Sepsis: multiple abnormalities, heterogeneous responses, and evolving understanding.

Sepsis represents the host's systemic inflammatory response to a severe infection. It causes substantial human morbidity resulting in hundreds of thousands of deaths each year. Despite decades of intense research, the basic mechanisms still remain elusive. In either experimental animal models of sepsis or human patients, there are substantial physiological changes, many of which may result in subsequent organ injury. Variations in age, gender, and medical comorbidities including diabetes and renal failure create additional complexity that influence the outcomes in septic patients. Specific system-based alterations, such as the coagulopathy observed in sepsis, offer both potential insight and possible therapeutic targets. Intracellular stress induces changes in the endoplasmic reticulum yielding misfolded proteins that contribute to the underlying pathophysiological changes. With these multiple changes it is difficult to precisely classify an individual's response in sepsis as proinflammatory or immunosuppressed. This heterogeneity also may explain why most therapeutic interventions have not improved survival. Given the complexity of sepsis, biomarkers and mathematical models offer potential guidance once they have been carefully validated. This review discusses each of these important factors to provide a framework for understanding the complex and current challenges of managing the septic patient. Clinical trial failures and the therapeutic interventions that have proven successful are also discussed.

Dextran Sulfate Sodium Colitis Facilitates Colonization with Shiga Toxin-Producing Escherichia coli: a Novel Murine Model for the Study of Shiga Toxicosis.

Shiga toxin-producing Escherichia coli (STEC) bacteria are globally important gastrointestinal pathogens causing hemorrhagic gastroenteritis with variable progression to potentially fatal Shiga toxicosis. Little is known about the potential effects of E. coli-derived Shiga-like toxins (STXs) on host gastrointestinal immune responses during infection, in part due to the lack of a reproducible immunocompetent-animal model of STEC infection without depleting the commensal microbiota. Here, we describe a novel and reproducible murine model utilizing dextran sulfate sodium (DSS) colitis to induce susceptibility to colonization with clinical-isolate STEC strains. After exposure to DSS and subsequent oral STEC challenge, all the mice were colonized, and 66% of STEC-infected mice required early euthanasia. Morbidity during STEC infection, but not infection with an isogenic STEC mutant with toxin deleted, was associated with increased renal transcripts of the injury markers KIM1 and NGAL, histological evidence of renal tubular injury, and increased renal interleukin 6 gene (IL-6) and CXCL1 inflammatory transcripts. Interestingly, the intestinal burden of STEC during infection was increased compared to its isogenic Shiga toxin deletion strain. Increased bacterial burdens during Shiga toxin production coincided with decreased induction of colonic IL-23 axis transcripts known to be critical for clearance of similar gastrointestinal pathogens in mice, suggesting a previously undescribed role for STEC Shiga toxins in suppressing host immune responses during STEC infection and survival. The DSS+STEC model establishes infection with clinical-isolate strains of STEC in immunocompetent mice without depleting the gastrointestinal microbiota, enabling characterization of the effects of STXs on the IL-23 axis and other gastrointestinal pathogen-host interactions.

Quiescent complement in nonhuman primates during E coli Shiga toxin-induced hemolytic uremic syndrome and thrombotic microangiopathy.

Enterohemorrhagic Escherichia coli (EHEC) produce ribosome-inactivating Shiga toxins (Stx1, Stx2) responsible for development of hemolytic uremic syndrome (HUS) and acute kidney injury (AKI). Some patients show complement activation during EHEC infection, raising the possibility of therapeutic targeting of complement for relief. Our juvenile nonhuman primate (Papio baboons) models of endotoxin-free Stx challenge exhibit full spectrum HUS, including thrombocytopenia, hemolytic anemia, and AKI with glomerular thrombotic microangiopathy. There were no significant increases in soluble terminal complement complex (C5b-9) levels after challenge with lethal Stx1 (n = 6) or Stx2 (n = 5) in plasma samples from T0 to euthanasia at 49.5 to 128 hours post-challenge. d-dimer and cell injury markers (HMGB1, histones) confirmed coagulopathy and cell injury. Thus, complement activation is not required for the development of thrombotic microangiopathy and HUS induced by EHEC Shiga toxins in these preclinical models, and benefits or risks of complement inhibition should be studied further for this infection.

Impaired function of the Tie-2 receptor contributes to vascular leakage and lethality in anthrax.

The anthrax lethal toxin (LT) enters host cells and enzymatically cleaves MAPKKs or MEKs. How these molecular events lead to death from anthrax remains poorly understood, but published reports suggest a direct effect of LT on vascular permeability. We have found that LT challenge in mice disrupts signaling through Tie-2, a tonically activated receptor tyrosine kinase in the endothelium. Genetic manipulations favoring Tie-2 activation enhanced interendothelial junctional contacts, prevented vascular leakage, and promoted survival following a lethal dose of LT. Cleavage of MEK1/2 was necessary for LT to induce endothelial barrier dysfunction, and activated Tie-2 signaled through the uncleaved fraction of MEKs to prevent LT's effects on the endothelium. Finally, primates infected with toxin-secreting Bacillus anthracis bacilli developed a rapid and marked imbalance in the endogenous ligands that signal Tie-2, similar to that seen in LT-challenged mice. Our results show that B. anthracis LT blunts signaling through Tie-2, thereby weakening the vascular barrier and contributing to lethality of the disease. Measurement of circulating Tie-2 ligands and manipulation of Tie-2 activity may represent future prognostic and therapeutic avenues for humans exposed to B. anthracis.

Distinct renal pathology and a chemotactic phenotype after enterohemorrhagic Escherichia coli shiga toxins in non-human primate models of hemolytic uremic syndrome.

Enterohemorrhagic Escherichia coli cause approximately 1.5 million infections globally with 176,000 cases occurring in the United States annually from ingesting contaminated food, most frequently E. coli O157:H7 in ground beef or fresh produce. In severe cases, the painful prodromal hemorrhagic colitis is complicated by potentially lethal hemolytic uremic syndrome (HUS), particularly in children. Bacterial Shiga-like toxins (Stx1, Stx2) are primarily responsible for HUS and the kidney and neurologic damage that ensue. Small animal models are hampered by the inability to reproduce HUS with thrombotic microangiopathy, hemolytic anemia, and acute kidney injury. Earlier, we showed that nonhuman primates (Papio) recapitulated clinical HUS after Stx challenge and that novel therapeutic intervention rescued the animals. Here, we present detailed light and electron microscopic pathology examination of the kidneys from these Stx studies. Stx1 challenge resulted in more severe glomerular endothelial injury, whereas the glomerular injury after Stx2 also included prominent mesangiolysis and an eosinophilic inflammatory infiltration. Both toxins induced glomerular platelet-rich thrombi, interstitial hemorrhage, and tubular injury. Analysis of kidney and other organs for inflammation biomarkers showed a striking chemotactic profile, with extremely high mRNA levels for IL-8, monocyte chemoattractant protein 1, and macrophage inflammatory protein 1α and elevated urine chemokines at 48 hours after challenge. These observations give unique insight into the pathologic consequences of each toxin in a near human setting and present potential pathways for therapeutic intervention.

The sepsis model: an emerging hypothesis for the lethality of inhalation anthrax.

Inhalation anthrax is often described as a toxin-mediated disease. However, the toxaemia model does not account for the high mortality of inhalation anthrax relative to other forms of the disease or for the pathology present in inhalation anthrax. Patients with inhalation anthrax consistently show extreme bacteraemia and, in contrast to animals challenged with toxin, signs of sepsis. Rather than toxaemia, we propose that death in inhalation anthrax results from an overwhelming bacteraemia that leads to severe sepsis. According to our model, the central role of anthrax toxin is to permit the vegetative bacteria to escape immune detection. Other forms of B. anthracis infection have lower mortality because their overt symptoms early in the course of disease cause patients to seek medical care at a time when the infection and its sequelae can still be reversed by antibiotics. Thus, the sepsis model explains key features of inhalation anthrax and may offer a more complete understanding of disease pathology for researchers as well as those involved in the care of patients.

Rescue from lethal Shiga toxin 2-induced renal failure with a cell-permeable peptide.

Intestinal infection with Shiga toxin (Stx)-producing E.coli is a leading cause of hemolytic uremic syndrome and acute renal injury in otherwise healthy children in the US. Antibiotics are contraindicated and a therapeutic priority is agents that act intracellularly against the bacterial toxins that drive kidney injury. Our aim was to evaluate whether intravenous administration of a cell-permeable peptide (TVP) that binds to Stx2 will reduce disease severity and rescue juvenile baboons from a lethal Stx2 dose (50 ng/kg). TVP (5 mg/kg) was delivered i.v. simultaneously with toxin (prevention protocol) or at 6 or 24 h after toxin with daily 1 mg/kg supplements up to day 4 (rescue protocols). Biomarkers were monitored in blood and urine up to 28 days. TVP therapy resulted in either absence of clinical signs of acute kidney injury and normal urine output (prevention), or delayed and reduced BUN and creatinine levels (rescue) with concomitant survival. Delayed peptide administration significantly reduced thrombocytopenia, but surprisingly did not alter anemia even when monitored for 28 days in rescued survivors. This is the first successful cell-permeable therapeutic that counteracts Stx2 lethality in an animal model, which recapitulates many of the human responses to enteric infection.

Infection of Immunocompetent Conventional Mice with Shiga Toxin-Producing E. coli: The DSS + STEC Model.

Previous methods of infecting mice with Shiga toxin-producing E. coli (STEC) required suppression of host immune function or ablation of the gut microbiota to induce susceptibility to gastrointestinal colonization. Consequently, many pathogen-host interactions occurring in immunocompetent hosts during STEC infection and Shiga toxicosis have remained unclear. The following protocol describes the use of dextran sulfate sodium (DSS) to induce a mild colitis in immunocompetent conventional C57BL/6 mice to facilitate susceptibility to STEC infection by oral gavage. STEC colonization in infected mice was confirmed by recovery of live STEC via fecal cultures and quantified via quantitative polymerase chain reaction of fecal DNA for the STEC-specific gene eae. DSS colitis is well established, broadly reproducible, and does not require specialized equipment or high levels of technical proficiency to be a useful method of inducing susceptibility to gastrointestinal STEC colonization. The DSS + STEC mouse model provides a novel and useful tool for the exploration of local STEC-host interactions in the gut environment and the pathogenesis of Shiga toxicosis.

Distinct physiologic and inflammatory responses elicited in baboons after challenge with Shiga toxin type 1 or 2 from enterohemorrhagic Escherichia coli.

Shiga toxin-producing Escherichia coli is a principal source of regional outbreaks of bloody diarrhea and hemolytic-uremic syndrome in the United States and worldwide. Primary bacterial virulence factors are Shiga toxin types 1 and 2 (Stx1 and Stx2), and we performed parallel analyses of the pathophysiologies elicited by the toxins in nonhuman primate models to identify shared and unique consequences of the toxemias. After a single intravenous challenge with purified Stx1 or Stx2, baboons (Papio) developed thrombocytopenia, anemia, and acute renal failure with loss of glomerular function, in a dose-dependent manner. Differences in the timing and magnitude of physiologic responses were observed between the toxins. The animals were more sensitive to Stx2, with mortality at lower doses, but Stx2-induced renal injury and mortality were delayed 2 to 3 days compared to those after Stx1 challenge. Multiplex analyses of plasma inflammatory cytokines revealed similarities (macrophage chemoattractant protein 1 [MCP-1] and tumor necrosis factor alpha [TNF-alpha]) and differences (interleukin-6 [IL-6] and granulocyte colony-stimulating factor [G-CSF]) elicited by the toxins with respect to the mediator induced and timing of the responses. Neither toxin induced detectable levels of plasma TNF-alpha. To our knowledge, this is the first time that the in vivo consequences of the toxins have been compared in a parallel and reproducible manner in nonhuman primates, and the data show similarities to patient observations. The availability of experimental nonhuman primate models for Stx toxemias provides a reproducible platform for testing antitoxin compounds and immunotherapeutics with outcome criteria that have clinical meaning.

Effects of Temperature on the Composition and Xanthine Oxidase Inhibitory Activities of Caffeic Acid Roasting Products.

The high-temperature treatment of caffeic acid by a model reaction for the processing of foods by roasting enhanced its xanthine oxidase (XO) inhibitory activity. The thermal reaction products included various oligomeric compounds, whose structures were determined as being produced via the intermediate 4-vinylcatechol. Measurements of their XO inhibitory activities were also carried out. Among the identified oligomers, the coupling products of caffeic acid and vinylcatechol, which were mainly produced at 140-170 °C, presented stronger XO inhibitory activities than the other types of oligomers produced. Further reacted compounds, which were mainly formed at 200 °C by the addition or elimination of catechol unit in the oligomers, displayed weaker activities. These results indicated that thermal enhancement of the XO inhibitory activity of caffeic acid can be explained by the differences in the XO inhibitory activities of the various constituents of the thermal reaction products. Caffeic acid and its derivatives are polyphenols found widely distributed in foods. Moreover, XO inhibition is closely related to the prevention of the life-style-related disease gout. The results suggest that a simple roasting process (170 °C) can lend useful human-health-related functionalities to caffeic acid containing foods such as coffee.

A computational solution to improve biomarker reproducibility during long-term projects.

Biomarkers are fundamental to basic and clinical research outcomes by reporting host responses and providing insight into disease pathophysiology. Measuring biomarkers with research-use ELISA kits is universal, yet lack of kit standardization and unexpected lot-to-lot variability presents analytic challenges for long-term projects. During an ongoing two-year project measuring plasma biomarkers in cancer patients, control concentrations for one biomarker (PF) decreased significantly after changes in ELISA kit lots. A comprehensive operations review pointed to standard curve shifts with the new kits, an analytic variable that jeopardized data already collected on hundreds of patient samples. After excluding other reasonable contributors to data variability, a computational solution was developed to provide a uniform platform for data analysis across multiple ELISA kit lots. The solution (ELISAtools) was developed within open-access R software in which variability between kits is treated as a batch effect. A defined best-fit Reference standard curve is modelled, a unique Shift factor "S" is calculated for every standard curve and data adjusted accordingly. The averaged S factors for PF ELISA kit lots #1-5 ranged from -0.086 to 0.735, and reduced control inter-assay variability from 62.4% to <9%, within quality control limits. S factors calculated for four other biomarkers provided a quantitative metric to monitor ELISAs over the 10 month study period for quality control purposes. Reproducible biomarker measurements are essential, particularly for long-term projects with valuable patient samples. Use of research-use ELISA kits is ubiquitous and judicious use of this computational solution maximizes biomarker reproducibility.

The membrane proteinase 3 expression on neutrophils was downregulated after treatment with infliximab in patients with rheumatoid arthritis.

Proteinase 3 (PR3) expression on neutrophils was examined in rheumatoid arthritis (RA) patients before and after antitumor necrosis factor (TNF)-alpha therapy. Membrane PR3 expression from patients with either an infection or RA significantly increased. Membrane PR3 expression on neutrophils from RA patients treated with infliximab (anti-TNF-alpha antibody) therapy was less than in those without such treatment in a resting state, but the expression later increased after stimulation in vitro. Membrane PR3 expression increased because of the stimulation of TNFalpha, whereas it was significantly suppressed by plasma or alpha(1)-proteinase inhibitor. The condition of patients with RA improved after treatment with infliximab. Membrane PR3 expression on neutrophils in RA patients was downregulated by infliximab. As a result, PR3 might play an important role in the neutrophil-mediated inflammatory reaction in patients with either RA or an infection.

Microfluidic detection of movements of Escherichia coli for rapid antibiotic susceptibility testing.

Various nanomechanical movements of bacteria provide a signature of bacterial viability. Most notably, bacterial movements have been observed to subside rapidly and dramatically when the bacteria are exposed to effective antibiotics. Thus, monitoring bacterial movements, if performed with high fidelity, could offer a path to various clinical microbiological applications, including antibiotic susceptibility tests. Here, we introduce a robust and ultrasensitive electrical transduction technique for detecting the nanomechanical movements of bacteria. The technique is based on measuring the electrical fluctuations in a microfluidic channel, which the bacteria populate. The swimming of planktonic bacteria and the random oscillations of surface-immobilized bacteria both cause small but detectable electrical fluctuations. We show that this technique provides enough sensitivity to detect even the slightest movements of a single cell; we also demonstrate an antibiotic susceptibility test in a biological matrix. Given that it lends itself to smooth integration with other microfluidic methods and devices, the technique can be developed into a functional antibiotic susceptibility test, in particular, for urinary tract infections.

Shiga Toxin Therapeutics: Beyond Neutralization.

Ribotoxic Shiga toxins are the primary cause of hemolytic uremic syndrome (HUS) in patients infected with Shiga toxin-producing enterohemorrhagic Escherichia coli (STEC), a pathogen class responsible for epidemic outbreaks of gastrointestinal disease around the globe. HUS is a leading cause of pediatric renal failure in otherwise healthy children, resulting in a mortality rate of 10% and a chronic morbidity rate near 25%. There are currently no available therapeutics to prevent or treat HUS in STEC patients despite decades of work elucidating the mechanisms of Shiga toxicity in sensitive cells. The preclinical development of toxin-targeted HUS therapies has been hindered by the sporadic, geographically dispersed nature of STEC outbreaks with HUS cases and the limited financial incentive for the commercial development of therapies for an acute disease with an inconsistent patient population. The following review considers potential therapeutic targeting of the downstream cellular impacts of Shiga toxicity, which include the unfolded protein response (UPR) and the ribotoxic stress response (RSR). Outcomes of the UPR and RSR are relevant to other diseases with large global incidence and prevalence rates, thus reducing barriers to the development of commercial drugs that could improve STEC and HUS patient outcomes.

Proteinase 3 expression on neutrophil membranes from patients with infectious disease.

Proteinase-3 (PR3) is an abundant serine proteinase stored in the azurophilic granules of neutrophils and released to the cell surface upon activation where it contributes to local tissue destruction and inflammation. The sub-population of membrane PR3 (mPR3) high expression (PR3-high) varies among individuals. There are many reports about PR3 in Wegener's granulomatosis, but few about PR3 expression in patients with common inflammatory disorders, such as sepsis. The mPR3 expression on neutrophils from 56 patients with inflammatory disorders and from 64 healthy volunteers was examined by flow cytometry. High variability in the percentage of PR3-high (%PR3-high) neutrophils was observed in healthy volunteers and patients with inflammatory disease, and the %PR3-high was significantly greater in the patients (72 +/- 19% vs 55 +/- 20%, P < 0.0001). Overall neutrophil PR3 expression in patients with infectious diseases, especially systemic inflammatory response syndrome (SIRS) was significantly high (P < 0.01) and showed a positive correlation with C-reactive protein (CRP). Even under inflammatory conditions not involving autoimmune vasculitis, there are significant increases in both the absolute surface expression of PR3 and the numbers of neutrophils expressing high levels of PR3 and these correlate with CRP levels. The data are consistent with a model in which neutrophil membrane expression of PR3 is greatly influenced by an in vivo inflammatory environment.

Shiga toxin 2-induced endoplasmic reticulum stress is minimized by activated protein C but does not correlate with lethal kidney injury.

Enterohemorrhagic Escherichia coli produce ribotoxic Shiga toxins (Stx), which are responsible for kidney injury and development of hemolytic uremic syndrome. The endoplasmic reticulum (ER) stress response is hypothesized to induce apoptosis contributing to organ injury; however, this process has been described only in vitro. ER stress marker transcripts of spliced XBP1 (1.78-fold), HSP40 (4.45-fold) and CHOP (7.69-fold) were up-regulated early in kidneys of Stx2 challenged mice compared to saline controls. Anti-apoptotic Bcl2 decreased (-2.41-fold vs. saline) and pro-apoptotic DR5 increased (6.38-fold vs. saline) at later time points. Cytoprotective activated protein C (APC) reduced early CHOP expression (-3.3-fold vs. untreated), increased later Bcl2 expression (5.8-fold vs. untreated), and had early effects on survival but did not alter DR5 expression. Changes in kidney ER stress and apoptotic marker transcripts were observed in Stx2-producing C. rodentium challenged mice compared to mice infected with a non-toxigenic control strain. CHOP (4.14-fold) and DR5 (2.81-fold) were increased and Bcl2 (-1.65-fold) was decreased. APC reduced CHOP expression and increased Bcl2 expression, but did not alter mortality. These data indicate that Stx2 induces renal ER stress and apoptosis in murine models of Stx2-induced kidney injury, but decreasing these processes alone was not sufficient to alter survival outcome.

Pro-Coagulant Endothelial Dysfunction Results from EHEC Shiga Toxins and Host Damage-Associated Molecular Patterns.

Hemolytic uremic syndrome (HUS) from enterohemorrhagic Escherichia coli infection is a leading cause of kidney failure in otherwise healthy U.S. children. The bacterial Shiga toxins (Stx) induce the characteristic coagulopathy of HUS, but the damage to toxin-receptor expressing cells and organ injury due to ischemia likely also releases inflammatory damage-associated molecular patterns (DAMPs), which may exacerbate injury along with the toxins. To examine this, human aortic and renal glomerular cell anti-coagulant and barrier functions were studied after in vitro challenge with Stx1, Stx2, and DAMPs. There was significant loss of surface anti-coagulant protein C pathway molecules, increased expression of pro-thrombotic PAR1 and reduced protein C activation capability by 15-27%. Histones nearly completely prevented the activated protein C protection of endothelial cells from thrombin-induced permeability. In mice, lethal Stx2 challenge elevated plasma HMGB1 (day 2, 321 ± 118%; p < 0.01) and extracellular histones (day 3, 158 ± 62%; p < 0.01). Mice colonized with Stx2-expressing Citrobacter rodentium developed increased HMGB1 (day 5, 155 ± 55%; p < 0.01) and histones (day 3, 378 ± 188%; p < 0.01). Anti-histone antibody reduced both DAMPs to baseline, but was not sufficient to improve survival outcome or kidney function. Together, these data suggest a potential role Stx to produce DAMPs, and DAMPs to produce endothelial injury and a pro-thrombotic environment.

Reduced neutrophil CD10 expression in nonhuman primates and humans after in vivo challenge with E. coli or lipopolysaccharide.

CD10, also known as neutral endopeptidase or CALLA, is a major metalloproteinase that regulates levels of biologically active peptides that initiate inflammatory, cardiovascular, and neurogenic responses. Relative tissue expression levels of CD10, its peptide substrates, and their receptors constitute the basic regulatory mechanism. Neutrophils contain abundant CD10 and are rapid responders to an inflammatory septic challenge. Expression of neutrophil surface antigens in response to inflammation was studied in the primate model of Escherichia coli-mediated sepsis and in human volunteers injected with lipopolysaccharide (LPS). There was a rapid and profound (up to 95%) reduced baboon neutrophil CD10 expression in response to E. coli injections of 5.71 x 106 CFU/kg to 2.45 x 109 CFU/kg that gradually resolved to preinjection levels. The reduction was both dose and time dependent. Reduced CD10 antigen on mature baboon neutrophils and bands was observed by immunohistochemistry. Human volunteers challenged with 4ng/kg LPS experienced transient chills, nausea, fever, and myalgia. Up to approximately 20% of their neutrophils had reduced CD10 expression, peaking at 2 to 8 h after injection. By 24 h, neutrophil CD10 expression resolved to preinjection levels. In contrast, in both the baboon and human studies, other neutrophil surface antigens were only slightly decreased (CD11a) or increased (CD11b, CD18, CD35, CD66b, and CD63). These data present the novel observation that neutrophil CD10 expression decreases significantly in response to in vivo inflammatory challenge. This decrease appears to be unique to CD10 and may contribute to a reduced regulation of bioactive peptides released in response to inflammatory challenge.

Complement, thrombotic microangiopathy and disseminated intravascular coagulation.

In the blurring boundaries between clinical practice and scientific observations, it is increasingly attractive to propose shared disease mechanisms that could explain clinical experience. With the advent of available therapeutic options for complement inhibition, there is a push for more widespread application in patients, despite a lack of clinically relevant research. Patients with disseminated intravascular coagulation (DIC) and thrombotic microangiopathies (TMA) frequently exhibit complement activation and share the clinical consequences of thrombocytopenia, microangiopathic hemolytic anemia, and microvascular thrombosis. However, they arise from very different molecular etiologies giving rise to cautious questions about inclusive treatment approaches because most clinical observations are associative and not cause-and-effect. Complement inhibition is successful in many cases of atypical hemolytic uremic syndrome, greatly reducing morbidity and mortality of patients by minimizing thrombocytopenia, microangiopathic hemolytic anemia, and microvascular thrombosis. But is this success due to targeting disease etiology or because complement is a sufficiently systemic target or both? These questions are important because complement activation and similar clinical features also are observed in many DIC patients, and there are mounting calls for systemic inhibition of complement mediators despite the enormous differences in the primary diseases complicated by DIC. We are in great need of thoughtful and standardized assessment with respect to both beneficial and potentially harmful consequences of complement activation in these patient populations. In this review, we discuss about what needs to be done in terms of establishing the strategy for complement inhibition in TMA and DIC, based on the current knowledge.