Anja Kipar

Circulating Histones Are Mediators of Trauma-associated Lung Injury

RATIONALE Acute lung injury is a common complication after severe trauma, which predisposes patients to multiple organ failure. This syndrome largely accounts for the late mortality that arises and despite many theories, the pathological mechanism is not fully understood. Discovery of histone-induced toxicity in mice presents a new dimension for elucidating the underlying pathophysiology. OBJECTIVES To investigate the pathological roles of circulating histones in trauma-induced lung injury. METHODS Circulating histone levels in patients with severe trauma were determined and correlated with respiratory failure and Sequential Organ Failure Assessment (SOFA) scores. Their cause-effect relationship was studied using cells and mouse models. MEASUREMENTS AND MAIN RESULTS In a cohort of 52 patients with severe nonthoracic blunt trauma, circulating histones surged immediately after trauma to levels that were toxic to cultured endothelial cells. The high levels were significantly associated with the incidence of acute lung injury and SOFA scores, as well as markers of endothelial damage and coagulation activation. In in vitro systems, histones damaged endothelial cells, stimulated cytokine release, and induced neutrophil extracellular trap formation and myeloperoxidase release. Cellular toxicity resulted from their direct membrane interaction and resultant calcium influx. In mouse models, cytokines and markers for endothelial damage and coagulation activation significantly increased immediately after trauma or histone infusion. Pathological examinations showed that lungs were the predominantly affected organ with edema, hemorrhage, microvascular thrombosis, and neutrophil congestion. An anti-histone antibody could reduce these changes and protect mice from histone-induced lethality. CONCLUSIONS This study elucidates a new mechanism for acute lung injury after severe trauma and proposes that circulating histones are viable therapeutic targets for improving survival outcomes in patients.

High Mobility Group Box-1 protein and Keratin-18, circulating serum proteins informative of acetaminophen-induced necrosis and apoptosis in vivo

Drug-induced hepatotoxicity represents a major clinical problem and an impediment to new medicine development. Serum biomarkers hold the potential to provide information about pathways leading to cellular responses within inaccessible tissues, which can inform the medicinal chemist and the clinician with respect to safe drug design and use. Hepatocyte apoptosis, necrosis, and innate immune activation have been defined as features of the toxicological response associated with the hepatotoxin acetaminophen (APAP). Within this investigation, we have unambiguously identified and characterized by liquid chromatography-tandem mass spectrometry differing circulating molecular forms of high-mobility group box-1 protein (HMGB1) and keratin-18 (K18), which are linked to the mechanisms and pathological changes induced by APAP in the mouse. Hypoacetylated HMGB1 (necrosis indicator), caspase-cleaved K18 (apoptosis indicator), and full-length K18 (necrosis indicator) present in serum showed strong correlations with the histological time course of cell death and was more sensitive than alanine aminotransferase activity. We have further identified a hyperacetylated form of HMGB1 (inflammatory indicator) in serum, which indicated that hepatotoxicity was associated with an inflammatory response. The inhibition of APAP-induced apoptosis and K18 cleavage by the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe) fluoromethyl ketone are associated with increased hepatic damage, by a shift to necrotic cell death only. These findings illustrate the initial verification of K18 and HMGB1 molecular forms as serum-based sensitive tools that provide insights into the cellular dynamics involved in APAP hepatotoxicity within an inaccessible tissue. Based on these findings, potential exists for the qualification and measurement of these proteins to further assist in vitro, in vivo, and clinical bridging in toxicological research.

Nitrite Regulates Hypoxic Vasodilation via Myoglobin-Dependent Nitric Oxide Generation

Background— Hypoxic vasodilation is a physiological response to low oxygen tension that increases blood supply to match metabolic demands. Although this response has been characterized for >100 years, the underlying hypoxic sensing and effector signaling mechanisms remain uncertain. We have shown that deoxygenated myoglobin in the heart can reduce nitrite to nitric oxide (NO·) and thereby contribute to cardiomyocyte NO· signaling during ischemia. On the basis of recent observations that myoglobin is expressed in the vasculature of hypoxia-tolerant fish, we hypothesized that endogenous nitrite may contribute to physiological hypoxic vasodilation via reactions with vascular myoglobin to form NO·. Methods and Results— We show in the present study that myoglobin is expressed in vascular smooth muscle and contributes significantly to nitrite-dependent hypoxic vasodilation in vivo and ex vivo. The generation of NO· from nitrite reduction by deoxygenated myoglobin activates canonical soluble guanylate cyclase/cGMP signaling pathways. In vivo and ex vivo vasodilation responses, the reduction of nitrite to NO·, and the subsequent signal transduction mechanisms were all significantly impaired in mice without myoglobin. Hypoxic vasodilation studies in myoglobin and endothelial and inducible NO synthase knockout models suggest that only myoglobin contributes to systemic hypoxic vasodilatory responses in mice. Conclusions— Endogenous nitrite is a physiological effector of hypoxic vasodilation. Its reduction to NO· via the heme globin myoglobin enhances blood flow and matches O2 supply to increased metabolic demands under hypoxic conditions.

Campylobacter jejuni Is Not Merely a Commensal in Commercial Broiler Chickens and Affects Bird Welfare

ABSTRACT Campylobacter jejuni is the leading cause of bacterial food-borne infection; chicken meat is its main source. C. jejuni is considered commensal in chickens based on experimental models unrepresentative of commercial production. Here we show that the paradigm of Campylobacter commensalism in the chicken is flawed. Through experimental infection of four commercial breeds of broiler chickens, we show that breed has a significant effect on C. jejuni infection and the immune response of the animals, although these factors have limited impact on the number of bacteria in chicken ceca. All breeds mounted an innate immune response. In some breeds, this response declined when interleukin-10 was expressed, consistent with regulation of the intestinal inflammatory response, and these birds remained healthy. In another breed, there was a prolonged inflammatory response, evidence of damage to gut mucosa, and diarrhea. We show that bird type has a major impact on infection biology of C. jejuni. In some breeds, infection leads to disease, and the bacterium cannot be considered a harmless commensal. These findings have implications for the welfare of chickens in commercial production where C. jejuni infection is a persistent problem. IMPORTANCE Campylobacter jejuni is the most common cause of food-borne bacterial diarrheal disease in the developed world. Chicken is the most common source of infection. C. jejuni infection of chickens had previously not been considered to cause disease, and it was thought that C. jejuni was part of the normal microbiota of birds. In this work, we show that modern rapidly growing chicken breeds used in intensive production systems have a strong inflammatory response to C. jejuni infection that can lead to diarrhea, which, in turn, leads to damage to the feet and legs on the birds due to standing on wet litter. The response and level of disease varied between breeds and is related to regulation of the inflammatory immune response. These findings challenge the paradigm that C. jejuni is a harmless commensal of chickens and that C. jejuni infection may have substantial impact on animal health and welfare in intensive poultry production. Campylobacter jejuni is the most common cause of food-borne bacterial diarrheal disease in the developed world. Chicken is the most common source of infection. C. jejuni infection of chickens had previously not been considered to cause disease, and it was thought that C. jejuni was part of the normal microbiota of birds. In this work, we show that modern rapidly growing chicken breeds used in intensive production systems have a strong inflammatory response to C. jejuni infection that can lead to diarrhea, which, in turn, leads to damage to the feet and legs on the birds due to standing on wet litter. The response and level of disease varied between breeds and is related to regulation of the inflammatory immune response. These findings challenge the paradigm that C. jejuni is a harmless commensal of chickens and that C. jejuni infection may have substantial impact on animal health and welfare in intensive poultry production.

Diet restriction inhibits apoptosis and HMGB1 oxidation and promotes inflammatory cell recruitment during acetaminophen hepatotoxicity.

Acetaminophen (APAP) overdose is a major cause of acute liver failure and serves as a paradigm to elucidate mechanisms, predisposing factors and therapeutic interventions. The roles of apoptosis and inflammation during APAP hepatotoxicity remain controversial. We investigated whether fasting of mice for 24 h can inhibit APAP-induced caspase activation and apoptosis through the depletion of basal ATP. We also investigated in fasted mice the critical role played by inhibition of caspase-dependent cysteine 106 oxidation within high mobility group box-1 protein (HMGB1) released by ATP depletion in dying cells as a mechanism of immune activation. In fed mice treated with APAP, necrosis was the dominant form of hepatocyte death. However, apoptosis was also observed, indicated by K18 cleavage, DNA laddering and procaspase-3 processing. In fasted mice treated with APAP, only necrosis was observed. Inflammatory cell recruitment as a consequence of hepatocyte death was observed only in fasted mice treated with APAP or fed mice cotreated with a caspase inhibitor. Hepatic inflammation was also associated with loss in detection of serum oxidized-HMGB1. A significant role of HMGB1 in the induction of inflammation was confirmed with an HMGB1-neutralizing antibody. The differential response between fasted and fed mice was a consequence of a significant reduction in basal hepatic ATP, which prevented caspase processing, rather than glutathione depletion or altered APAP metabolism. Thus, the inhibition of caspase-driven apoptosis and HMGB1 oxidation by ATP depletion from fasting promotes an inflammatory response during drug-induced hepatotoxicity/liver pathology.

Morphologic Features and Development of Granulomatous Vasculitis in Feline Infectious Peritonitis

Feline infectious peritonitis (FIP) is a fatal, Coronavirus (CoV)-induced systemic disease in cats, characterized by granulomas in organs and granulomatous vasculitis. This study describes the morphologic features of granulomatous vasculitis in FIP as well as its development in the course of monocyte-associated feline CoV (FCoV) viremia in five naturally infected Domestic Shorthair cats with FIP. Monocyte-associated FCoV viremia was demonstrated by immunohistology, RNA in situ hybridization, and electron micropscopy. Granulomatous phlebitis at different stages of development was observed. Vasculitic processes ranged from attachment and emigration of FCoV-infected monocytes to vascular/perivascular granulomatous infiltrates with destruction of the vascular basal lamina. Monocytes as well as perivascular macrophages were activated because they were strongly positive for CD18 and expressed cytokines (tumor necrosis factor-α and interleukin-1β) and matrix metalloproteinase-9. In addition, general activation of endothelial cells, represented by major histocompatibility complex II upregulation, was observed in all cases. These results confirm FIP as a monocyte-triggered systemic disease and demonstrate the central role of activated monocytes in FIP vasculitis.

Cellular composition, coronavirus antigen expression and production of specific antibodies in lesions in feline infectious peritonitis

Abstract Twenty-three cats with spontaneous feline infectious peritonitis (FIP) were examined by light microscopy including immunohistology and histochemistry in order to determine the cellular composition and the expression of viral antigen in lesions in FIP. Furthermore, the presence of plasma-cells producing coronavirus-specific antibodies was evaluated in situ. Macrophages and neutrophils were demonstrated by an antibody against calprotectin (leukocyte protein L1, myeloid/histiocyte antigen), neutrophils were recognized due to their chloroacetate esterase activity, and B- and T-lymphocytes were identified by antibodies against the CD3 antigen and the CD45R antigen, respectively. Expression of viral antigen was immunohistologically demonstrated by a monoclonal antibody (mAb) against coronavirus while coronavirus-specific antibodies in situ were identified by the application of feline coronavirus prior to the coronavirus antibody. Lesions were classified as diffuse alterations at serosal surfaces, granulomas with areas of necrosis, granulomas without extended necrosis, focal and perivascular lymphoplasmocytic infiltrates, and granulomatous-necrotizing vasculitis. Diffuse alterations on serosal surfaces were represented either by activated mesothelial cells with single coronavirus antigen-bearing macrophages or by layers of precipitated exudate containing single to numerous granulomas with areas of necrosis. In liver and spleen, the exudate was often underlaid by a small band of subcapsular B-cells with an occasional plasma-cell producing coronavirus-specific antibodies. In other locations, a variably broad band of B-cells and plasma-cells, often infiltrating between underlying muscle fibers, separated the exudate from the unaltered tissue. Some of these plasma-cells were positive for coronavirus-specific antibodies. In granulomas with areas of necrosis, the central necrosis was surrounded by macrophages usually expressing considerable amounts of viral antigen. Few B-cells and plasma-cells were found in the periphery. In granulomas without extended necrosis, the number of macrophages were lower. Only few macrophages expressing low amounts of viral antigen were present. B-cells and plasma-cells formed a broad rim. Few plasma-cells stained positive for coronavirus-specific antibodies. In both types of granulomas, few neutrophils were found between macrophages. Few T-cells were seen scattered throughout the lesions. Focal and perivascular lymphoplasmocytic infiltrates were mainly seen in omentum and leptomeninx. B-cells were the predominant cells; some plasma-cells were positive for coronavirus-specific antibodies. Viral antigen was not readily detected in these alterations. Granulomatous-necrotizing vasculitis was occasionally found in kidneys and leptomeninx. It was dominated by macrophages which often stained strongly positive for coronavirus antigen. Different types of alteration were often seen in the same animal and even the same tissue. There was no obvious correlation between the cats age, gross pathological changes, and the histological types of alteration. Single plasma-cells positive for coronavirus-specific antibodies were found around blood vessels distant from inflammatory alterations, within the lung parenchyma, as infiltrating cells in the mucosa of the small intestine, and in spleen and mesenteric lymph node. Results show that alterations in FIP are heterogeneous concerning cellular composition and expression of viral antigen. The dominance of B-cells in part of the lesions together with the presence of plasma-cells positive for coronavirus-specific antibodies indicate that these cells may play a role in the maintenance of inflammatory processes in FIP.

Lethal outbreak of disease associated with feline calicivirus infection in cats

Recently, in the USA, virulent mutants of feline calicivirus (FCV) have been identified as the cause of a severe and acute virulent systemic disease, characterised by jaundice, oedema and high mortality in groups of cats. This severe manifestation of FCV disease has so far only been reported in the USA. However, in 2003, an outbreak of disease affected a household of four adult cats and an adult cat from a neighbouring household in the UK. Three of the adult cats in the household and the neighbouring cat developed clinical signs including pyrexia (39·5 to 40·5°C), lameness, voice loss, inappetence and jaundice. One cat was euthanased in extremis, two died and one recovered. A postmortem examination of one of the cats revealed focal cellulitis around the right hock and right elbow joints. The principal finding of histopathological examinations of selected organs from two of the cats was disseminated hepatocellular necrosis with mild inflammatory infiltration. Immunohistology identified FCV antigen in parenchymal and Kupffer cells in the liver of both animals and in alveolar macrophages of one of them. In addition, calicivirus-like particles were observed by electron microscopy within the hepatocytes of one cat. FCV was isolated from two of the dead cats and from the two surviving cats. Sequence analysis showed that they were all infected with the same strain of virus, but that it was different from strains of FCV associated with the virulent systemic disease in cats in the USA. The outbreak was successfully controlled by quarantine in the owner’s house.

Immunohistochemical Characterization of Inflammatory Cells in Brains of Dogs with Granulomatous Meningoencephalitis

The inflammatory cells of eleven dogs with canine granulomatous meningoencephalitis were characterized immunohistochemically. Macrophages were identified by antibodies directed against lysozyme and the DH82 antigen (expressed by cells of a malignant histiocytosis). T cells were demonstrated by CD3, CD43, and CD45R antigen, and B cells by immunoglobulin G and immunoglobulin M expression. Furthermore, staining for the major histocompatibility complex (MHC) class II antigen was evaluated. Diseased animals ranged from 1 to 9 years of age. Small and medium-sized breeds were affected predominantly. Lesions were widespread and localized mainly in the brain stem, less frequently in the cerebrum or cerebellum. Alterations were represented by perivascular cuffs, parenchymal granulomas, and leptomeningeal infiltrates. Lymphocytes and macrophages comprised the dominant cell populations; their percentage varied substantially between different animals and between sections from the same individual. Immunohistochemically, the bulk of lymphocytes were CD3 antigen-positive T cells, while only a few cells were CD43 and CD45R antigen-positive or were classified as B cells. The majority of macrophages expressed both lysozyme and DH82 antigen; however, some were positive for only one antigen. MHC class II antigen-expression, observed only within and in close proximity to the lesions, was found on all inflammatory cells, pericytes/endothelial cells, and microglia. Results were negative for canine distemper virus antigen and nucleoprotein mRNA, rabies virus antigen, fungi, bacteria, and protozoal agents. This immunomorphologic study reveals that inflammatory lesions in canine granulomatous meningoencephalitis consist of a heterogeneous population of MHC class II antigen-positive macrophages and predominantly CD3 antigen-positive lymphocytes. The data suggest a T cell-mediated delayed-type hypersensitivity of an organ-specific autoimmune disease as a possible pathogenic mechanism for this unique canine brain lesion.

Regulation and role of REST and REST4 variants in modulation of gene expression in in vivo and in vitro in epilepsy models.

Repressor element-1 silencing transcription factor (REST) is a candidate modulator of gene expression during status epilepticus in the rodent. In such models, full-length REST and the truncated REST4 variant are induced and can potentially direct differential gene expression patterns. We have addressed the regulation of these REST variants in rodent hippocampal seizure models and correlated this with expression of the proconvulsant, substance P encoding, PPT-A gene. REST and REST4 were differentially regulated following kainic acid stimulus both in in vitro and in vivo models. REST4 was more tightly regulated than REST in both models and its transient expression correlated with that of the differential regulation of PPT-A. Consistent with this, overexpression of a truncated REST protein (HZ4, lacking the C-terminal repression domain) increased expression of the endogenous PPT-A gene. Similarly the proximal PPT-A promoter reporter gene construct was differentially regulated by the distinct REST isoforms in hippocampal cells with HZ4 being the major inducer of increased reporter expression. Furthermore, REST and REST4 proteins were differentially expressed and compartmentalized within rat hippocampal cells in vitro following noxious stimuli. This differential localization of the REST isoforms was confirmed in the CA1 region following perforant path and kainic acid induction of status epilepticus in vivo. We propose that the interplay between REST and REST4 alter the expression of proconvulsant genes, as exemplified by the PPT-A gene, and may therefore regulate the progression of epileptogenesis.