mocetinostat and Orthomyxoviridae-Infections

Topics: Coinfection; Communicable Diseases; Humans; Influenza, Human; Lung; Neutrophils; Orthomyxoviridae; Orthomyxoviridae Infections; Peroxidase; Pseudomonas aeruginosa; Pseudomonas Infections

Influenza A virus is an important human pathogen that causes 3 to 5 million severe cases of influenza worldwide each year. An aberrant innate immune response, particularly hypercytokinemia, is thought to play an important role in the disease, although the pathogenesis of severe influenza virus infection remains unclear and no specific and efficacious immunotherapy is available. This study reports dysregulated complement activation in mice after infection with A/Puerto Rico/8/34 (PR8). C5aR1-deficient mice and mice treated with an anti-C5aR1 antibody were used as models to study the C5a-C5aR1 axis during acute lung injury (ALI) induced by influenza virus infection. The results showed that blocking the C5a-C5aR1 axis alleviated ALI by inhibiting endothelial cell activation and dampening the host immune response (i.e., reduced TNF-α, IL-1β, IL-6, IP-10, MCP-1, IL-12p70, and IFN-γ concentrations in plasma), particularly CTL-mediated immunopathology. Furthermore, blockade of the C5a-C5aR1 axis inhibited viral replication in lung tissue. Taken together, the results indicate that the C5a-C5aR1 axis plays an important role in the outcome of ALI induced by influenza virus infection and that regulation of complement activation, particularly the C5aR1 inhibition, is a promising intervention and adjunctive treatment.

Topics: Acute Lung Injury; Animals; Antibodies, Monoclonal; Complement Activation; Complement C5a; Cytokines; Female; Influenza A virus; Intercellular Adhesion Molecule-1; Lung; Mice, Inbred BALB C; Mice, Knockout; Orthomyxoviridae Infections; Peroxidase; Receptor, Anaphylatoxin C5a; Virus Replication

The immunological mechanisms of secondary bacterial infection followed by influenza virus infection were examined. When mice were intranasally infected with influenza virus A and then infected with P. aeruginosa at 4 days after viral infection, bacterial clearance in the lung significantly decreased compared to that of non-viral infected mice. Neutrophils from viral infected mice showed impaired digestion and/or killing of phagocytized bacteria due to reduced myeloperoxidase (MPO) activity. G-CSF production in the lungs of viral infected mice was lower than that of non-viral infected mice after secondary bacterial infection. When viral infected mice were injected with G-CSF before secondary bacterial infection, the MPO activity of viral infected mice restored to the same level as that of non-infected mice. Bacteria clearance in viral infected mice was also recovered by G-CSF administration. Thus, neutrophil dysfunction caused by influenza virus is attributed to insufficient G-CSF production, which induces a secondary bacterial infection.

Topics: Animals; Bacterial Load; Coinfection; Cytokines; Female; Granulocyte Colony-Stimulating Factor; Inflammation Mediators; Influenza A virus; Influenza A Virus, H1N1 Subtype; Mice; Neutrophils; Orthomyxoviridae Infections; Peroxidase; Phagocytosis; Pneumonia, Bacterial; Pseudomonas aeruginosa; Risk

Parenchymal lung inflammation and airway and alveolar epithelial cell apoptosis are associated with cigarette smoke exposure (CSE), which contributes to chronic obstructive pulmonary disease (COPD). Epidemiological studies indicate that people exposed to chronic cigarette smoke with or without COPD are more susceptible to influenza A virus (IAV) infection. We found increased p53, PAI-1 and apoptosis in AECs, with accumulation of macrophages and neutrophils in the lungs of patients with COPD. In Wild-type (WT) mice with passive CSE (PCSE), p53 and PAI-1 expression and apoptosis were increased in AECs as was lung inflammation, while those lacking p53 or PAI-1 resisted AEC apoptosis and lung inflammation. Further, inhibition of p53-mediated induction of PAI-1 by treatment of WT mice with caveolin-1 scaffolding domain peptide (CSP) reduced PCSE-induced lung inflammation and reversed PCSE-induced suppression of eosinophil-associated RNase1 (EAR1). Competitive inhibition of the p53-PAI-1 mRNA interaction by expressing p53-binding 3'UTR sequences of PAI-1 mRNA likewise suppressed CS-induced PAI-1 and AEC apoptosis and restored EAR1 expression. Consistent with PCSE-induced lung injury, IAV infection increased p53, PAI-1 and apoptosis in AECs in association with pulmonary inflammation. Lung inflammation induced by PCSE was worsened by subsequent exposure to IAV. Mice lacking PAI-1 that were exposed to IAV showed minimal viral burden based on M2 antigen and hemagglutination analyses, whereas transgenic mice that overexpress PAI-1 without PCSE showed increased M2 antigen and inflammation after IAV infection. These observations indicate that increased PAI-1 expression promotes AEC apoptosis and exacerbates lung inflammation induced by IAV following PCSE.

Topics: Alveolar Epithelial Cells; Animals; Apoptosis; Caveolin 1; Humans; Influenza A virus; Influenza, Human; Leukocyte Elastase; Luciferases; Lung; Lung Injury; Mice, Inbred C57BL; Orthomyxoviridae Infections; Peptide Fragments; Peroxidase; Plasminogen Activator Inhibitor 1; Promoter Regions, Genetic; Pulmonary Disease, Chronic Obstructive; Smoking; Tumor Suppressor Protein p53

The antioxidant N-acetyl-l-cysteine (NAC) had been shown to inhibit replication of seasonal human influenza A viruses. Here, the effects of NAC on H9N2 swine influenza virus-induced acute lung injury (ALI) were investigated in mice. BALB/c mice were inoculated intranasally with 10(7) 50% tissue culture infective doses (TCID(50)) of A/swine/HeBei/012/2008/(H9N2) viruses with or without NAC treatments to induce ALI model. The result showed that pulmonary inflammation, pulmonary edema, MPO activity, total cells, neutrophils, macrophages, TNF-α, IL-6, IL-1β and CXCL-10 in BALF were attenuated by NAC. Moreover, our data showed that NAC significantly inhibited the levels of TLR4 protein and TLR4 mRNA in the lungs. Pharmacological inhibitors of TLR4 (E5564) exerted similar effects like those determined for NAC in H9N2 swine influenza virus-infected mice. These results suggest that antioxidants like NAC represent a potential additional treatment option that could be considered in the case of an influenza A virus pandemic.

Topics: Acetylcysteine; Acute Lung Injury; Animals; Antioxidants; Cytokines; Disease Models, Animal; Humans; Inflammation Mediators; Influenza A Virus, H9N2 Subtype; Influenza, Human; Lipid A; Lung; Male; Mice; Mice, Inbred BALB C; Orthomyxoviridae Infections; Peroxidase; Swine; Toll-Like Receptor 4; Virus Replication

Highly pathogenic avian influenza H5N1 (HPAI H5N1) viruses can infect mammals, including humans, causing severe systemic disease with the inhibition of the immune system and a high mortality rate. In conditions of lymphoid tissue depletion, the liver plays an important role in host defence against viruses. The changes in mice liver infected with HPAI H5N1 virus A/goose/Krasnoozerskoye/627/05 have been studied. It has been shown that the virus persistence in the liver leads to the expression of proinflammatory cytokines (TNF- α , IL-6) and intracellular proteases (lysozyme, cathepsin D, and myeloperoxidase) by Kupffer cells. Defective antiviral response exacerbates destructive processes in the liver accelerating the development of liver failure.

Topics: Animals; Antigens, Viral; Influenza A Virus, H5N1 Subtype; Interleukin-6; Kupffer Cells; Liver; Male; Mice; Orthomyxoviridae Infections; Peroxidase; Tumor Necrosis Factor-alpha

Because the pathogenesis of acute respiratory distress syndrome (ARDS) induced by influenza virus infection remains unknown, we can only improve on existing therapeutic interventions. To approach the subject, we investigated immunological etiology focused on cytokines and an acute lung damage factor in influenza-induced ARDS by using a PR-8 (A/H1N1)-infected mouse model. The infected mouse showed fulminant severe pneumonia with leukocyte infiltration, claudin alteration on tight junctions, and formation of hyaline membranes. In addition to interferon (IFN)-α, plenty of keratinocyte-derived chemokines (KC), macrophage inflammatory protein 2 (MIP-2), regulated on activation normal T-cell expressed and secreted (RANTES), and monocyte chemotactic protein 1 (MCP-1) were significantly released into bronchoalveolar lavage fluid (BALF) of the model. We focused on neutrophil myeloperoxidase (MPO) as a potent tissue damage factor and examined its contribution in influenza pneumonia by using mice genetically lacking in MPO. The absence of MPO reduced inflammatory damage with suppression of leakage of total BALF proteins associated with alteration of claudins in the lung. MPO(-/-) mice also suppressed viral load in the lung. The present study suggests that MPO-mediated OCl(-) generation affects claudin molecules and leads to protein leakage and viral spread as a damage factor in influenza-induced ARDS.

Topics: Animals; Cytokines; Female; Influenza A Virus, H1N1 Subtype; Mice; Mice, Inbred BALB C; Mice, Knockout; Neutrophils; Orthomyxoviridae Infections; Peroxidase; Pneumonia, Viral; Respiratory Distress Syndrome

Acute respiratory distress syndrome, a severe form of acute lung injury (ALI), is a major cause of death during influenza pneumonia. We have provided evidence for the involvement of recruited neutrophils, their toxic enzymes such as myeloperoxidase and matrix metalloproteinases (MMPs), and neutrophil extracellular traps in aggravating alveolar-capillary damage. In this study, we investigated the effects of doxycycline (DOX), an inhibitor of MMPs, on influenza-induced ALI. BALB/c mice were infected with a sublethal dose of mouse-adapted virulent influenza A/Aichi/2/68 (H3N2) virus, and administered daily with 20mg/kg or 60 mg/kg DOX orally. The effects of DOX on ALI were determined by measuring inflammation, capillary leakage, and MMP activities. Furthermore, levels of T1-α (a membrane protein of alveolar type I epithelium) and thrombomodulin (an endothelial protein) in the bronchoalveolar lavage fluid were evaluated by Western blot analysis. Our results demonstrate significantly decreased inflammation and protein leakage in the lungs after DOX treatment. Levels of MMP-2 and MMP-9 activity, T1-α and thrombomodulin were also diminished in the DOX-treated group. These findings were corroborated by histopathologic analyses, which demonstrated significant reduction in lung damage. Although DOX treatment reduced ALI, there were no effects on virus titers and body weights. Taken together, these results demonstrate that DOX may be useful in ameliorating ALI during influenza pneumonia. Further studies are warranted to determine whether DOX can be used in combination with anti-viral agents to alleviate severe influenza pneumonia.

Topics: Acute Lung Injury; Animals; Anti-Bacterial Agents; Blotting, Western; Doxycycline; Female; Humans; Influenza A Virus, H3N2 Subtype; Lung; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Neutrophil Infiltration; Orthomyxoviridae Infections; Peroxidase; Reverse Transcriptase Polymerase Chain Reaction; Thrombomodulin

C57Bl/6 mice were intranasally infected with influenza virus A/H5N1 A/goose/Krasnoozerskoye/627/05. The mortality rate of animals reached 70% on day 14 of the disease. The lungs of animals were characterized by necroses, destruction of vessels, hemorrhagic and thrombotic complications, edematous syndrome, and early fibrosis of the interstitium. On days 6-10 after infection, fibrosis was found in the zones of postnecrotic inflammatory infiltration. The expression of lysozyme and myeloperoxidase by pulmonary macrophages was initially increased, but decreased on day 10 of the study. The number of cathepsin D-expressing macrophages was elevated up to the 10th day of examination.

Topics: Animals; Cathepsin D; Disease Models, Animal; Fas-Associated Death Domain Protein; Influenza A Virus, H5N1 Subtype; Interleukin-6; Lung; Macrophages, Alveolar; Male; Mice; Mice, Inbred C57BL; Muramidase; Nitric Oxide Synthase Type II; Orthomyxoviridae Infections; Peroxidase; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

The expression of myeloperoxidase (MPO) was examined in the swine influenza virus (SIV)-infected neutrophils in the lungs of pigs experimentally infected with swine influenza virus (SIV) subtype H1N2 by immunohistochemistry. Five pigs each from the infected and non-infected group were euthanized 1, 3, 5, 7, and 10 days post-inoculation (dpi). Immunohistochemical reactivity was mainly seen in neutrophils. The score for pulmonary histopathological lesions correlated with the score for MPO immunohistochemical reactivity (r ( s ) = 0.962, P < 0.01). In addition, the score for in situ hybridization of SIV nucleic acid correlated with the score for MPO immunohistochemical reactivity (r ( s ) = 0.976, P < 0.01). These results suggest neutrophils are one of the primary effector cells in the early phase of SIV infection in pigs.

Topics: Animals; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Immunohistochemistry; Influenza A Virus, H1N2 Subtype; Lung; Lung Diseases; Neutrophils; Orthomyxoviridae Infections; Peroxidase; Polymerase Chain Reaction; Random Allocation; Sus scrofa; Swine; Swine Diseases

Complications of acute respiratory distress syndrome (ARDS) are common among critically ill patients infected with highly pathogenic influenza viruses. Macrophages and neutrophils constitute the majority of cells recruited into infected lungs, and are associated with immunopathology in influenza pneumonia. We examined pathological manifestations in models of macrophage- or neutrophil-depleted mice challenged with sublethal doses of influenza A virus H1N1 strain PR8. Infected mice depleted of macrophages displayed excessive neutrophilic infiltration, alveolar damage, and increased viral load, later progressing into ARDS-like pathological signs with diffuse alveolar damage, pulmonary edema, hemorrhage, and hypoxemia. In contrast, neutrophil-depleted animals showed mild pathology in lungs. The brochoalveolar lavage fluid of infected macrophage-depleted mice exhibited elevated protein content, T1-α, thrombomodulin, matrix metalloproteinase-9, and myeloperoxidase activities indicating augmented alveolar-capillary damage, compared to neutrophil-depleted animals. We provide evidence for the formation of neutrophil extracellular traps (NETs), entangled with alveoli in areas of tissue injury, suggesting their potential link with lung damage. When co-incubated with infected alveolar epithelial cells in vitro, neutrophils from infected lungs strongly induced NETs generation, and augmented endothelial damage. NETs induction was abrogated by anti-myeloperoxidase antibody and an inhibitor of superoxide dismutase, thus implying that NETs generation is induced by redox enzymes in influenza pneumonia. These findings support the pathogenic effects of excessive neutrophils in acute lung injury of influenza pneumonia by instigating alveolar-capillary damage.

Topics: Acute Lung Injury; Animals; Blotting, Western; Bronchoalveolar Lavage Fluid; Cells, Cultured; Dogs; Female; Immunoenzyme Techniques; Influenza A Virus, H1N1 Subtype; Kidney; Macrophages; Mice; Mice, Inbred BALB C; Neutrophils; Orthomyxoviridae Infections; Peroxidase; Pneumonia; Pulmonary Alveoli; Respiratory Distress Syndrome; Superoxide Dismutase

Recent studies have demonstrated an essential role of alveolar macrophages during influenza virus infection. Enhanced mortalities were observed in macrophage-depleted mice and pigs after influenza virus infection, but the basis for the enhanced pathogenesis is unclear. This study revealed that blocking macrophage recruitment into the lungs in a mouse model of influenza pneumonitis resulted in enhanced alveolar epithelial damage and apoptosis, as evaluated by histopathology, immunohistochemistry, Western blot, RT-PCR, and TUNEL assays. Abrogation of macrophage recruitment was achieved by treatment with monoclonal antibody against monocyte chemoattractant protein-1 (MCP-1) after sub-lethal challenge with mouse-adapted human influenza A/Aichi/2/68 virus. Interestingly, elevated levels of hepatocyte growth factor (HGF), a mitogen for alveolar epithelium, were detected in bronchoalveolar lavage samples and in lung homogenates of control untreated and nonimmune immunoglobulin (Ig)G-treated mice after infection compared with anti-MCP-1-treated infected mice. The lungs of control animals also displayed strongly positive HGF staining in alveolar macrophages as well as alveolar epithelial cell hyperplasia. Co-culture of influenza virus-infected alveolar epithelial cells with freshly isolated alveolar macrophages induced HGF production and phagocytic activity of macrophages. Recombinant HGF added to mouse lung explants after influenza virus infection resulted in enhanced BrdU labeling of alveolar type II epithelial cells, indicating their proliferation, in contrast with anti-HGF treatment showing significantly reduced epithelial regeneration. Our data indicate that inhibition of macrophage recruitment augmented alveolar epithelial damage and apoptosis during influenza pneumonitis, and that HGF produced by macrophages in response to influenza participates in the resolution of alveolar epithelium.

Topics: Animals; Antibodies, Monoclonal; Apoptosis; Blotting, Western; Body Weight; Bronchoalveolar Lavage Fluid; Cell Proliferation; Cells, Cultured; Chemokine CCL2; Chemokine CXCL1; Chemotaxis, Leukocyte; Coculture Techniques; Disease Models, Animal; Female; Hepatocyte Growth Factor; Humans; Hyperplasia; Immunohistochemistry; In Situ Nick-End Labeling; Influenza A virus; Injections, Intraperitoneal; Macrophages, Alveolar; Mice; Mice, Inbred BALB C; Orthomyxoviridae Infections; Peroxidase; Pneumonia, Viral; Pulmonary Alveoli; Recombinant Proteins; Respiratory Mucosa; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Viral Load

Severe pneumonia is found in simultaneous influenza pneumonia and bacterial infection, and suggests a relationship with immunological mechanisms. Here, we performed two-dimensional gel electrophoresis to detect immunological molecules related to the fulminant pneumonia caused by influenza virus and Streptococcus pneumoniae co-infection in mice. We found two spots that were expressed strongly in co-infected mouse lungs, compared with S. pneumoniae or influenza virus singly infected mouse lungs. The spots were analysed by mass spectrometry, and identified as alpha-1 anti-trypsin (A1AT), known as an anti-protease for neutrophil-derived proteolytic enzymes, and creatine kinase, which reflects a greater degree of lung damage and cell death. A1AT expression was increased significantly, and proteolytic enzymes from neutrophils, such as neutrophil elastase, myeloperoxidase and lysozyme, were also secreted abundantly in influenza virus and S. pneumoniae co-infected lungs compared with S. pneumoniae or influenza virus singly infected lungs. These data suggest that A1AT may play a central role as a molecule with broad anti-inflammatory properties, and regulation of the neutrophil-mediated severe lung inflammation is important in the pathogenesis of co-infection with influenza virus and bacteria.

Topics: alpha 1-Antitrypsin; Animals; Bronchoalveolar Lavage Fluid; Chemokine CXCL2; Creatine Kinase; Disease Susceptibility; Electrophoresis, Gel, Two-Dimensional; Influenza A virus; Leukocyte Elastase; Lung; Male; Mice; Mice, Inbred CBA; Muramidase; Orthomyxoviridae Infections; Peroxidase; Pneumonia, Pneumococcal; Pneumonia, Viral

Studies have shown that cigarette smoke impacts respiratory host defense mechanisms; however, it is poorly understood how these smoke-induced changes impact the overall ability of the host to deal with pathogenic agents.. The objective of this study was to investigate the impact of mainstream cigarette smoke exposure on immune inflammatory responses and viral burden after respiratory infection with influenza A.. C57BL/6 mice were sham- or smoke-exposed for 3 to 5 mo and infected with either 2.5 x 10(3) pfu (low dose) or 2.5 x 10(5) pfu (high dose) influenza virus.. Although smoke exposure attenuated the airway's inflammatory response to low-dose infection, we observed increased inflammation in smoke-exposed compared with sham-exposed mice after infection with high-dose influenza, despite a similar rate of viral clearance. The heightened inflammatory response was associated with increased expression of tumor necrosis factor-alpha, interleukin-6, and type 1 IFN in the airway, and increased mortality. Importantly, smoke exposure did not interfere with the development of influenza-specific memory responses; sham- and smoke-exposed animals were equally protected upon viral rechallenge.. Our study suggests that, in mice, cigarette smoke affects primary antiviral immune-inflammatory responses, whereas secondary immune protection remains intact.

Topics: Animals; Female; Flow Cytometry; Inflammation; Influenza A virus; Interleukin-6; Mice; Mice, Inbred C57BL; Orthomyxoviridae Infections; Peroxidase; Smoking; Tumor Necrosis Factor-alpha; Viral Load

We established a mouse model in which fatal pneumonia was induced by pneumococcal superinfection following influenza virus infection in chronic Pseudomonas aeruginosa infected mice. In this mouse model, influenza virus infection caused a significant increase in inflammatory cells, cytokines and severe tissue damage in the lungs of these P. aeruginosa infected mice, before pneumococcal infection. Intrapulmonary virus titres were significantly increased in mice with chronic P. aeruginosa infection, compared with control mice. Neutrophil function analysis showed significant reduction of myeloperoxidase (MPO) activity and lysozyme secretion by influenza virus infection in these mice. Our results suggest that influenza virus infection may play an important role in inducing pneumococcal pneumonia in chronic P. aeruginosa infected mice. Our results suggested that our mouse model is useful for investigating the pathogenesis of influenza virus infection in patients with chronic lung infection.

Topics: Acute Disease; Animals; Chronic Disease; Colony Count, Microbial; Cytokines; Disease Models, Animal; Disease Susceptibility; Lung; Lung Diseases, Parasitic; Male; Mice; Mice, Inbred Strains; Muramidase; Orthomyxoviridae Infections; Peroxidase; Pneumococcal Infections; Pneumonia, Pneumococcal; Pseudomonas Infections; Superinfection

Mice lacking surfactant protein SP-A [SP-A(-/-)] and wild type SP-A(+/+) mice were infected with influenza A virus (IAV) by intranasal instillation. Decreased clearance of IAV was observed in SP-A(-/-) mice and was associated with increased pulmonary inflammation. Treatment of SP-A(-/-) mice with exogenous SP-A enhanced viral clearance and decreased lung inflammation. Uptake of IAV by alveolar macrophages was similar in SP-A(-/-) and SP-A(+/+) mice. Myeloperoxidase activity was reduced in isolated bronchoalveolar lavage neutrophils from SP-A(-/-) mice. B lymphocytes and activated T lymphocytes were increased in the lung and spleen, whereas T helper (Th) 1 responses were increased [interferon-gamma, interleukin (IL)-2, and IgG(2a)] and Th2 responses were decreased (IL-4, and IL-10, and IgG(1)) in the lungs of SP-A(-/-) mice 7 days after IAV infection. In the absence of SP-A, impaired viral clearance was associated with increased lung inflammation, decreased neutrophil myeloperoxidase activity, and increased Th1 responses. Because the airway is the usual portal of entry for IAV and other respiratory pathogens, SP-A is likely to play a role in innate defense and adaptive immune responses to IAV.

Topics: Adaptation, Physiological; Alphainfluenzavirus; Animals; Bronchoalveolar Lavage Fluid; CD4 Lymphocyte Count; CD8-Positive T-Lymphocytes; Cytokines; Hemagglutination; Immunoglobulins; Lung; Lung Diseases; Lymphocyte Count; Lymphocytes; Macrophages; Mice; Mice, Knockout; Neutrophils; Orthomyxoviridae Infections; Peroxidase; Phagocytosis; Proteolipids; Pulmonary Surfactant-Associated Protein A; Pulmonary Surfactant-Associated Proteins; Pulmonary Surfactants; Spleen; Viral Load

Mice lacking surfactant protein surfactant protein D (SP-D(-/-)) and wild-type mice (SP-D(+/+)) were infected with influenza A virus (IAV) by intranasal instillation. IAV infection increased the endogenous SP-D concentration in wild-type mice. SP-D-deficient mice showed decreased viral clearance of the Phil/82 strain of IAV and increased production of inflammatory cytokines in response to viral challenge. However, the less glycosylated strain of IAV, Mem/71, which is relatively resistant to SP-D in vitro, was cleared efficiently from the lungs of SP-D(-/-) mice. Viral clearance of the Phil/82 strain of IAV and the cytokine response were both normalized by the coadministration of recombinant SP-D. Since the airway is the usual portal of entry for influenza A virus and other respiratory pathogens, SP-D is likely to play an important role in innate defense responses to IAV.

Topics: Animals; Bronchoalveolar Lavage Fluid; Cytokines; Glycoproteins; Influenza A virus; Lung; Lymphocyte Count; Macrophages, Alveolar; Mice; Mice, Knockout; Neutrophils; Orthomyxoviridae Infections; Peroxidase; Phagocytosis; Pulmonary Surfactant-Associated Protein D; Pulmonary Surfactants; T-Lymphocytes