Batf-Dependent Th17 cells act through IL23R pathway to promote prostate adenocarcinoma initiation and progression.

BACKGROUND: The role of Th17 cells in prostate cancer (PCa) is not fully understood. The transcription factor BATF controls the differentiation of Th17 cells. Mice deficient in Batf do not produce Th17 cells. METHODS: In this study, we aimed to characterize the role of Batf-dependent Th17 cells in PCa by crossbreeding Batf knockout (Batf-/-) mice with mice conditionally mutant for Pten. We found that Batf-/- mice had changes in the morphology of prostate epithelial cells compared to normal mice, and Batf-/- mice deficient in Pten (named Batf-) had smaller prostate size and developed fewer invasive prostate adenocarcinomas than Pten-deficient mice with Batf expression (named Batf+). The prostate tumors in Batf- mice showed reduced proliferation, increased apoptosis, decreased angiogenesis and inflammatory cell infiltration, and activation of NF-κB signaling. Moreover, Batf- mice showed significantly reduced IL-23/IL-23R signaling. In the prostate stroma of Batf- mice, IL-23R-positive cells were decreased considerably compared to Batf+ mice. Splenocytes and prostate tissues from Batf- mice cultured under Th17 differentiation conditions expressed reduced IL-23/IL-23R than cultured cells from Batf+ mice. Anti-IL23p19 antibody treatment of Pten-deficient mice reduced prostate tumors and angiogenesis compared to control IgG-treated mice. In human prostate tumors, BATF mRNA level was positively correlated with IL23A and IL-23R but not RORC. CONCLUSION: Our novel findings underscore the crucial role of IL-23/IL23R signaling in mediating the function of Batf-dependent Th17 cells, thereby promoting PCa initiation and progression. This highlights the Batf-IL-23R axis as a promising target for the development of innovative strategies for PCa prevention and treatment.

Fungal chitin is not an independent mediator of allergic fungal asthma severity.

Chitin, a polysaccharide found in the fungal cell wall and the exoskeletons of house dust mites and cockroaches, has garnered attention as a potential immunoreactive allergen. Mammals have evolved to express chitin-degrading chitinases (acidic mammalian chitinase/AMCase and chitotriosidase) that may modulate immune responses to chitin. We have previously reported that mice deficient in AMCase (Chia-/-) demonstrated better lung function during allergic fungal asthma. As expected, we show that mice overexpressing AMCase (SPAM mice) had worse airway hyperreactivity (AHR) during allergic fungal asthma. We further demonstrate that chitin-positive Aspergillus fumigatus conidia are detectable in the allergic lung during chronic exposure. Lung function in Chia-/- and SPAM mice directly correlated with the level of chitinase activity during chronic fungal exposure (Chia-/- mice, negligible chitinase activity, lower AHR; SPAM mice, heightened chitinase activity, higher AHR), suggesting that the breakdown of chitin promoted AHR. However, chronic exposure of normal mice to purified A. fumigatus chitin resulted in only moderate inflammatory changes in the lung which were not sufficient to induce AHR. Moreover, despite having dramatic differences in chitinase activity, chronic exposure of Chia-/- and SPAM mice to purified A. fumigatus chitin likewise did not modulate AHR. Collectively, these results indicate that chronic exposure to fungal chitin alone is incapable of driving AHR. Furthermore, our data suggests that the chitinase-mediated degradation of chitin associated with A. fumigatus conidia may facilitate unmasking and/or liberation of other fungal cell wall components that drive inflammatory responses which contribute to AHR.

Protein-Protein interactive networks identified in bronchoalveolar lavage of severe compared to nonsevere asthma.

INTRODUCTION: Previous bronchoalveolar lavage fluid (BALF) proteomic analysis has evaluated limited numbers of subjects for only a few proteins of interest, which may differ between asthma and normal controls. Our objective was to examine a more comprehensive inflammatory biomarker panel in quantitative proteomic analysis for a large asthma cohort to identify molecular phenotypes distinguishing severe from nonsevere asthma. METHODS: Bronchoalveolar lavage fluid from 48 severe and 77 nonsevere adult asthma subjects were assessed for 75 inflammatory proteins, normalized to BALF total protein concentration. Validation of BALF differences was sought through equivalent protein analysis of autologous sputum. Subjects' data, stratified by asthma severity, were analysed by standard statistical tests, principal component analysis and 5 machine learning algorithms. RESULTS: The severe group had lower lung function and greater health care utilization. Significantly increased BALF proteins for severe asthma compared to nonsevere asthma were fibroblast growth factor 2 (FGF2), TGFα, IL1Ra, IL2, IL4, CCL8, CCL13 and CXCL7 and significantly decreased were platelet-derived growth factor a-a dimer (PDGFaa), vascular endothelial growth factor (VEGF), interleukin 5 (IL5), CCL17, CCL22, CXCL9 and CXCL10. Four protein differences were replicated in sputum. FGF2, PDGFaa and CXCL7 were independently identified by 5 machine learning algorithms as the most important variables for discriminating severe and nonsevere asthma. Increased and decreased proteins identified for the severe cluster showed significant protein-protein interactions for chemokine and cytokine signalling, growth factor activity, and eosinophil and neutrophil chemotaxis differing between subjects with severe and nonsevere asthma. CONCLUSION: These inflammatory protein results confirm altered airway remodelling and cytokine/chemokine activity recruiting leukocytes into the airways of severe compared to nonsevere asthma as important processes even in stable status.

Elevated cardiac hemoglobin expression is associated with a pro-oxidative and inflammatory environment in primary mitral regurgitation.

BACKGROUND: Primary mitral regurgitation (PMR) is associated with oxidative and inflammatory myocardial damage. We reported greater exosome hemoglobin (Hb) in pericardial fluid (PCF) versus plasma, suggesting a cardiac source of Hb. OBJECTIVE: Test the hypothesis that Hb is produced in the PMR heart and is associated with increased inflammation. METHODS AND RESULTS: Hb gene expression for subunits alpha (HBA) and beta (HBB) was assessed in right atria (RA), left atria (LA) and left ventricular (LV) tissue from donor hearts (n = 10) and PMR patient biopsies at surgery (n = 11). PMR patients (n = 22) had PCF and blood collected for macrophage markers, pro-inflammatory cytokines, and matrix metalloproteinases (MMPs). In-situ hybridization for HBA mRNA and immunohistochemistry for Hb-alpha (Hbα) and Hb-beta (Hbß) protein was performed on PMR tissue. RESULTS: HBA and HBB genes are significantly increased (>4-fold) in RA, LA, and LV in PMR vs. normal hearts. In PMR tissue, HBA mRNA is expressed in both LV cardiomyocytes and interstitial cells by in-situ hybridization; however, Hbα and Hbß protein is only expressed in interstitial cells by immunohistochemistry. PCF oxyHb is significantly increased over plasma along with low ratios (<1.0) of haptoglobin:oxyHb and hemopexin:heme supporting a highly oxidative environment. Macrophage chemotactic protein-1, tumor necrosis factor-α, interleukin-6, and MMPs are significantly higher in PCF vs. plasma. CONCLUSION: There is increased Hb production in the PMR heart coupled with the inflammatory state of the heart, suggests a myocardial vulnerability of further Hb delivery and/or production during cardiac surgery that could adversely affect LV functional recovery.

Chitinase-3-like 1 regulates TH2 cells, TFH cells and IgE responses to helminth infection.

Introduction: Data from patient cohorts and mouse models of atopic dermatitis, food allergy and asthma strongly support a role for chitinase-3-like-1 protein (CHI3L1) in allergic disease. Methods: To address whether Chi3l1 also contributes to TH2 responses following nematode infection, we infected Chi3l1 -/- mice with Heligmosomoides polygyrus (Hp) and analyzed T cell responses. Results: As anticipated, we observed impaired TH2 responses in Hp-infected Chi3l1 -/- mice. However, we also found that T cell intrinsic expression of Chi3l1 was required for ICOS upregulation following activation of naïve CD4 T cells and was necessary for the development of the IL-4+ TFH subset, which supports germinal center B cell reactions and IgE responses. We also observed roles for Chi3l1 in TFH, germinal center B cell, and IgE responses to alum-adjuvanted vaccination. While Chi3l1 was critical for IgE humoral responses it was not required for vaccine or infection-induced IgG1 responses. Discussion: These results suggest that Chi3l1 modulates IgE responses, which are known to be highly dependent on IL-4-producing TFH cells.

Loss of the mammalian G-protein coupled receptor, G2A, modulates severity of invasive pulmonary aspergillosis.

Background: Aspergillus fumigatus is a well-known opportunistic pathogen that causes a range of diseases including the often-fatal disease, invasive pulmonary aspergillosis (IPA), in immunocompromised populations. The severity of IPA is dependent on both host- and pathogen-derived signaling molecules that mediate host immunity and fungal growth. Oxylipins are bioactive oxygenated fatty acids known to influence host immune response and Aspergillus developmental programs. Aspergillus synthesizes 8-HODE and 5,8-diHODE that have structural similarities to 9-HODE and 13-HODE, which are known ligands of the host G-protein-coupled receptor G2A (GPR132). Materials and methods: Oxylipins were extracted from infected lung tissue to assess fungal oxylipin production and the Pathhunter ß-arrestin assay was used to assess agonist and antagonist activity by fungal oxylipins on G2A. An immunocompetent model of A. fumigatus infection was used to assess changes in survival and immune responses for G2A-/- mice. Results: Here we report that Aspergillus oxylipins are produced in lung tissue of infected mice and in vitro ligand assays suggest 8-HODE is a G2A agonist and 5,8-diHODE is a partial antagonist. To address the hypothesis that G2A could be involved in the progression of IPA, we assessed the response of G2A-/- mice to A. fumigatus infection. G2A-/- mice showed a survival advantage over wild-type mice; this was accompanied by increased recruitment of G2A-/- neutrophils and increased levels of inflammatory markers in A. fumigatus-infected lungs. Conclusions: We conclude that G2A suppresses host inflammatory responses to Aspergillus fumigatus although it remains unclear if fungal oxylipins are involved in G2A activities.

Feasibility study of intraoperative pericardial fluid biomarkers and length of stay after cardiac surgery.

Objective: Pericardial fluid biomarkers reflect the physiologic state of the myocardium. Previously, we showed a sustained increase in pericardial fluid biomarkers compared with blood in the 48 hours after cardiac surgery. We assess the feasibility of analyzing 9 common cardiac biomarkers from pericardial fluid collected during cardiac surgery and test a preliminary hypothesis of association between the most common biomarkers, troponin and brain natriuretic peptide, and length of stay after surgery. Methods: We prospectively enrolled 30 patients aged 18 years or more undergoing coronary artery or valvular surgery. Patients with ventricular assist devices, atrial fibrillation surgery, thoracic aorta surgery, redo surgery, concomitant noncardiac surgery, and preoperative inotropic support were excluded. Before pericardial excision during surgery, a 1-cm pericardial incision was made to insert an 18-gauge catheter and collect 10 mL of pericardial fluid. Concentrations of 9 established biomarkers of cardiac injury or inflammation including brain natriuretic peptide and troponin were measured. Zero truncated Poisson regression adjusted for Society of Thoracic Surgery Preoperative Risk of Mortality tested for a preliminary association between pericardial fluid biomarkers and length of stay. Results: Pericardial fluid was collected and pericardial fluid biomarkers resulted for all patients. Adjusted for Society of Thoracic Surgery risk, brain natriuretic peptide, and troponin were associated with increased intensive care unit and overall hospital length of stay. Conclusions: In 30 patients, pericardial fluid was obtained and analyzed for cardiac biomarkers. Adjusting for Society of Thoracic Surgery risk, pericardial fluid troponin and brain natriuretic peptide were preliminarily associated with increased length of stay. Further investigation is needed to validate this finding and to investigate the potential clinical utility of pericardial fluid biomarkers.

Impact of early pericardial fluid chymase activation after cardiac surgery.

Introduction: Chymase is a highly destructive serine protease rapidly neutralized in the circulation by protease inhibitors. Here we test whether pericardial fluid (PCF) chymase activation and other inflammatory biomarkers determine intensive care unit length of stay, and explore mechanisms of chymase delivery by extracellular vesicles to the heart. Methods: PCF was collected from adult patients (17 on-pump; 13 off-pump) 4 h after cardiac surgery. Extracellular vesicles (EVs) containing chymase were injected into Sprague-Dawley rats to test for their ability to deliver chymase to the heart. Results: The mean intensive care unit (ICU) stay and mean total length of stay was 2.17 ± 3.8 days and 6.41 ± 1.3 days respectively. Chymase activity and 32 inflammatory markers did not differ in on-pump vs. off-pump cardiac surgery. Society of Thoracic Surgeons Predicted Risk of Morbidity and Mortality Score (STS-PROM), 4-hour post-surgery PCF chymase activity and C-X-C motif chemokine ligand 6 (CXCL6) were all independent predictors of ICU and total hospital length of stay by univariate analysis. Mass spectrometry of baseline PCF shows the presence of serine protease inhibitors that neutralize chymase activity. The compartmentalization of chymase within and on the surface of PCF EVs was visualized by immunogold labeling and transmission electron microscopy. A chymase inhibitor prevented EV chymase activity (0.28 fmol/mg/min vs. 14.14 fmol/mg/min). Intravenous injection of PCF EVs obtained 24 h after surgery into Sprague Dawley rats shows diffuse human chymase uptake in the heart with extensive cardiomyocyte damage 4 h after injection. Discussion: Early postoperative PCF chymase activation underscores its potential role in cardiac damage soon after on- or off-pump cardiac surgery. In addition, chymase in extracellular vesicles provides a protected delivery mechanism from neutralization by circulating serine protease inhibitors.

Hematopoietic 12/15-lipoxygenase activity negatively contributes to fungal-associated allergic asthma.

Asthma is one of the most common noncommunicable diseases in the world. Approximately 30% of severe cases are associated with fungal sensitization, often associated with allergy to the opportunistic mold Aspergillus fumigatus. Leukotrienes, immunopathogenic mediators derived from the metabolism of arachidonic acid (AA) by 5-lipoxygenase (5-LOX), are often elevated in severe asthma. As such, these mediators are Food and Drug Administration-approved therapeutic targets of the antiasthmatic drugs Zileuton/Zyflo and Singulair/Montelukast. A second enzyme involved in AA metabolism is 12/15-lipoxygenase (12/15-LOX; Alox15). Here, C57BL/6 wild-type (WT) mice subjected to experimental fungal asthma had increased expression of Alox15 mRNA and increased levels of 12-HETE, a product of 12/15-LOX activity, in the lung when compared with naïve and vehicle-treated mice. Mice deficient in 12/15-LOX (Alox15-/-) demonstrated better lung function, as measured by airway hyperresponsiveness (AHR), during fungal asthma. Histological assessment revealed reduced inflammation in the lungs of Alox15-/- mice compared with WT mice, which was corroborated by flow cytometric analysis of multiple myeloid (eosinophils and neutrophils) and lymphoid (CD4+ T and γδ T) cell populations. This was further supported by decreased levels of specific chemokines that promote the recruitment of these cells. Likewise, type 1 and 2, but not type 17 cytokines, were significantly lower in the lungs of Alox15-/- mice. Bone marrow chimera studies revealed that the presence of 12/15-LOX in hematopoietic cells contributed to AHR during fungal asthma. Taken together, our data support the hypothesis that hematopoietic-associated 12/15-LOX contributes to type 1 and 2 responses and exacerbation of allergic fungal asthma.NEW & NOTEWORTHY Humans with asthma sensitized to fungi often have more severe asthma than those who are not sensitized to fungi. Products of arachidonic acid generated via 5-lipoxygenase are often elevated in severe asthma and are successful FDA-approved drug targets. Less understood is the role of products generated via 12/15-lipoxygenase. We demonstrate that 12/15-lipoxygenase expression in hematopoietic cells contributes to type 1 and 2 responses and impaired lung function during allergic fungal asthma.

EXOSOMES AND MICROVESICLES FROM ADIPOSE-DERIVED MESENCHYMAL STEM CELLS PROTECTS THE ENDOTHELIAL GLYCOCALYX FROM LPS INJURY.

ABSTRACT: Introduction: Endothelial glycocalyx damage occurs in numerous pathological conditions and results in endotheliopathy. Extracellular vesicles, including exosomes and microvesicles, isolated from adipose-derived mesenchymal stem cells (ASCs) have therapeutic potential in multiple disease states; however, their role in preventing glycocalyx shedding has not been defined. We hypothesized that ASC-derived exosomes and microvesicles would protect the endothelial glycocalyx from damage by LPS injury in cultured endothelial cells. Methods : Exosomes and microvesicles were collected from ASC conditioned media by centrifugation (10,000 g for microvesicles, 100,000 g for exosomes). Human umbilical vein endothelial cells (HUVECs) were exposed to 1 µg/mL lipopolysaccharide (LPS). LPS-injured cells (n = 578) were compared with HUVECS with concomitant LPS injury plus 1.0 µg/mL of exosomes (n = 540) or microvesicles (n = 510) for 24 hours. These two cohorts were compared with control HUVECs that received phosphate-buffered saline only (n = 786) and HUVECs exposed to exosomes (n = 505) or microvesicles (n = 500) alone. Cells were fixed and stained with FITC-labeled wheat germ agglutinin to quantify EGX. Real-time quantitative reverse-transcription polymerase chain reaction was used on HUVECs cell lystate to quantify hyaluron synthase-1 (HAS1) expression. Results: Exosomes alone decreased endothelial glycocalyx staining intensity when compared with control (4.94 vs. 6.41 AU, P < 0.001), while microvesicles did not cause a change glycocalyx staining intensity (6.39 vs. 6.41, P = 0.99). LPS injury resulted in decreased glycocalyx intensity as compared with control (5.60 vs. 6.41, P < 0.001). Exosomes (6.85 vs. 5.60, P < 0.001) and microvesicles (6.35 vs. 5.60, P < 0.001) preserved endothelial glycocalyx staining intensity after LPS injury. HAS1 levels were found to be higher in the exosome (1.14 vs. 3.67 RE, P = 0.02) and microvesicle groups (1.14 vs. 3.59 RE, P = 0.02) when compared with LPS injury. Hyaluron synthase-2 and synthase-3 expressions were not different in the various experimental groups. Conclusions: Exosomes alone can damage the endothelial glycocalyx. However, in the presence of LPS injury, both exosomes and microvesicles protect the glycocalyx layer. This effect seems to be mediated by HAS1. Level of Evidence : Basic science study.

Glycocalyx degradation and the endotheliopathy of viral infection.

The endothelial glycocalyx (EGX) contributes to the permeability barrier of vessels and regulates the coagulation cascade. EGX damage, which occurs in numerous disease states, including sepsis and trauma, results in endotheliopathy. While influenza and other viral infections are known to cause endothelial dysfunction, their effect on the EGX has not been described. We hypothesized that the H1N1 influenza virus would cause EGX degradation. Human umbilical vein endothelial cells (HUVECs) were exposed to varying multiplicities of infection (MOI) of the H1N1 strain of influenza virus for 24 hours. A dose-dependent effect was examined by using an MOI of 5 (n = 541), 15 (n = 714), 30 (n = 596), and 60 (n = 653) and compared to a control (n = 607). Cells were fixed and stained with FITC-labelled wheat germ agglutinin to quantify EGX. There was no difference in EGX intensity after exposure to H1N1 at an MOI of 5 compared to control (6.20 vs. 6.56 Arbitrary Units (AU), p = 0.50). EGX intensity was decreased at an MOI of 15 compared to control (5.36 vs. 6.56 AU, p<0.001). The degree of EGX degradation was worse at higher doses of the H1N1 virus; however, the decrease in EGX intensity was maximized at an MOI of 30. Injury at MOI of 60 was not worse than MOI of 30. (4.17 vs. 4.47 AU, p = 0.13). The H1N1 virus induces endothelial dysfunction by causing EGX degradation in a dose-dependent fashion. Further studies are needed to characterize the role of this EGX damage in causing clinically significant lung injury during acute viral infection.

Neuroimmune Responses in a New Experimental Animal Model of Cerebral Aspergillosis.

Exposure of immunosuppressed individuals to the opportunistic fungal pathogen Aspergillus fumigatus may result in invasive pulmonary aspergillosis (IPA), which can lead to the development of cerebral aspergillosis (CA), a highly lethal infection localized in the central nervous system (CNS). There are no experimental models of CA that effectively mimic human disease, resulting in a considerable knowledge gap regarding mechanisms of neurological pathogenicity and neuroimmune responses during infection. In this report, immunosuppressed mice (via acute, high-dose corticosteroid administration) challenged with A. fumigatus resting conidia intranasally, followed a day later by a 70-fold lower inoculum of pre-swollen conidia intravenously (IN + IV + steroid), demonstrated increased weight loss, signs of severe clinical disease, increased fungal burden in the brain, and significant reduction in survival compared to immunosuppressed mice challenged intranasally only (IN + steroid) or non-immunosuppressed mice challenged both intranasally and intravenously (IN + IV). The IN + IV + steroid group demonstrated significant decreases in monocytes, eosinophils, dendritic cells (DCs), and invasive natural killer T (iNKT) cells, but not neutrophils or γδ T cells, in the brain compared to the IN + IV group. Likewise, the IN + IV + steroid group had significantly lower levels of interleukin (IL)-1ß, IL-6, IL-17A, CC motif chemokine ligand 3 (CCL3), CXC chemokine ligand 10 (CXCL10), and vascular endothelial growth factor (VEGF) in the brain compared to the IN + IV group. IN + IV + steroid was superior to both IN + IV + chemotherapy (cytarabine + daunorubicin) and IN + IV + neutropenia for the development of CA. In conclusion, we have developed a well-defined, physiologically relevant model of disseminated CA in corticosteroid-induced immunosuppressed mice with a primary pulmonary infection. This model will serve to advance understanding of disease mechanisms, identify immunopathogenic processes, and help define the protective neuroinflammatory response to CA. IMPORTANCE Invasive fungal infections (IFIs) result in significant mortality in immunosuppressed individuals. Of these, invasive pulmonary aspergillosis (IPA), caused by the opportunistic mold Aspergillus fumigatus, is the most lethal. Lethality in IPA is due to two main factors: destruction of the lung leading to compromised pulmonary function, and dissemination of the organism to extrapulmonary organs. Of these, the CNS is the most common site of dissemination. However, very little is known regarding the pathogenesis of or immune response during cerebral aspergillosis, which is directly due to the lack of an animal model that incorporates immunosuppression, lung infection, and consistent dissemination to the CNS/brain. In this report, we have developed a new experimental animal model of CA which includes the above parameters and characterized the neuroimmune response. We further compared this disseminated CA model to two additional immunosuppressive strategies. Overall, this model of disseminated CA following IPA in an immunosuppressed host provides a novel platform for studying the efficacy of antifungal drugs and immunotherapies for improving disease outcomes.

A Systematic Review to Assess the Relationship between Disseminated Cerebral Aspergillosis, Leukemias and Lymphomas, and Their Respective Therapeutics.

Disseminated disease following invasive pulmonary aspergillosis (IPA) remains a significant contributor to mortality amongst patients with hematologic malignancies (HMs). At the highest risk of mortality are those with disseminated disease to the central nervous system, known as cerebral aspergillosis (CA). However, little is known about the risk factors contributing to disease amongst HM patients. A systematic review using PRISMA guidelines was undertaken to define HM patient subgroups, preventative measures, therapeutic interventions, and outcomes of patients with disseminated CA following IPA. The review resulted in the identification of 761 records, of which 596 articles were screened, with the final inclusion of 47 studies and 76 total patients. From included articles, the proportion of CA was assessed amongst HM patient subgroups. Further, pre-and post-infection characteristics, fungal species, and mortality were evaluated for the total population included and HM patient subgroups. Patients with acute myeloid leukemia and acute lymphoid lymphoma, patients receiving corticosteroids as a part of their HM therapeutic regimen, and anti-fungal prophylaxis constitute the top identified patient populations at risk for disseminated CA. Overall, information presented here indicates that measures for the prevention of IPA should be taken in higher-risk HM patient subgroups. Specifically, the type of anti-fungal therapy used should be carefully considered for those patients with IPA and increased risk for cerebral dissemination. Additional reports detailing patient characteristics are needed to define further the risk of developing disseminated CA from IPA in patients with HMs.

A Novel Controlled PTEN-Knockout Mouse Model for Prostate Cancer Study.

Prostate cancer (PCa) is associated with advanced age, but how age contributes to prostate carcinogenesis remains unknown. The prostate-specific Pten conditional knockout mouse model closely imitates human PCa initiation and progression. To better understand how age impacts PCa in an experimental model, we have generated a spatially and temporally controlled Pten-null PCa murine model at different ages (aged vs. non-aged) of adult mice. Here, we present a protocol to inject the Cre-expressing adenovirus with luciferin tag, intraductally, into the prostate anterior lobes of Pten-floxed mice; Pten-loss will be triggered post-Cre expression at different ages. In vivo imaging of luciferin signal following viral infection confirmed successful delivery of the virus and Cre activity. Immunohistochemical staining confirmed prostate epithelial-specific expression of Cre recombinase and the loss of Pten and activation of P-Akt, P-S6, and P-4E-BP1. The Cre-expression, Pten ablation, and activated PI3K/AKT/mTOR pathways were limited to the prostate epithelium. All mice developed prostatic epithelial hyperplasia within 4 weeks after Pten ablation and prostatic intraepithelial neoplasia (PIN) within 8 weeks post-Pten ablation. Some PINs had progressed to invasive adenocarcinoma at 8-16 weeks post-Pten ablation. Aged mice exhibited significantly accelerated PI3K/AKT/mTOR signaling and increased PCa onset and progression compared to young mice. The viral infection success rate is ∼80%. This model will be beneficial for investigations of cancer-related to aging.

A Comparison of Growth Factors and Cytokines in Fresh Frozen Plasma and Never Frozen Plasma.

BACKGROUND: Fresh frozen plasma (FFP) contains proinflammatory mediators released from cellular debris during frozen storage. In addition, recent studies have shown that transfusion of never-frozen plasma (NFP), instead of FFP, may be superior in trauma patients. We hypothesized that FFP would have higher levels of inflammatory mediators when compared to NFP. MATERIALS AND METHODS: FFP (n = 8) and NFP (n = 8) samples were obtained from an urban, level 1 trauma center blood bank. The cytokines in these samples were compared using a Milliplex (Milliplex Sigma) human cytokine magnetic bead panel multiplex assay for 41 different biomarkers. RESULTS: Growth factors that were higher in NFP included platelet-derived growth factor-AA (PDGF-AA; 8.09 versus 108.00 pg/mL, P < 0.001) and PDGF-AB (0.00 versus 215.20, P= 0.004). Soluble CD40-ligand (sCD40L), a platelet activator and pro-coagulant, was higher in NFP (31.81 versus 80.45 pg/mL, P< 0.001). RANTES, a leukocyte chemotactic cytokine was higher in NFP (26.19 versus 1418.00 pg/mL, P< 0.001). Interleukin-4 (5.70 versus 0.00 pg/mL, P= 0.03) and IL-8 (2.20 versus 0.52 pg/ml, P= 0.03) levels were higher in were higher in FFP. CONCLUSIONS: Frozen storage of plasma may result in decrease of several growth factors and/or pro-coagulants found in NFP. In addition, the freezing and thawing process may induce release of pro-inflammatory chemokines. Further studies are needed to determine if these cytokines result in improved outcomes with NFP over FFP in transfusion of trauma patients.

The chemokine CX3CL1/fractalkine regulates immunopathogenesis during fungal-associated allergic airway inflammation.

Individuals that present with difficult-to-control asthma and sensitivity to one or more fungal species are categorized as a subset of severe asthma patients belonging to a group herein referred to as severe asthma with fungal sensitization (SAFS). We have previously reported the identification of numerous cytokines and chemokines that were elevated in human asthmatics that were sensitized to fungi vs. nonfungal sensitized asthmatics. Here, we show that the unique chemokine CX3CL1 (fractalkine) is elevated in both bronchoalveolar lavage fluid and sputum from human asthmatics sensitized to fungi, implicating an association with CX3CL1 in fungal asthma severity. In an experimental model of fungal-associated allergic airway inflammation, we demonstrate that the absence of CX3CR1 signaling unexpectedly resulted in a profound impairment in lung function. Histological assessment of lung tissue revealed an unrestricted inflammatory response that was subsequently characterized by enhanced levels of neutrophils, eosinophils, and inflammatory monocytes. Neutrophilic inflammation correlated with elevated IL-17A, proinflammatory cytokines (TNF-α, IL-1α, and IL-1ß), neutrophil survival factors (granulocyte colony-stimulating factor), and neutrophil-targeting chemokines (CCL3 and CCL4). Eosinophilia correlated with elevated type 2 responses (IL-5 and IL-13) whereas inflammatory monocyte levels correlated with elevated type 1 responses (IFN-γ and CXCL9) and survival factors (macrophage colony-stimulating factor). Despite enhanced inflammatory responses, the immunoregulatory cytokine IL-10 and the natural inhibitor of IL-1 signaling, IL-1RA, were significantly elevated rather than impaired. Regulatory T-cell levels were unchanged, as were levels of the anti-inflammatory cytokines IL-35 and IL-38. Taken together, the CX3CL1/CX3CR1 axis preserves lung function during fungal-associated allergic airway inflammation through a nonclassical immunoregulatory mechanism.

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and receptors in type 1, type 2 and type 17 inflammation in cross-sectional asthma study.

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) reportedly promotes, or conversely, resolves inflammation in asthma. In this study of TRAIL and cell receptors in sputum, bronchoalveolar lavage and biopsy from subjects in the Severe Asthma Research Program at Wake Forest, the high TRAIL group had significant increases in all leucocytes, and was associated with increased type 1, type 2 and type 17 cytokines, but not type 9 interleukin 9. Two variants at loci in the TRAIL gene were associated with higher sputum levels of TRAIL. Increased TRAIL decoy receptor R3/DcR1 was observed on sputum leucocytes compared with death receptor R1/DR4, suggesting reduced apoptosis and prolonged cellular inflammation.

CD4+ T helper 17 cell response of aged mice promotes prostate cancer cell migration and invasion.

BACKGROUND: Aging is the most important risk factor for prostate cancer (PCa), but how age contributes to PCa is poorly understood. Aging is characterized by low-grade systemic inflammation (i.e., inflammaging) that is often attributed to the progressive activation of immune cells over time, which may play an important role in prostate carcinogenesis. Th17 response is elevated in aging humans and mice, but it remains unknown whether it is increased in prostate tissue or contributes to prostate carcinogenesis during aging. In this study, we aimed to determine the role of age-related Th17 response in PCa cell growth, migration, and invasion. METHODS: C57BL/6J (B6) mouse was used as an aging animal model and the prostate histopathology during aging was analyzed. Splenic CD4+ T cells were isolated from young (16-20 weeks old) and aged (96-104 weeks old) mice, and cultured in the presence of plate-bound anti-CD3/anti-CD28, with or without Th17 differentiation conditions. The cells were collected and used for subsequent flow cytometry or quantitative reverse transcription polymerase chain reaction. The supernatant was collected and used to treat PCa cell lines. The treated PCa cells were analyzed for cell viability, migration, invasion, and nuclear factor kappa B (NF-κB) signaling. RESULTS: Aged mice had enlarged prostate glands and increased morphological alterations, with not only increased inflammatory cell infiltration but also increased Th17 cytokines in prostate tissue, compared to young mice. Naïve CD4+ T cells from aged mice differentiated increased interleukin (IL)-17-expressing cells. CD4+ T cells from aged mice spleen had increased Th17 cells, Th17 cytokines and Th17/Treg ratio compared to young mice. Factors secreted from aged CD4+ T cells, especially from ex vivo differentiated Th17 cells, not only promoted PCa cell viability, migration, and invasion but also activated the NF-κB signaling in PCa cells compared to young mice. CONCLUSIONS: These results indicate that age-related CD4+ T cells, especially Th17 cells-secreted factors have the potential to contribute to prostate carcinogenesis. Our work could prompt further research using autochthonous PCa mouse models at different ages to elucidate the functional role of Th17 response in prostate carcinogenesis during aging.

12/15-Lipoxygenase Deficiency Impairs Neutrophil Granulopoiesis and Lung Proinflammatory Responses to Aspergillus fumigatus.

Development of invasive aspergillosis correlates with impairments in innate immunity. We and others have recently shown that arachidonic acid metabolism pathways, specifically the cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) pathways, participate in the induction of protective innate immune responses during invasive aspergillosis. Based on the high degree of cooperation and interconnection within the eicosanoid network, we hypothesized that 12/15-LOX is also active during invasive aspergillosis. We report in this study that mice deficient in the gene encoding 12/15-LOX (Alox15) are profoundly susceptible to invasive aspergillosis. Decreased survival correlated with increased fungal burden and evidence of increased lung damage. These defects were associated with very early (6 and 12 h) 12/15-LOX-dependent inflammatory cytokine (IL-1α, IL-1ß, and TNF-α) and chemokine (CCL3 and CCL4) production. Neutrophil levels in the lung were blunted in the absence of 12/15-LOX, although neutrophil antifungal activity was intact. However, lower neutrophil levels in the lungs of Alox15 -/- mice were not a result of impaired recruitment or survival; rather, Alox15 -/- mice demonstrated impaired neutrophil granulopoiesis in the bone marrow intrinsically and after fungal exposure. Employing a lower inoculum to allow for better survival allowed the identification of 12/15-LOX-dependent induction of IL-17A and IL-22. Impaired IL-17A and IL-22 production correlated with reduced invariant NKT cell numbers as well as lower IL-23 levels. Together, these data indicate that 12/15-LOX is a critical player in induction of the earliest aspects of the innate immune response to Aspergillus fumigatus.