Jordan P. Metcalf

Corticosteroids as adjunctive therapy for diffuse alveolar hemorrhage associated with bone marrow transplantation

BACKGROUND Diffuse alveolar hemorrhage is a frequent complication of treating malignancies with high-dose chemotherapy and bone marrow transplantation and is associated with very high mortality. This disorders association with pulmonary inflammation, its coincidence with marrow recovery, and the usefulness of corticosteroids for treating other pulmonary hemorrhage syndromes provided the rationale for this study. METHODS We retrospectively studied 65 episodes of diffuse alveolar hemorrhage that has occurred in 63 of 603 consecutively treated patients who had undergone high-dose chemotherapy with bone marrow transplantation. Patients were divided into three groups according to the therapy they had received for diffuse alveolar hemorrhage: supportive therapy alone (n = 12); low-dose corticosteroids (30 mg or less of methylprednisolone or its equivalent; n = 10); and high-dose corticosteroids (more than 30 mg methylprednisolone or its equivalent; n = 43). The primary outcome measures were overall survival and survival to hospital discharge, occurrence of respiratory failure requiring intubation, and development of infections subsequent to the diagnosis of diffuse alveolar hemorrhage. RESULTS Overall survival at the end of the follow-up period was significantly higher for the high-dose corticosteroid group compared with the supportive therapy group (P = 0.005); however, treatment with low-dose steroids did not increase survival over supportive therapy alone (P = 0.198). In addition, survival to discharge was significantly increased for the high-dose group compared with the other two groups combined (33% versus 9.1%, P = 0.038). Respiratory failure after the diagnosis of diffuse alveolar hemorrhage developed in only 12 of the 22 unintubated patients in the high-dose group compared with 9 of the 10 initially unintubated patients in the other two groups (P = 0.056). Although the incidence of infections was high (40%) subsequent to diffuse alveolar hemorrhage, neither high-dose nor low-dose corticosteroid treatment significantly increased the risk of infections (P > 0.4, all comparisons). CONCLUSIONS In this study, high-dose corticosteroid therapy for diffuse alveolar hemorrhage related to bone marrow transplantation was associated with improved total survival and survival to hospital discharge, and decreased development of respiratory failure in these patients. These results suggest the therapy is beneficial, and further prospective studies are warranted to verify the effectiveness of the treatment.

Adenovirus Type 7 Induces Interleukin-8 Production via Activation of Extracellular Regulated Kinase 1/2

ABSTRACT Infection with adenovirus serotype 7 (Ad7) frequently causes lower respiratory pneumonia and is associated with severe lung inflammation and neutrophil infiltration. Earlier studies indicated release of proinflammatory cytokines, specifically interleukin-8 (IL-8), by pulmonary epithelial cells following infection by Ad7. However, the mechanism of IL-8 induction by Ad7 is unclear. We have explored the role of the Ras/Raf/MEK/Erk pathway in the Ad7-associated induction of IL-8 using a model system of A549 epithelial cells. We found that Ad7 infection induced a rapid activation of epithelial cell-derived Erk. The MEK-specific inhibitors PD98059 and U0126 blocked Erk activation and release of IL-8 following infection with Ad7. Treatment with PD98059 is cytostatic and not cytotoxic, as treated cells regain the ability to phosphorylate Erk and secrete IL-8 after removal of the drug. The expression of a mutated form of Ras in A549 epithelial cells blocked the induction of IL-8 promoter activity, and MEK inhibitor blocked induction of IL-8 mRNA. These results suggest that the Ras/Raf/MEK/Erk pathway is necessary for the Ad7 induction of IL-8 and that induction occurs at the level of transcription. Further, the kinetics of Erk activation and IL-8 induction suggest that an early viral event, such as receptor binding, may be responsible for the observed inflammatory response.

Innate Immune Response to H3N2 and H1N1 Influenza Virus Infection in a Human Lung Organ Culture Model

We studied cytokine responses to influenza virus PR8 (H1N1) and Oklahoma/309/06 (OK/06, H3N2) in a novel human lung tissue model. Exposure of the model to influenza virus rapidly activated the mitogen-activated protein kinase signaling (MAPK) pathways ERK, p38 and JNK. In addition, RNase protection assay demonstrated the induction of several cytokine and chemokine mRNAs by virus. This finding was reflected at the translational level as IL-6, MCP-1, MIP-1 alpha/beta, IL-8 and IP-10 proteins were induced as determined by ELISA. Immunohistochemistry for IP-10 and MIP-1 alpha revealed that alveolar epithelial cells and macrophages were the source of these two cytokines. Taken together, both PR8 and OK/06 cause similar induction of cytokines in human lung, although OK/06 is less effective at inducing the chemokines MCP-1 and IL-8. This human organ culture model should thus provide a relevant platform to study the biological responses of human lung to influenza virus infection.

Innate Immune Response to Adenovirus

ABSTRACT Adenovirus is a common infectious pathogen in both children and adults. It is a significant cause of morbidity in immunocompetent people living in crowded living conditions and of mortality in immunocompromised hosts. It has more recently become a popular vehicle for gene therapy applications. The host response to wild-type infection and gene therapy vector exposure involves both the innate and adaptive immune systems. The initial innate immune response is associated with the severe acute manifestations of adenovirus infection and also plays a significant role in acute toxicity owing to adenovirus vector exposure. This review discusses the innate immune response primarily during wild-type adenovirus infection because this serves as the basis for understanding the response during both natural infection and exposure to adenovirus vectors.

Innate immune response to influenza virus.

Purpose of review The recent pandemic of a novel H1N1 influenza virus has stressed the importance of effective approaches to prevent viral infection. The innate immune system is our first line of defense against invading viruses. This review aims to give a brief summary of recent findings on the response of the innate immune system to influenza virus. Recent findings Three families of pattern recognition receptors, toll-like receptors (TLRs), retinoic acid-inducible gene 1 protein like helicases (RLRs) and nucleotide-binding domain and leucine-rich-repeat-containing proteins (NLRs), are involved in recognition of influenza virus and they cooperatively operate to respond to the virus in cell culture or mouse models. Influenza virus mainly induces two types of innate immune cytokine responses: a proinflammatory response and an antiviral response. Recently, the NLRP3 inflammasome has proved to be an essential component in the host defense against influenza infection. The mitochondrion, traditionally recognized for its key role in respiration, metabolism and apoptosis, is becoming recognized as an important organelle for regulation of innate immune responses to influenza virus. Summary The NLRP3 inflammasome is an essential component in the host defense against influenza infection. Further investigations are required to elucidate whether NLRP3 is associated with the adaptive response and to identify the components of influenza virus that activate this important mediator. The role of mitochondria as a potential central platform of innate response is becoming appreciated.

Cigarette smoke extract suppresses the RIG-I-initiated innate immune response to influenza virus in the human lung

Cigarette smoking is the major cause of chronic obstructive pulmonary disease (COPD) and predisposes subjects to severe respiratory tract infections. Epidemiological studies have shown that cigarette smokers are seven times more likely to contract influenza infection than nonsmokers. The mechanisms underlying this increased susceptibility are poorly characterized. Retinoic acid-inducible gene (RIG)-I is believed to play an important role in the recognition of, and response to, influenza virus and other RNA viruses. Our study focused on how cigarette smoke extract (CSE) alters the influenza-induced proinflammatory response and suppresses host antiviral activity in the human lung using a unique lung organ culture model. We first determined that treatment with 2-20% CSE did not induce cytotoxicity as assessed by LDH release. However, CSE treatment inhibited influenza-induced IFN-inducible protein 10 protein and mRNA expression. Induction of the major antiviral cytokine IFN-β mRNA was also decreased by CSE. CSE also blunted viral-mediated RIG-I mRNA and protein expression. Inhibition of viral-mediated RIG-I induction by CSE was prevented by the antioxidants N-acetyl-cysteine and glutathione. These findings show that CSE suppresses antiviral and innate immune responses in influenza virus-infected human lungs through oxidative inhibition of viral-mediated induction of the pattern recognition receptor RIG-I. This immunosuppressive effect of CSE may play a role in the enhanced susceptibility of smokers to serious influenza infection in the lung.

Bacillus anthracis spores stimulate cytokine and chemokine innate immune responses in human alveolar macrophages through multiple mitogen-activated protein kinase pathways.

ABSTRACT Contact with the human alveolar macrophage plays a key role in the innate immune response to Bacillus anthracis spores. Because there is a significant delay between the initial contact of the spore with the host and clinical evidence of disease, there appears to be temporary containment of the pathogen by the innate immune system. Therefore, the early macrophage response to Bacillus anthracis exposure is important in understanding the pathogenesis of this disease. In this paper, we studied the initial events after exposure to spores, beginning with the rapid internalization of spores by the macrophages. Spore exposure rapidly activated the mitogen-activated protein kinase signaling pathways extracellular signal-regulated kinase, c-Jun-NH2-terminal kinase, and p38. This was followed by the transcriptional activation of cytokine and primarily monocyte chemokine genes as determined by RNase protection assays. Transcriptional induction is reflected at the translational level, as interleukin-1α (IL-1α), IL-1β, IL-6, and tumor necrosis factor alpha (TNF-α) cytokine protein levels were markedly elevated as determined by enzyme-linked immunosorbent assay. Induction of IL-6 and TNF-α, and, to a lesser extent, IL-1α and IL-1β, was partially inhibited by the blockade of individual mitogen-activated protein kinases, while the complete inhibition of cytokine induction was achieved when multiple signaling pathway inhibitors were used. Taken together, these data clearly show activation of the innate immune system in human alveolar macrophages by Bacillus anthracis spores. The data also show that multiple signaling pathways are involved in this cytokine response. This report is the first comprehensive examination of this process in primary human alveolar macrophages.

Adenovirus Type 7 Induces Interleukin-8 in a Lung Slice Model and Requires Activation of Erk

ABSTRACT Adenovirus (Ad), particularly Ad type 7 (Ad7), causes severe lung infection and pneumonia. Initially, Ad causes neutrophilic inflammation of the distal airways and alveoli. Interleukin-8 (IL-8) is the major lung neutrophil chemotaxin, and we have shown that Ad7 induces IL-8 release from the A549 alveolar epithelial cell line. We sought to determine whether ex vivo human and bovine lung tissue containing primary pneumocytes could be used as a more accurate and relevant model to study Ad acute inflammation. We found that cultured lung tissue preserved normal lung architecture for more than 10 days. IL-8 was generated upon exposure of the lung organ culture to Ad7. IL-8 production required activation of the Ras/Erk pathway, since a pharmacological inhibitor blocked the appearance of IL-8 in the medium. Both human and bovine lung explants supported replication of Ad7, and immunohistochemistry experiments demonstrated the presence of the Ad hexon antigen within alveolar epithelial cells. These findings show that our novel human lung organ culture accurately reproduces the in vivo infectious disease process. Thus, this organ culture model represents a valuable tool for studying the acute innate immune response to respiratory infections.

Bacillus anthracis Peptidoglycan Stimulates an Inflammatory Response in Monocytes through the p38 Mitogen-Activated Protein Kinase Pathway

We hypothesized that the peptidoglycan component of B. anthracis may play a critical role in morbidity and mortality associated with inhalation anthrax. To explore this issue, we purified the peptidoglycan component of the bacterial cell wall and studied the response of human peripheral blood cells. The purified B. anthracis peptidoglycan was free of non-covalently bound protein but contained a complex set of amino acids probably arising from the stem peptide. The peptidoglycan contained a polysaccharide that was removed by mild acid treatment, and the biological activity remained with the peptidoglycan and not the polysaccharide. The biological activity of the peptidoglycan was sensitive to lysozyme but not other hydrolytic enzymes, showing that the activity resides in the peptidoglycan component and not bacterial DNA, RNA or protein. B. anthracis peptidoglycan stimulated monocytes to produce primarily TNFα; neutrophils and lymphocytes did not respond. Peptidoglycan stimulated monocyte p38 mitogen-activated protein kinase and p38 activity was required for TNFα production by the cells. We conclude that peptidoglycan in B. anthracis is biologically active, that it stimulates a proinflammatory response in monocytes, and uses the p38 kinase signal transduction pathway to do so. Given the high bacterial burden in pulmonary anthrax, these findings suggest that the inflammatory events associated with peptidoglycan may play an important role in anthrax pathogenesis.

Human Lung Innate Immune Response to Bacillus anthracis Spore Infection

ABSTRACT Bacillus anthracis, the causative agent of inhalational anthrax, enters a host through the pulmonary system before dissemination. We have previously shown that human alveolar macrophages participate in the initial innate immune response to B. anthracis spores through cell signal-mediated cytokine release. We proposed that the lung epithelia also participate in the innate immune response to this pathogen, and we have developed a human lung slice model to study this process. Exposure of our model to B. anthracis (Sterne) spores rapidly activated the mitogen-activated protein kinase signaling pathways ERK, p38, and JNK. In addition, an RNase protection assay showed induction of mRNA of several cytokines and chemokines. This finding was reflected at the translational level by protein peak increases of 3-, 25-, 9-, 34-, and 5-fold for interleukin-6 (IL-6), tumor necrosis factor alpha, IL-8, macrophage inflammatory protein 1α/β, and monocyte chemoattractant protein 1, respectively, as determined by an enzyme-linked immunosorbent assay. Inhibition of individual pathways by UO126, SP600125, and SB0203580 decreased induction of chemokines and cytokines by spores, but this depended on the pathways inhibited and the cytokines and chemokines induced. Combining all three inhibitors reduced induction of all cytokines and chemokines tested to background levels. An immunohistochemistry analysis of IL-6 and IL-8 revealed that alveolar epithelial cells and macrophages and a few interstitial cells are the source of the cytokines and chemokines. Taken together, these data showed the activation of the pulmonary epithelium in response to B. anthracis spore exposure. Thus, the lung epithelia actively participate in the innate immune response to B. anthracis infection through cell signal-mediated elaboration of cytokines and chemokines.