidylic acid (poly (I : C)), a ligand for TLR3. Our results revealed that the enhanced TLR activity was primarily induced by secretory phospholipase A2 (sPLA2), a component of BV (BV-sPLA2). PLA2 mediates the hydrolysis of membrane phospholipids into lysophospholipids and free fatty acids. We demonstrated that BV-sPLA2 increased the intracellular uptake of poly (I : C), phosphorylation of the nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs), and poly (I : C)-mediated interleukin 8 (IL-8) production in human keratinocytes. We further showed that the enzymatic activity of BV-sPLA2 was essential for the increased uptake of poly (I : C). These findings suggest that BV-sPLA2 may induce a modification of the cell membrane structure, leading to enhanced poly (I : C) uptake in keratinocytes. BV-sPLA2 might be able to promote wound healing by enhancing TLR3 responses.
We present a case of 71-year-old man who developed severe septic shock following transurethral resection of prostate (TURP) and cystolitholapaxy (CLL), showing favorable response after multidisciplinary treatment consisting PMX-DHP and CHDF. The patient underwent TURP for prostatic hypertrophy and CLL for bladder stone. The operation was completed with no intraoperative complications. 3 days after operation, a 39.8°C fever, blood pressure decreased and clouding of the consciousness were observed. The patient was transferred to the intensive care unit (ICU) and treated with mechanical ventilation and appropriate drug therapy. 5 hours after admission to the ICU, first PMX-DHP treatment was performed for 16 hours, but hemodynamics was not improved and became no urination. Second PMX-DHP and CHDF treatment performed 9 hours after first PMX-DHP. After second PMX-DHP the patient got stable hemodynamics and sufficient urine volume. Mechanical ventilation was ended 9 days after the ICU admission and moved to the general ward.
Due to the impaired peripheral perfusion during hemodynamic crisis, all toes and 2 fingers were necrosis and amputated. After 6 months of rehabilitation, he discharged to home.
Limulus amebocyte lysate (LAL) is widely used for detection of endotoxin, one of the most potent pyrogen. Recent studies revealed the mechanism of activation of Factor C and Factor B, endotoxin binding proteins in LAL cascade. It is well known that Factor C is the first factor to bind endotoxin aggregates. The second coagulation factor, Factor B is important to achieve specificity of LAL to endotoxin because activated Factor C on endotoxin aggregates are essential for its activation. The endotoxin-specific signal is amplified after the Factor B activation in LAL. On the other hand, recombinant Factor C reagents rely on only the specificity of Factor C to endotoxin, and amplifies the trypsin-like activity of activated Factor C that may not be specific to endotoxin. This mechanism seems not to be as specific to endotoxin as LAL. More evaluation and improvement are necessary for recombinant reagents for endotoxin measurement as a safety test.
Cellular senescence is associated with the induction of a proinflammatory phenotype. Previous studies revealed that senescent endothelial cells are localized at the sites of atherosclerotic lesions, suggesting the involvement of endothelial cell senescence in atherogenesis. Importantly, bacterial infection has been speculated to contribute to the pathogenesis of atherosclerosis ; however the effect of bacterial components or host-derived antimicrobial substances on senescent endothelial cells has not fully understood. Here, we investigated the effects of Gram-negative bacterial lipopolysaccharide (LPS) and antimicrobial peptide LL-37, which is deposited at the atherosclerotic loci, on senescent endothelial cells using serially passaged human umbilical vein endothelial cells (HUVECs). The results indicated that senescent endothelial cells basally exhibited proinflammatory phenotype, as evidenced by increased expression of intercellular adhesion molecule-1 (ICAM-1) and activation of NF-κB (phosphorylation of p65), compared with non-senescent cells. Of note, senescent endothelial cells more potently responded to the stimulation of LPS or LL-37, as evidenced by further enhanced ICAM-1 expression and NF-κB activation, compared with non-senescent cells. Moreover, expression levels of the receptors for LPS (TLR4) and LL-37 (purinergic receptor P2X7) were upregulated in senescent endothelial cells, suggesting that both LPS and LL-37 enhance the ICAM-1 expression and NF-κB activation in senescent endothelial cells possibly via the upregulated TLR4 and P2X7. Together these observations indicate that senescent endothelial cells may contribute to the pathogenesis of atherosclerosis via the basal proinflammatory phenotype and the enhanced inflammatory responses against atherogenic factors including bacterial LPS and host-derived antimicrobial LL-37.
The immunological activity of LPS is closely related with the localization of fatty acids and the acyloxyacyl structures formed by them in lipid A molecules. MALDI-TOF mass spectrometry (MS) is commonly used to determine the fatty acid localization by detecting oxonium ions derived from non-reducing end glucosamine by the split of glucosamine disaccharide of lipid A. However, alternative method is required because the sensitivity in MS is reduced when the purity of lipid A preparation is not enough. To solve this problem, we developed the method of periodate oxidation. The glucosamine disaccharide of lipid A was split by sodium periodate after reduction with NaBH4 and hemiacetal ring of reducing end glucosamine was opened. Through this oxidation, non-reducing end glucosamine with fatty acids bound to it remained, and could be detected by MALDI-TOF MS. This method was applied to the wild-type lipid A, and the modified lipid A of Escherichia coli strains KGU0485 and KGU0496 constructed in the previous study by the introduction of Klebsiella acyltransferase gene. The results indicated that the method was useful for the determination of fatty acid-localization in lipid A molecules.
Tuberculosis (TB) remains an important infectious disease, causing ten million new cases and 1.4 million deaths a year in worldwide. Elucidating pathogenicity of Mycobacterium tuberculosis, the causative agent of TB, will contribute to development of new drugs, vaccines and treatments. Proline-glutamic acid (PE) /proline-proline-glutamic acid (PPE) family accounts for approximately 10% of the coding region of M. tuberculosis genome and its functions are largely unknown. PE proteins having polymorphic GC-rich repetitive sequences (PGRS) in carboxyl-terminal are members of PE_PGRS family. PE_PGRS62 and PE_PGRS30 are members of PE_PGRS family and homologues of MAG24, the virulence factor of M. marinum. We are in the process of analyzing the functions of PE_PGRS62 and PE_PGRS30, and have results suggesting that PE_PGRS62 regulates autophagy, whereas PE_PGRS30 induces cell death.
It is widely recognized that an early detection of life-threatening sepsis or septic shock using biomarkers is of great importance for successful treatment of diseases. Bacterial endotoxins in human blood are one of the target analytes to identify sepsis patients. It has been half a century since endotoxin determination techniques with Limulus Amebocyte Lysate (LAL) were applied to human plasma using the gel-clot method. Thereafter, chromogenic and turbidimetric techniques with high specificity to endotoxin or β-D-glucan were successfully developed, and these technologies enable detection of trace amounts of the analytes in blood. With reference to FDA-approved β-D-glucan detection, it became a global standard to aim for early diagnosis of invasive fungal diseases. A number of questions and problems with endotoxin detection, however, still remain unsolved from the standpoint of clinical significance.
In this review, firstly we focus on development history, recent advances and limitations of LAL techniques coupled with extraction methodologies from blood, and secondly, we suggest a new approach to the solution of the above for biomarker-guided therapy and clinical investigations.
Evidence for endotoxin removal therapy with polymyxin B immobilized column (PMX) has been accumulated these more than two decades. However, the evidence level is still weak. RCT is currently on going in US. The targeted patient’s population with high, but some definitive range of blood endotoxin level and organ dysfunction score who is likely to benefit from PMX therapy could be clarified through this trial.
Recently, PMX has been used for the different kind of patients’ population such as acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF) and septic shock due to viral infection such as H1N1. Improvement of hemodynamics and oxygenation have been reported in these patients’ population as same as the case of endotoxemic septic shock. Under the current SARS-CoV-2 pandemic, it is reported PMX was applied for COVID-19 patient. The reduction of inflammatory state of the patient and the improvement of chest radiographic findings were observed.
Interestingly, the effective action of PMX therapy looks happening in a similar fashion for these different kinds of patient’s population. The comprehensive mechanism of action to explain this clinical effectiveness is urgently needed to be studied.
PMX has a multi-faceted action such as endotoxin removal, removal of pro- and anti-inflammatory mediators, improvement of coagulation abnormality, removal of proapoptotic factor and immunomodulatory effect due to monocyte and neutrophil removal. It is required to study how these actions could lead to the improvement of each organ dysfunction.
Presepsin (P-SEP), an approximately 70-amino-acid fragment of CD14 : a lipopolysaccharide (LPS) receptor that transmits signals through CD14-MD-2/TLR4 to cells, was first discovered as a sepsis marker in 2002 when high concentrations of a protein with soluble CD14-like immunoreactivity were detected in the blood of sepsis patients. We used an immunoassay to measure the serum concentrations of the new peptide, which was initially named soluble CD14-subtype (sCD14-ST) and later renamed presepsin.
Rabbit sepsis models revealed that presepsin is induced by the cecal ligation and punctual (CLP) sepsis model but not by the LPS injection model. In vitro experiments using human monocytes and neutrophils suggested that presepsin is produced when bacteria are phagocytosed by immune cells. The first clinical study was initiated by Professor Shigeatsu Endo (Iwate Medical University, Japan) ; it demonstrated that sepsis patients have higher presepsin levels compared with Systemic Inflammatory Response Syndrome (SIRS) patients, indicating that presepsin could be useful for sepsis diagnosis. This interesting marker is not only helpful for the diagnosis of sepsis but is also indicative of disease severity, as measured by the Sequential Organ Failure Assessment (SOFA) score.
More research remains necessary, as understanding of the fundamental aspects of presepsin is still limited. Presepsin is elevated in high mortality elective cardiac surgery and in severe COVID-19 patients, but the mechanism by which presepsin production is increased during severe COVID-19 disease is unknown. In this article, presepsin biochemistry is described, then the application of presepsin in clinical diagnosis is discussed.
Sepsis Panel of Verigene® System is an automated multi-item genetic testing system that is able to simultaneously detect bacteria and drug resistance genes for the purpose of selecting therapeutic agents for patients with sepsis.
Verigene® System started selling in 2016 and is covered by insurance in June 2017.
Compared to the conventional method that took a long time, the Verigene® system enables 24-48 hours earlier than the existing method to identify major bacteria and to rapidly detect drug resistance genes. This enables faster and more effective and appropriate antibacterial drug selection in the treatment for sepsis.