Protocol for TRAUMADORNASE: a prospective, randomized, multicentre, double-blinded, placebo-controlled clinical trial of aerosolized dornase alfa to reduce the incidence of moderate-to-severe hypoxaemia in ventilated trauma patients.

BACKGROUND: Acute respiratory distress syndrome continues to drive significant morbidity and mortality after severe trauma. The incidence of trauma-induced, moderate-to-severe hypoxaemia, according to the Berlin definition, could be as high as 45%. Its pathophysiology includes the release of damage-associated molecular patterns (DAMPs), which propagate tissue injuries by triggering neutrophil extracellular traps (NETs). NETs include a DNA backbone coated with cytoplasmic proteins, which drive pulmonary cytotoxic effects. The structure of NETs and many DAMPs includes double-stranded DNA, which prevents their neutralization by plasma. Dornase alfa is a US Food and Drug Administration-approved recombinant DNase, which cleaves extracellular DNA and may therefore break up the backbone of NETs and DAMPs. Aerosolized dornase alfa was shown to reduce trauma-induced lung injury in experimental models and to improve arterial oxygenation in ventilated patients. METHODS: TRAUMADORNASE will be an institution-led, multicentre, double-blinded, placebo-controlled randomized trial in ventilated trauma patients. The primary trial objective is to demonstrate a reduction in the incidence of moderate-to-severe hypoxaemia in severe trauma patients during the first 7 days from 45% to 30% by providing aerosolized dornase alfa as compared to placebo. The secondary objectives are to demonstrate an improvement in lung function and a reduction in morbidity and mortality. Randomization of 250 patients per treatment arm will be carried out through a secure, web-based system. Statistical analyses will include a descriptive step and an inferential step using fully Bayesian techniques. The study was approved by both the Agence Nationale de la Sécurité du Médicament et des Produits de Santé (ANSM, on 5 October 2018) and a National Institutional Review Board (CPP, on 6 November 2018). Participant recruitment began in March 2019. Results will be published in international peer-reviewed medical journals. DISCUSSION: If early administration of inhaled dornase alfa actually reduces the incidence of moderate-to-severe hypoxaemia in patients with severe trauma, this new therapeutic strategy may be easily implemented in many clinical trauma care settings. This treatment may facilitate ventilator weaning, reduce the burden of trauma-induced lung inflammation and facilitate recovery and rehabilitation in severe trauma patients. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03368092. Registered on 11 December 2017.

[50 Years ago: First Successful Treatment of Acute Allograft Rejection after a Human Heart Transplantation]. / Vor 50 Jahren: Erste erfolgreiche Behandlung einer akuten Herztransplantat-Abstoßungskrise.

The first clinical use of the "Munich antilymphocyte globulin" (ALG) at the occasion of the first successful human heart transplantation is briefly described. The cardiac transplantation was carried out by Christiaan Barnard and his team in Cape Town, South Africa, in 1968. The patient developed an acute allograft rejection which could be successfully reversed within three weeks using the intravenous administration of ALG. This event can be regarded as the beginning of a success story of ALG in its use as a powerful immunosuppressive agent in all categories of clinical organ transplantation.

Origin and Consequences of Necroinflammation.

When cells undergo necrotic cell death in either physiological or pathophysiological settings in vivo, they release highly immunogenic intracellular molecules and organelles into the interstitium and thereby represent the strongest known trigger of the immune system. With our increasing understanding of necrosis as a regulated and genetically determined process (RN, regulated necrosis), necrosis and necroinflammation can be pharmacologically prevented. This review discusses our current knowledge about signaling pathways of necrotic cell death as the origin of necroinflammation. Multiple pathways of RN such as necroptosis, ferroptosis, and pyroptosis have been evolutionary conserved most likely because of their differences in immunogenicity. As the consequence of necrosis, however, all necrotic cells release damage associated molecular patterns (DAMPs) that have been extensively investigated over the last two decades. Analysis of necroinflammation allows characterizing specific signatures for each particular pathway of cell death. While all RN-pathways share the release of DAMPs in general, most of them actively regulate the immune system by the additional expression and/or maturation of either pro- or anti-inflammatory cytokines/chemokines. In addition, DAMPs have been demonstrated to modulate the process of regeneration. For the purpose of better understanding of necroinflammation, we introduce a novel classification of DAMPs in this review to help detect the relative contribution of each RN-pathway to certain physiological and pathophysiological conditions.

Molecular and Translational Classifications of DAMPs in Immunogenic Cell Death.

The immunogenicity of malignant cells has recently been acknowledged as a critical determinant of efficacy in cancer therapy. Thus, besides developing direct immunostimulatory regimens, including dendritic cell-based vaccines, checkpoint-blocking therapies, and adoptive T-cell transfer, researchers have started to focus on the overall immunobiology of neoplastic cells. It is now clear that cancer cells can succumb to some anticancer therapies by undergoing a peculiar form of cell death that is characterized by an increased immunogenic potential, owing to the emission of the so-called "damage-associated molecular patterns" (DAMPs). The emission of DAMPs and other immunostimulatory factors by cells succumbing to immunogenic cell death (ICD) favors the establishment of a productive interface with the immune system. This results in the elicitation of tumor-targeting immune responses associated with the elimination of residual, treatment-resistant cancer cells, as well as with the establishment of immunological memory. Although ICD has been characterized with increased precision since its discovery, several questions remain to be addressed. Here, we summarize and tabulate the main molecular, immunological, preclinical, and clinical aspects of ICD, in an attempt to capture the essence of this phenomenon, and identify future challenges for this rapidly expanding field of investigation.

The Role of Damage-Associated Molecular Patterns (DAMPs) in Human Diseases: Part II: DAMPs as diagnostics, prognostics and therapeutics in clinical medicine.

This article is the second part of a review that addresses the role of damage-associated molecular patterns (DAMPs) in human diseases by presenting examples of traumatic (systemic inflammatory response syndrome), cardiovascular (myocardial infarction), metabolic (type 2 diabetes mellitus), neurodegenerative (Alzheimer's disease), malignant and infectious diseases. Various DAMPs are involved in the pathogenesis of all these diseases as they activate innate immune machineries including the unfolded protein response and inflammasomes. These subsequently promote sterile autoinflammation accompanied, at least in part, by subsequent adaptive autoimmune processes. This review article discusses the future role of DAMPs in routine practical medicine by highlighting the possibility of harnessing and deploying DAMPs either as biomarkers for the appropriate diagnosis and prognosis of diseases, as therapeutics in the treatment of tumours or as vaccine adjuncts for the prophylaxis of infections. In addition, this article examines the potential for developing strategies aimed at mitigating DAMPs-mediated hyperinflammatory responses, such as those seen in systemic inflammatory response syndrome associated with multiple organ failure.

The Role of Damage-Associated Molecular Patterns in Human Diseases: Part I - Promoting inflammation and immunity.

There is increasing interest by physicians in the impact of the innate immune system on human diseases. In particular, the role of the molecules that initiate and amplify innate immune pathways, namely damage-associated molecular patterns (DAMPs), is of interest as these molecules are involved in the pathogenesis of many human disorders. The first part of this review identifies five classes of cell stress/tissue injury-induced DAMPs that are sensed by various recognition receptor-bearing cells of the innate immune system, thereby mounting inflammation, promoting apoptosis and shaping adaptive immune responses. The DAMPs activate and orchestrate several innate immune machineries, including inflammasomes and the unfolded protein response that synergistically operates to induce inflammatory, metabolic and adaptive immune pathologies. Two examples of autoimmune diseases are discussed as they represent a typical paradigm of the intimate interplay between innate and adaptive immune responses.

Danger signals - damaged-self recognition across the tree of life.

Multicellular organisms suffer injury and serve as hosts for microorganisms. Therefore, they require mechanisms to detect injury and to distinguish the self from the non-self and the harmless non-self (microbial mutualists and commensals) from the detrimental non-self (pathogens). Danger signals are "damage-associated molecular patterns" (DAMPs) that are released from the disrupted host tissue or exposed on stressed cells. Seemingly ubiquitous DAMPs are extracellular ATP or extracellular DNA, fragmented cell walls or extracellular matrices, and many other types of delocalized molecules and fragments of macromolecules that are released when pre-existing precursors come into contact with enzymes from which they are separated in the intact cell. Any kind of these DAMPs enable damaged-self recognition, inform the host on tissue disruption, initiate processes aimed at restoring homeostasis, such as sealing the wound, and prepare the adjacent tissues for the perception of invaders. In mammals, antigen-processing and -presenting cells such as dendritic cells mature to immunostimulatory cells after the perception of DAMPs, prime naïve T-cells and elicit a specific adaptive T-/B-cell immune response. We discuss molecules that serve as DAMPs in multiple organisms and their perception by pattern recognition receptors (PRRs). Ca(2+)-fluxes, membrane depolarization, the liberation of reactive oxygen species and mitogen-activated protein kinase (MAPK) signaling cascades are the ubiquitous molecular mechanisms that act downstream of the PRRs in organisms across the tree of life. Damaged-self recognition contains both homologous and analogous elements and is likely to have evolved in all eukaryotic kingdoms, because all organisms found the same solutions for the same problem: damage must be recognized without depending on enemy-derived molecules and responses to the non-self must be directed specifically against detrimental invaders.

Transfusion-Related Acute Lung Injury: The Work of DAMPs.

Current notions in immunology hold that not only pathogen-mediated tissue injury but any injury activates the innate immune system. In principle, this evolutionarily highly conserved, rapid first-line defense system responds to pathogen-induced injury with the creation of infectious inflammation, and non-pathogen-induced tissue injury with 'sterile' tissue inflammation. In this review, evidence has been collected in support of the notion that the transfusion-related acute lung injury induces a 'sterile' inflammation in the lung of transfused patients in terms of an acute innate inflammatory disease. The inflammatory response is mediated by the patient's innate immune cells including lung-passing neutrophils and pulmonary endothelial cells, which are equipped with pattern recognition receptors. These receptors are able to sense injury-induced, damage-associated molecular patterns (DAMPs) generated during collection, processing, and storage of blood/blood components. The recognition process leads to activation of these innate cells. A critical role for a protein complex known as the NLRP3 inflammasome has been suggested to be at the center of such a scenario. This complex undergoes an initial 'priming' step mediated by 1 class of DAMPs and then an 'activating' step mediated by another class of DAMPs to activate interleukin-1beta and interleukin-18. These 2 cytokines then promote, via transactivation, the formation of lung inflammation.

Injury-induced allograft rejection: A rendezvous with evolution.

Modern immunology, in many ways, is based on three major paradigms: the clonal selection theory, the pattern recognition theory, and the danger/injury theory. The last theory holds that any cell stress and tissue injury, including allograft injury, via induction of damage-associated molecular patterns, induces immunity, including alloimmunity, leading to allograft rejection. On the other hand, the concept precludes that non-self per se induces immunity as proposed by the two former theories. Recently, the danger/injury model has gained considerable acceptance by immunologists, in particular as promoted by new insights into the function of the mammalian gut microbiota, representing a huge assemblage of non-self. Harboring microbiota by hosts is characterized by the fact that harmless noninjurious commensal microbes are protected by innate immunity-based tolerance, whereas intestinal injury-causing pathogenic microbes are immunologically attacked. Plausibility and validity of the danger/injury concept is stringently supported by observations of similar phenomena across the tree of life: the ability of the immune system to discriminate between harmful life-threatening non-self to induce immunity and harmless beneficial non-self to induce tolerance has apparently emerged during evolution. Immune defense responses to injuring/injured non-self (e.g., as reflected by plant resistance to biotic and abiotic stresses on one hand, and allograft rejection on the other hand) as well as immunity-controlled protection of beneficial non-self (e.g., as reflected by microbiota and the fetus of placental mammals) are processes in the interest of evolution and, thus, evolved under pressure across the phylogenetic tree.

Future immunosuppression in organ transplantation: treating the innate immune system of the deceased donor--start tomorrow.

This article, based in part on an invited talk at the Annual International Conference of Saudi Society of Nephrology & Transplantation in 2012, reviews current notions of the emerging field of innate alloimmunity by highlighting novel thoughts regarding future immunosuppressive therapy in organ transplantation. In light of new insights into the mechanisms of innate immunity on one hand and the essential role of regulatory T cells in controlling alloimmune responses on the other hand, potential clinical tools to generate tolerogenic dendritic cells are explored. These cells have been shown to promote induction of regulatory T cells that possess the potential to prevent acute and chronic allograft rejection. Experimental findings from both research areas are discussed in support of the notion that presentation of alloantigens under subimmunogenic noninflammatory conditions, achieved by vigorous inhibition of oxidative injury-induced allograft inflammation (known to occur in both the deceased donor and the recipient during allograft reperfusion), may lead to the induction of tolerogenic dendritic cell-mediated regulatory T cells, thereby offering a realistic opportunity to induce allotolerance in transplant recipients. However, before planning clinical trials in recipients, the start of such a novel therapeutic strategy to prevent allograft rejection could consist of designing and performing a quadruple drug treatment of deceased (brain-dead) donors aimed at generating donor-derived tolerogenic dendritic cells. The combination use of (1) an antioxidant, (2) a complement-inhibiting agent, (3) an IL-1ß inhibitor, and (4) a polyclonal antilymphocytic preparation is recommended as the preferred choice of such a donor treatment. If proven successful in organ donors, similar therapeutic modalities should subsequently be considered to apply to the recipient during allograft reperfusion under strict study conditions.

Emerging role of innate immunity in organ transplantation part III: the quest for transplant tolerance via prevention of oxidative allograft injury and its consequences.

In parts I and II of this tripartite review, the innate immune system was briefly described, focusing on its emerging role in organ transplantation and by emphasizing the oxidative injury-induced allograft inflammation that promotes the generation of immunostimulatory donor- and recipient-derived dendritic cells (DCs) translating innate immune events into adaptive alloimmunity. This part III of the review discusses the possibility to induce adaptive antigen-specific T regulatory cells (Tregs) in the clinical situation by harnessing donor- and recipient-derived tolerogenic DCs (tolDCs) as inducers for alloantigen-specific adaptive Tregs, an effort that follows current trends to harnessing DCs for immunotherapy. This challenge is based on accumulating evidence from basic immunological work in support of the notion that presentation of antigens, including weak transplantation antigens under subimmunogenic conditions within a noninflammatory microenvironment, promotes generation of tolDCs. With respect to these basic immunological studies, which are expressively reviewed, prevention of oxidative allograft injury can be regarded as an efficient tool in the clinical situation to present alloantigens under subimmunogenic conditions within an intragraft noninflammatory milieu, thereby potentially generating tolDCs able to induce Tregs-mediated innate allotolerance. Various therapeutic strategies to prevent oxidative allograft injury occurring in both the brain-dead donor before/during organ removal and the recipient during/after donor organ reperfusion as well as therapeutic options to inhibit injury-induced molecular and cellular consequences are finally discussed.

Emerging role of innate immunity in organ transplantation: part I: evolution of innate immunity and oxidative allograft injury.

The innate immune system is an evolutionarily highly conserved first rapid line of host defense against tissue injury and consists of a whole family of mobile and sessile cells such as antigen-presenting cells, innate lymphocytes, neutrophils, and vascular cells-dendritic cells representing the bridge to development of an adaptive immune response. The system depends on molecules collectively known as pattern recognition receptors to survey the extracellular space and the cytoplasm for the presence of exogenous pathogen-associated molecular patterns derived from microorganisms as well as damage-associated molecular patterns derived from endogenous tissue injury. Among various pattern recognition receptors, Toll-like receptors play central roles in sensing tissue damage and activating the innate immune system following any injury including postischemic reperfusion injury. This part I of a review focuses on reactive oxygen species-mediated reperfusion injury in terms of a canonical injury to every solid allograft that metamorphoses it into an acutely inflamed organ. Sources and mechanisms of reactive oxygen species production during postischemic reperfusion injury are reviewed by stressing a role of reactive oxygen species-producing enzyme systems as well as differentiating between reactive oxygen species production in donor-derived vascular cells and recipient-derived neutrophils. Finally, a potential role of hypoxia-sensing enzyme systems in the generation of reactive oxygen species is being discussed as an evolutionary principle and the critical cause of oxidative injury to allografts during reperfusion in the recipient.

Emerging role of innate immunity in organ transplantation part II: potential of damage-associated molecular patterns to generate immunostimulatory dendritic cells.

Part 2 of the review focuses on the potential of oxidative injury-induced damage-associated molecular patterns (DAMPs) to generate immunostimulatory dendritic cells (DCs) translating innate to adaptive immunity. Four different classes of DAMPs are defined, and their potential role in mediating pathways contributing to maturation of immunostimulatory DCs is explored and discussed. Accordingly, injury-induced molecules are divided into (1) class I DAMPs that, when recognized by pattern recognition receptors of DCs, trigger their activation; (2) class II DAMPs that are recognized by special activating receptors on innate lymphocytes that, after activation, contribute to maturation of DCs; (3) class III DAMPs that are recognized by pattern recognition receptors involved in the activation of inflammasomes, that is, molecular platforms that trigger the activation of proinflammatory cytokines promoting maturation of DCs; and (4) class IV DAMPs in terms of neoantigens that are recognized by preexisting natural immunoglobulin M antibodies, which-via complement activation-are able to aggravate the oxidative tissue injury and, thereby, may indirectly promote maturation of DCs. These new insights into mechanisms of oxidative injury-mediated generation of immunostimulatory DCs are finally discussed by addressing possible novel therapeutic strategies with the aim to prevent the capacity of oxidative injury to induce DAMPs in the donor organ. The ultimate goal of those strategies will be to induce transplant tolerance by avoiding oxidative injury in the donor and the recipient and thereby inhibiting activation of immunostimulatory DCs but promoting activation of tolerogenic DCs.

Role of heat shock protein 70 in innate alloimmunity.

This article briefly describes our own experience with the proven demonstration of heat shock protein 70 (HSP70) in reperfused renal allografts from brain-dead donors and reflects about its potential role as a typical damage-associated molecular pattern (DAMP) in the setting of innate alloimmunity. In fact, our group was able to demonstrate a dramatic up-regulation of HSP70 expression after postischemic reperfusion of renal allografts. Of note, up-regulation of this stress protein expression, although to a lesser extent, was already observed after cold storage of the organ indicating that this molecule is already induced in the stressed organism of a brain-dead donor. However, whether or not the dramatic up-regulation of HSP70 expression contributes to mounting an innate alloimmune response cannot be judged in view of these clinical findings. Nevertheless, HSP70, since generated in association with postischemic reperfusion-induced allograft injury, can be called a typical DAMP - as can every molecule be termed a DAMP that is generated in association with any stressful tissue injury regardless of its final positive or negative regulatory function within the innate immune response elicited by it. In fact, as we discuss in this article, the context-dependent, even contradistinctive activities of HSP70 reflect the biological phenomenon that, throughout evolution, mammals have developed an elaborate network of positive and negative regulatory mechanisms, which provide balance between defensive and protective measures against unwarranted destruction of the host. In this sense, up-regulated expression of HSP70 in an injured allograft might reflect a pure protective response against the severe oxidative injury of a reperfused donor organ. On the other hand, up-regulated expression of this stress protein in an injured allograft might reflect a (futile) attempt of the innate immune system to restore homeostasis with the aim to eliminate the "unwanted foreign allograft invader" by contributing to development of an adaptive alloimmune response. However, this adaptive immune response against donor histocompatibility alloantigens - in its evolutionary sense aimed to restore homeostasis - is by no means protective from a recipient's view point but tragically ends up with allograft rejection. Indeed: in this sense, allograft rejection is the result of a fateful confusion by the immune system of danger and benefit!

The role of postischemic reperfusion injury and other nonantigen-dependent inflammatory pathways in transplantation.

The Injury Hypothesis, first published in 1994 and modified several times between 1996 and 2002, holds that the reactive oxygen species-mediated reperfusion injury to allografts initiates and induces the alloimmune response and contributes to alloatherogenesis. Recent experimental and clinical evidence in support of the concept is presented suggesting that (1) reactive oxygen species-mediated allograft injury activates the innate immune system of the donor and recipient; (2) injury-induced putative endogenous ligands of Toll-like receptors (TLRs) of host origin such as heat shock proteins interact with and activate TLR4-bearing dendritic cells that mature and induce the adaptive alloimmune response (acute rejection), and interact with and activate TLR4-bearing vascular cells contributing to the development of alloatherosclerosis (chronic rejection); and (3) TLR4-triggered signaling, involved in the establishment of a reperfusion injury, seems to use myeloid differentiation marker 88-independent, Toll/interleukin-1 receptor domain containing adaptor inducing interferon-beta-dependent pathways that are associated with the maturation of dendritic cells and induction of interferon-inducible genes.