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Up to 20% of idiopathic interstitial lung disease is familial, referred to as Familial Pulmonary Fibrosis (FPF). We performed an integrated analysis of FPF genetic risk by comprehensively evaluating for genetic rare variants (RVs) in a large cohort of FPF kindreds.We performed whole-exome sequencing and/or candidate gene sequencing from affected individuals in 569 FPF kindreds, followed by co-segregation analysis in large kindreds, gene burden analysis, gene-based risk scoring, cell type enrichment analysis, and co-expression network construction.We found that 14.9% - 23.4% of genetic risk in kindreds could be explained by RVs in genes previously linked to FPF, predominantly telomere related genes. We identified new candidate genes in a small number of families, including SYDE1, SERPINB8, GPR87, and NETO1, and developed tools for evaluation and prioritization of RV-containing genes across kindreds. Several pathways were enriched for RV-containing genes in FPF, including focal adhesion and mitochondrial complex I assembly. By combining single cell transcriptomics with prioritized candidate genes, we discovered that expression of RV-containing genes was enriched in smooth muscle cells, type II alveolar epithelial cells, and endothelial cells.In the most comprehensive FPF genetic study to date, we defined the prevalence of RVs in known FPF-related genes and identified new candidate genes and pathways relevant to FPF. However, we did not identify new RV-containing genes shared across multiple kindreds, thereby suggesting that heterogeneous genetic variants involving a variety of genes and pathways mediate genetic risk in most FPF kindreds.

Rationale: The preclinical natural history of progressive lung fibrosis is poorly understood.Objectives: Our goals were to identify risk factors for interstitial lung abnormalities (ILA) on high-re...

Pharmacologic evidence suggests that activation of A(2B) adenosine receptors results in proinflammatory effects relevant to the progression of asthma, a chronic lung disease associated with elevated interstitial adenosine concentrations in the lung. This concept has been challenged by the finding that genetic removal of A(2B) receptors leads to exaggerated responses in models of acute inflammation. Therefore, the goal of our study was to determine the effects of A(2B) receptor gene ablation in the context of ovalbumin-induced chronic pulmonary inflammation. We found that repetitive airway allergen challenge induced a significant increase in adenosine levels in fluid recovered by bronchoalveolar lavage. Genetic ablation of A(2B) receptors significantly attenuated allergen-induced chronic pulmonary inflammation, as evidenced by a reduction in the number of bronchoalveolar lavage eosinophils and in peribronchial eosinophilic infiltration. The most striking difference in the pulmonary inflammation induced in A(2B) receptor knockout (A(2B)KO) and wild-type mice was the lack of allergen-induced IL-4 release in the airways of A(2B)KO animals, in line with a significant reduction in IL-4 protein and mRNA levels in lung tissue. In addition, attenuation of allergen-induced transforming growth factor-beta release in airways of A(2B)KO mice correlated with reduced airway smooth muscle and goblet cell hyperplasia/hypertrophy. In conclusion, genetic removal of A(2B) adenosine receptors in mice leads to inhibition of allergen-induced chronic pulmonary inflammation and airway remodeling. These findings are in agreement with previous pharmacologic studies suggesting a deleterious role for A(2B) receptor signaling in chronic lung inflammation.

Nuclear factor kappa B (NF-kappa B) is a critical transcription factor for the production of many inflammatory cytokines. It is activated in the airway epithelium of human asthmatics and in mice after allergic stimulation. To examine the role of NF-kappa B activation in allergic inflammation, the authors generated transgenic mouse lines that allowed for the inducible stimulation of NF-kappa B in airway epithelial cells. After allergic sensitization with ovalbumin and alum, mice were challenged daily with ovalbumin aerosols and NF-kappa B was activated in airway epithelium by administration of doxycycline. Enhancement of airway epithelial NF-kappa B expression alone did not lead to increased airway responsiveness to methacholine. However, induction of epithelial NF-kappa B during allergic inflammation caused airway hyperresponsiveness, increased airway neutrophilic and lymphocytic inflammation and goblet cell hyperplasia. Accompanying the exaggerated inflammation was an increase in the cytokines granulocyte colony-stimulating factor (G-CSF), interleukin (IL)-15, and KC. Interestingly, the counter regulatory interleukin, IL-10, was suppressed by NF-kappa B activation. The epithelial NF-kappa B dependent modulation of these cytokines provides a plausible explanation for the increased inflammation seen with overexpression of NF-kappa B. Modulation of airway epithelial NF-kappa B activation enhances the airway hyperresponsiveness and mucus secretion found in the mouse lung during allergic inflammation. NF-kappa B represents a potential target for pharmacologic intervention in human asthma.

IL-4 contributes to immunopathology induced in mice by primary respiratory syncytial virus (RSV) infection. However, the cellular source of IL-4 in RSV infection is unknown. We identified CD3−CD49b+ cells as the predominant source of IL-4 in the lungs of RSV-infected BALB/c mice. We ruled out T cells, NK cells, NKT cells, mast cells, and eosinophils as IL-4 expressors in RSV infection by flow cytometry. Using IL4 GFP reporter mice (4get) mice, we identified the IL-4-expressing cells in RSV infection as basophils (CD3−CD49b+FcεRI+c-kit−). Because STAT1−/− mice have an enhanced Th2-type response to RSV infection, we also sought to determine the cellular source and role of IL-4 in RSV-infected STAT1−/− mice. RSV infection resulted in significantly more IL-4-expressing CD3−CD49b+ cells in the lungs of STAT1−/− mice than in BALB/c mice. CD49b+IL-4+ cells sorted from the lungs of RSV-infected STAT1−/− mice and stained with Wright-Giemsa had basophil characteristics. As in wild-type BALB/c mice, IL-4 contributed to lung histopathology in RSV-infected STAT1−/− mice. Depletion of basophils in RSV-infected STAT1−/− mice reduced lung IL-4 expression. Thus, we show for the first time that a respiratory virus (RSV) induced basophil accumulation in vivo. Basophils were the primary source of IL-4 in the lung in RSV infection, and STAT1 was a negative regulator of virus-induced basophil IL-4 expression.

BackgroundActivation of coagulation by expression of tissue factor (TF) in the airspace is a hallmark of acute lung injury (ALI) but the timing of TF activation in relationship to increases in lung permeability and inflammation are unknown. MethodsTo test the hypothesis that TF is upregulated early in the course of acute bleomycin lung injury and precedes increased permeability and inflammation we studied the early course of bleomycin‐induced ALI in mice. Mice were treated with 0.04U intratracheal bleomycin or vehicle control and bronchoalveolar lavage (BAL) and lung tissue were collected daily for 7 days. Whole lung TF mRNA was determined by QT‐PCR. TF protein was assessed by ELISA and immunostaining. BAL procoagulant activity was measured by BAL clot time and thrombin‐antithrombin complexes. Inflammation was assessed by BAL cell count, differentials and CXCL1/KC concentration. Lung permeability was assessed by BAL protein and lung wet to dry weight ratio. ResultsExpression of CXCL1 occurred by day 1. BAL protein and lung wet‐to‐dry weight ratio increased significantly by day 3. TF mRNA and BAL procoagulant activity peaked on day 4 while whole lung TF protein peaked on day 6. Changes in permeability and procoagulant activity preceded inflammatory cell influx which was maximal at day 6 while whole lung TF protein peaked along with inflammation. ConclusionThese data demonstrate that cytokine upregulation is the earliest response to bleomycin administration, followed by increased lung permeability, upregulation of TF, and recruitment of inflammatory cells.

Background Fibrosing mediastinitis (FM) is an idiosyncratic reaction to infection with Histoplasma capsulatum with a prevalence of 3:100,000 people infected. The rarity of post-histoplasmosis fibrosing mediastinitis (PHFM) in areas where H. capsulatum is endemic suggests that an abnormal immunological host response may be responsible for the development of fibrosis. Our group previously reported an association between subjects with PHFM and human leukocyte antigen (HLA)-A*02. We sought to confirm or extend those findings with application of high resolution HLA typing in a cohort of subjects with PHFM. Methods High-resolution HLA typing was performed on DNA samples from a new cohort 34 patients with PHFM. Control cohorts included 707 subjects from the “European American” subset of the National Marrow Donor Program® (NMDP) and 700 subjects from Dialysis Clinic, Inc. (DCI). The carriage frequencies of the HLA alleles identified in the PHFM, NMDP, and DCI cohorts were calculated and then all were compared. Results We found an increase in the carriage frequency of HLA-DQB1*04:02 in PHFM subjects relative to the controls (0.15 versus 0.07 in DCI and 0.05 in NMDP; p = 0.08 and 0.03). Multiple logistic regression showed that DQB1*04:02 was statistically significant (p = 0.04), while DQB1*03:02 and C*03:04 had point estimates of OR > 1, though they did not reach statistical significance. The HLA-A*02 association was not replicated. Conclusions HLA-DQB1*04:02 is associated with PHFM, which supports the premise that an aberrant host immune response contributes to the development of PHFM.

Rationale: Up to 20% of cases of idiopathic interstitial pneumonia cluster in families, comprising the syndrome of familial interstitial pneumonia (FIP); however, the genetic basis of FIP remains uncertain in most families.Objectives: To determine if new disease-causing rare genetic variants could be identified using whole-exome sequencing of affected members from FIP families, providing additional insights into disease pathogenesis.Methods: Affected subjects from 25 kindreds were selected from an ongoing FIP registry for whole-exome sequencing from genomic DNA. Candidate rare variants were confirmed by Sanger sequencing, and cosegregation analysis was performed in families, followed by additional sequencing of affected individuals from another 163 kindreds.Measurements and Main Results: We identified a potentially damaging rare variant in the gene encoding for regulator of telomere elongation helicase 1 (RTEL1) that segregated with disease and was associated with very short telomeres in peripheral blood ...

The allergic inflammation occurring in asthma is believed to be accompanied by the production of free radicals. To investigate the role of free radicals and the cells affected we turned to a murine model of allergic inflammation produced by sensitization to ovalbumin with subsequent aerosol challenge. We examined oxidant stress by measuring and localizing the sensitive and specific marker of lipid peroxidation, the F2-isoprostanes. F2-isoprostanes in whole lung increased from 0.30 +/- 0.08 ng/lung at baseline to a peak of 0.061 +/- 0.09 ng/lung on the ninth day of daily aerosol allergen challenge. Increased immunoreactivity to 15-F2t-IsoP (8-iso-PGF2alpha) or to isoketal protein adducts was found in epithelial cells 24 h after the first aerosol challenge and at 5 days in macrophages. Collagen surrounding airways and blood vessels, and airway and vascular smooth muscle, also exhibited increased immunoreactivity after ovalbumin challenge. Dietary vitamin E restriction in conjunction with allergic inflammation led to increased whole lung F2-isoprostanes while supplemental vitamin E suppressed their formation. Similar changes in immunoreactivity to F2-isoprostanes were seen. Airway responsiveness to methacholine was also increased by vitamin E depletion and decreased slightly by supplementation with the antioxidant. Our findings indicate that allergic airway inflammation in mice is associated with an increase in oxidant stress, which is most striking in airway epithelial cells and macrophages. Oxidant stress plays a role in the production of airway responsiveness.

We developed a novel mouse model of malignant pleural effusion (MPE) by injecting Lewis lung cancer (LLC) cells directly into the pleural space of syngeneic C57B/6 mice. The pleural effusions in this model share common cellular and biochemical features with human MPEs. Implantation and growth of pleural tumors triggers a host inflammatory response characterized by a mixed inflammatory cell influx into the pleural fluid. LLC cells exhibited high basal nuclear factor (NF)-kappaB activity in vitro and in vivo, which we used to drive expression of a NF-kappaB-dependent green fluorescent protein-firefly luciferase fusion reporter construct. NF-kappaB-dependent reporter expression allowed intravital tracing of pleural tumors. Inhibition of NF-kappaB in LLC cells did not affect cell viability in culture; however, injection of LLC cells expressing a dominant NF-kappaB inhibitor resulted in decreased tumor burden, decreased pleural effusion volume, and decreased pleural effusion TNF-alpha levels. These studies indicate that tumor NF-kappaB activity regulates pleural tumor progression. This reproducible model of MPE can be used to further study the influence of specific host and tumor factors on the pathogenesis of MPE and evaluate new therapeutic strategies.

Background: Smooth muscle contraction is one of the hallmarks of asthma. A recently developed pyridine derivative, Y-27632, a selective Rho kinase inhibitor, has been reported to inhibit the smooth muscle contraction of human and animal trachea in ex vivo systems but its effect in animal models of airway hyperresponsiveness (AHR) has not been examined. The purpose of this study was to evaluate the effect of Y-27632 in a murine model of allergic and virally induced AHR. Methods: Baseline lung resistance and methacholine induced AHR were measured in mice sensitised to ovalbumin (OVA) and also in mice infected with respiratory syncytial virus (RSV) following ovalbumin sensitisation (OVA/RSV). Results: Time course and dose ranging experiments indicated that 30 mg/kg Y-27632 given by gavage 2 hours before methacholine challenge significantly reduced baseline lung resistance and prevented AHR in OVA sensitised mice. Y-27632 also suppressed AHR induced by the bronchospastic agent serotonin in OVA sensitised mice and prevented methacholine induced AHR in OVA/RSV mice. Conclusions: These results suggest that the signalling pathway mediated through Rho kinase may have an important role in bronchial smooth muscle tone in allergen induced and virus induced AHR and should be considered as a novel target for asthma treatment.

Nonselective cyclooxygenase (COX) inhibition during allergic sensitization with ovalbumin in a murine model leads to an increase in the Type 2 cytokines interleukin-5 (IL-5) and IL-13; however, the effect of selective COX-1 and COX-2 inhibitors on these cytokines is unknown. We found that COX-1 protein was constitutively expressed in lung tissue. Expression of COX-1 protein did not increase with ovalbumin sensitization, but expression of COX-2 protein did. Ovalbumin-sensitized mice treated with either selective COX-1 inhibitor SC58560 (OVA-COX-1 inhibitor) or selective COX-2 inhibitor SC58236 (OVA-COX-2 inhibitor) had significantly greater airway hyperresponsiveness (p < 0.05) and higher levels of IL-13 (p < 0.05) in lung supernatants than did untreated mice that were ovalbumin sensitized (OVA). Lung mRNA levels for the chemokine receptors CCR1 through CCR5 (expressed on eosinophils, basophils, lymphocytes, and dendritic cells) were increased in the OVA-COX-2 inhibitor and OVA-indomethacin groups. We conclude that in the BALB/c mouse, COX inhibition with either a COX-1 or COX-2 inhibitor during allergen sensitization augments production of IL-13 and increases airway hyperresponsiveness.

Rationale: Asymptomatic relatives of patients with familial interstitial pneumonia (FIP), the inherited form of idiopathic interstitial pneumonia, carry increased risk for developing interstitial lung disease. Objectives: Studying these at-risk individuals provides a unique opportunity to investigate early stages of FIP pathogenesis and develop predictive models of disease onset. Methods: Seventy-five asymptomatic first-degree relatives of FIP patients (mean age, 50.8 yr) underwent blood sampling and high-resolution chest computed tomography (HRCT) scanning in an ongoing cohort study; 72 consented to bronchoscopy with bronchoalveolar lavage (BAL) and transbronchial biopsies. Twenty-seven healthy individuals were used as control subjects. Measurements and Main Results: Eleven of 75 at-risk subjects (14%) had evidence of interstitial changes by HRCT, whereas 35.2% had abnormalities on transbronchial biopsies. No differences were noted in inflammatory cells in BAL between at-risk individuals and control subjects. At-risk subjects had increased herpesvirus DNA in cell-free BAL and evidence of herpesvirus antigen expression in alveolar epithelial cells (AECs), which correlated with expression of endoplasmic reticulum stress markers in AECs. Peripheral blood mononuclear cell and AEC telomere length were shorter in at-risk individuals than healthy control subjects. The minor allele frequency of the Muc5B rs35705950 promoter polymorphism was increased in at-risk subjects. Levels of several plasma biomarkers differed between at-risk subjects and control subjects, and correlated with abnormal HRCT scans. Conclusions: Evidence of lung parenchymal remodeling and epithelial dysfunction was identified in asymptomatic individuals at risk for FIP. Together, these findings offer new insights into the early pathogenesis of idiopathic interstitial pneumonia and provide an ongoing opportunity to characterize presymptomatic abnormalities that predict progression to clinical disease.

Severe respiratory syncytial virus (RSV)-induced disease is associated with childhood asthma and atopy. We combined murine models of allergen-sensitization and RSV infection to explore the interaction of allergic and virus-induced airway inflammation and its impact on airway hyperresponsiveness (AHR). We found that RSV infection during ova-sensitization (OVA/RSV) increased and prolonged AHR compared to mice only RSV-infected (RSV) or ova-sensitized (OVA). AHR is known to be associated with an increase in Type 2 cytokines (IL-4, IL-5, and IL-13) in allergen-sensitized mice. Therefore, we hypothesized that RSV-induced enhancement of AHR was a result of potentiating the Type 2 cytokine profile promoted by ova-sensitization. Surprisingly, we found that Type 2 cytokines induced by ova-sensitization were not increased by RSV infection despite the increase in AHR, and in some cases were diminished. RNAse protection assay revealed no difference in IL-4 and IL-5 mRNA levels between the OVA and OVA/RSV groups, and IL-13 mRNA was significantly decreased in the OVA/RSV mice compared to the OVA group. Flow cytometric analysis of Type 2 cytokines demonstrated the same frequency of IL-4 and IL-5 production in lung-derived T lymphocytes from the OVA/RSV and OVA groups. Direct cytokine ELISA measurements of lung supernatant showed the level of IL-13 was significantly decreased in the OVA/RSV group compared to OVA mice, while there was no difference in either IL-4 or IL-5 between these two groups. These data indicate that the enhanced and prolonged AHR caused by the interaction of allergic airway inflammation and virus-induced immune responses is a complex process that can not be explained simply by augmented production of Type 2 cytokines.

Respiratory syncytial virus (RSV)-induced disease is associated with recurrent episodes of wheezing in children, and an effective vaccine currently is not available. The use of 2 immunizations (a formalin-inactivated, alum-precipitated RSV vaccine [FI-RSV] given intramuscularly and live RSV given intranasally [LVIN]), with a control immunization, were compared in a well-characterized model of RSV challenge, with or without concomitant allergic sensitization with ovalbumin. FI-RSV caused a significant increase in airway hyperresponsiveness in mice after RSV infection during allergic sensitization, and this was associated with an increase in type 2 cytokine production. In contrast, immunization with LVIN did not change type 2 cytokine production and protected against RSV-induced airway hyperresponsiveness in the setting of allergic sensitization. This study suggests that immune modulation with RSV vaccination can have profound effects on RSV-induced airway disease and that prevention of airway hyperresponsiveness is an important end point in vaccine development.

The immunomodulatory role of arachidonic acid metabolites in allergic sensitization is undefined. Prostaglandin E(2) (PGE(2)), a product of arachidonic acid metabolism through the cyclooxygenase pathway, has been reported to favor Type 2-like cytokine secretion profiles in murine and human CD4(+) T cells by inhibiting the production of Type 1-associated cytokines. On the basis of these in vitro data, we hypothesized that indomethacin, a nonselective cyclooxygenase inhibitor, would diminish allergen-induced production of Type 2 cytokines in mice, and protect against airway hyperresponsiveness (AHR) to methacholine. We found that ovalbumin-sensitized mice that were treated with indomethacin (OVA-indomethacin mice) had significantly greater AHR (p < 0.05) and higher levels of IL-5 (176 +/- 52 versus 66 +/- 4 pg/ml) and IL-13 (1,226 +/- 279 versus 475 +/- 65 pg/ml) in lung supernatants than mice sensitized with ovalbumin alone (OVA mice), while levels of IL-4 and serum IgE were not different. Lung mRNA expression of the C-C chemokine MCP-1 was increased in OVA-indomethacin mice, while there was no difference between the two groups in lung mRNA expression of eotaxin, MIP-1alpha, MIP-1beta, or MIP-2. Histologic examination revealed greater pulmonary interstitial eosinophilia in OVA-indomethacin mice as well. Contrary to our expectations, we conclude that in the BALB/c mouse, cyclooxygenase inhibition during allergen sensitization increases AHR, production of IL-5 and IL-13, and interstitial eosinophilia.

T cell function is a critical determinant of immune responses as well as susceptibility to allergic diseases. Activated T cells can differentiate into effectors whose cytokine profile is limited to type 1 (IFN-γ-dominant) or type 2 (IL-4-, IL-5-dominant) patterns. To investigate mechanisms that connect extracellular stimuli with the regulation of effector T cell function, we have measured immune responses of transgenic mice whose NF-κB/Rel signaling pathway is inhibited in T cells. Surprisingly, these mice developed type 2 T cell-dependent responses (IgE and eosinophil recruitment) in a model of allergic pulmonary inflammation. In contrast, type 1 T cell responses were severely impaired, as evidenced by markedly diminished delayed-type hypersensitivity responses, IFN-γ production, and Ag-specific IgG2a levels. Taken together, these data indicate that inhibition of NF-κB can lead to preferential impairment of type 1 as compared with type 2 T cell-dependent responses.

Severe respiratory syncytial virus (RSV)-induced disease is associated with childhood asthma and atopy. We combined models of allergen sensitization and RSV infection to begin exploring the immunologic interactions between allergic and virus-induced airway inflammation and its impact on airway hypersensitivity. Airway resistance was measured after methacholine challenge in tracheally intubated mice by whole body plethysmography. Lung inflammation was assessed by bronchoalveolar lavage (BAL) and histopathology. RSV infection alone did not cause significant airway hyperresponsiveness (AHR) to methacholine. Ovalbumin (OVA)-induced AHR lasted only a few days past the discontinuance of OVA aerosol in mice that were ovalbumin sensitized and mock infected. In contrast, OVA-sensitized mice infected with RSV during the OVA aerosol treatments (OVA/RSV) had AHR for more than 2 weeks after infection. However, 2 weeks after either RSV or mock infection, OVA/RSV mice had significantly more lymphocytes found during BAL than OVA mice, whereas the OVA and OVA/RSV groups had the same number of eosinophils. Histopathologic analysis confirmed an increased inflammation in the lungs of OVA/RSV mice compared with OVA mice. In addition, OVA/RSV mice had a more widespread distribution of mucus in their airways with increased amounts of intraluminal mucus pools compared with the other groups. Thus, prolonged AHR in RSV-infected mice during ovalbumin-sensitization correlates with increased numbers of lymphocytes in BAL fluid, increased lung inflammation, and mucus deposition in the airways, but not with airway eosinophilia. A further understanding of the immunologic consequences of combined allergic and virus-induced airway inflammation will impact the management of diseases associated with airway hyperreactivity.

Short telomeres are frequently identified in patients with idiopathic pulmonary fibrosis (IPF) and its inherited form, familial interstitial pneumonia (FIP). We identified a kindred with FIP with short telomeres who did not carry a mutation in known FIP genes TERT or hTR . We performed targeted sequencing of other telomere-related genes to identify the genetic basis of FIP in this kindred. The proband was a 69 year-old man with dyspnea, restrictive pulmonary function test results, and reticular changes on high-resolution CT scan. An older male sibling had died from IPF. The proband had markedly shortened telomeres in peripheral blood and undetectably short telomeres in alveolar epithelial cells. Polymerase chain reaction-based sequencing of NOP10 , TINF2 , NHP2 , and DKC1 revealed that both affected siblings shared a novel A to G 1213 transition in DKC1 near the hTR binding domain that is predicted to encode a Thr405Ala amino acid substitution. hTR levels were decreased out of proportion to DKC1 expression in the T405A DKC1 proband, suggesting this mutation destabilizes hTR and impairs telomerase function. This DKC1 variant represents the third telomere-related gene identified as a genetic cause of FIP. Further investigation into the mechanism by which dyskerin contributes to the development of lung fibrosis is warranted.

Glycosylphophatidylinositol (GPI)-anchored proteins play important roles in many biological processes, and mutations affecting proteins involved in the synthesis of the GPI anchor are reported to cause a wide spectrum of intellectual disabilities (IDs) with characteristic additional phenotypic features. Here, we describe a total of five individuals (from three unrelated families) in whom we identified mutations in PGAP3, encoding a protein that is involved in GPI-anchor maturation. Three siblings in a consanguineous Pakistani family presented with profound developmental delay, severe ID, no speech, psychomotor delay, and postnatal microcephaly. A combination of autozygosity mapping and exome sequencing identified a 13.8 Mb region harboring a homozygous c.275G>A (p.Gly92Asp) variant in PGAP3 region 17q11.2-q21.32. Subsequent testing showed elevated serum alkaline phosphatase (ALP), a GPI-anchored enzyme, in all three affected children. In two unrelated individuals in a cohort with developmental delay, ID, and elevated ALP, we identified compound-heterozygous variants c.439dupC (p.Leu147Profs(∗)16) and c.914A>G (p.Asp305Gly) and homozygous variant c.314C>G (p.Pro105Arg). The 1 bp duplication causes a frameshift and nonsense-mediated decay. Further evidence supporting pathogenicity of the missense mutations c.275G>A, c.314C>G, and c.914A>G was provided by the absence of the variants from ethnically matched controls, phylogenetic conservation, and functional studies on Chinese hamster ovary cell lines. Taken together with recent data on PGAP2, these results confirm the importance of the later GPI-anchor remodelling steps for normal neuronal development. Impairment of PGAP3 causes a subtype of hyperphosphatasia with ID, a congenital disorder of glycosylation that is also referred to as Mabry syndrome.

Idiopathic pulmonary fibrosis (IPF) is characterized by interstitial lung infiltrates, dyspnea, and progressive respiratory failure. Reports linking telomerase mutations to familial interstitial pneumonia (FIP) suggest that telomerase activity and telomere length maintenance are important in disease pathogenesis. To investigate the role of telomerase in lung fibrotic remodeling, intratracheal bleomycin was administered to mice deficient in telomerase reverse transcriptase (TERT) or telomerase RNA component (TERC) and to wild-type controls. TERT-deficient and TERC-deficient mice were interbred to the F6 and F4 generation, respectively, when they developed skin manifestations and infertility. Fibrosis was scored using a semiquantitative scale and total lung collagen was measured using a hydroxyprolinemicroplate assay. Telomere lengths were measured in peripheral blood leukocytes and isolated type II alveolar epithelial cells (AECs). Telomerase activity in type II AECs was measured using a real-time polymerase chain reaction (PCR)-based system. Following bleomycin, TERT-deficient and TERC-deficient mice developed an equivalent inflammatory response and similar lung fibrosis (by scoring of lung sections and total lung collagen content) compared to controls, a pattern seen in both early (F1) and later (F6 TERT and F4 TERC) generations. Telomere lengths were reduced in peripheral blood leukocytes and isolated type II AECs from F6 TERT-deficient and F4 TERC-deficient mice compared to controls. Telomerase deficiency in a murine model leads to telomere shortening, but does not predispose to enhanced bleomycin-induced lung fibrosis. Additional genetic or environmental factors may be necessary for development of fibrosis in the presence of telomerase deficiency.

The diagnosis of acute lung injury (ALI) is based on a consensus clinical definition. Despite the simplicity of this definition, ALI remains underdiagnosed and undertreated. Severe trauma is a well-described cause of ALI that represents a relatively homogeneous subset of patients with ALI. The aims of this study were to develop a panel of plasma biomarkers to facilitate diagnosis of trauma-induced ALI and to enhance our understanding of the pathogenesis of human ALI.A retrospective nested case control of 192 patients admitted to the trauma intensive care unit at a university hospital between 2002 and 2006. We compared 107 patients with ALI to 85 patients without ALI. Plasma was collected within 72 hours of intensive care unit admission. Twenty-one plasma biomarkers were measured in duplicate in each plasma sample.Patients with ALI had higher severity of illness scores, more days of mechanical ventilation, longer hospital stays, and higher mortality versus controls. Seven biomarkers (receptor for advanced glycation end products, procollagen peptide III, brain natriuretic peptide, angiopoietin-2, interleukin-10, tumor necrosis factor alpha, and interleukin-8) had a high diagnostic accuracy as reflected by the area under the receiver operating characteristic curve of 0.86 (95% confidence interval, 0.82-0.92) in differentiating ALI from controls.A model using seven plasma biomarkers had a high diagnostic accuracy in differentiating patients with trauma-induced ALI from trauma patients without ALI. In addition, use of a panel of biomarkers provides insight into the likely importance of alveolar epithelial injury in the pathogenesis of early ALI.

Nonselective cyclooxygenase (COX) inhibition during the development of allergic disease in a murine model causes an increase in type 2 cytokines and lung eosinophilia; however, the mechanisms responsible for this augmented allergen-induced inflammation have not been examined. Ab depletion of CD4 and CD8 cells revealed that the heightened allergic inflammation caused by COX inhibition was CD4, but not CD8, dependent. Allergen sensitization and airway challenge alone led to undetectable levels of IL-5 and IL-13 in the lungs of IL-4, IL-4Rα, and STAT6 knockout (KO) mice, but COX inhibition during the development of allergic inflammation resulted in wild-type levels of IL-5 and IL-13 and heightened airway eosinophilia in each of the three KO mice. These results indicate that the effect of COX inhibition was independent of signaling through IL-4, IL-4Rα, and STAT6. However, whereas COX inhibition increased IgE levels in allergic wild-type mice, IgE levels were undetectable in IL-4, IL-4Rα, and STAT6 KO mice, suggesting that IL-13 alone is not a switch factor for IgE synthesis in this model. These results illustrate the central role played by products derived from the COX pathway in the regulation of allergic immune responses.

Background: Microbial infections are associated with the initial susceptibility to and flares of asthma. However, immunologic mechanisms whereby infections might alter the asthmatic phenotype are lacking. Objective: To test the hypothesis that memory T cells specific both for a viral antigen and an allergen could influence the pathogenesis of allergic disease in vivo. Methods: We developed a system in which 2 distinct T-cell receptors coexist on the T-cell surface, 1 specific for a virus and the other for an inhaled antigen. Results: We show that a population of dual-receptor T cells, polarized through a virus-specific T-cell receptor to contain TH 1o r TH2 cells, can be reactivated through an unrelated T-cell receptor in recall responses in vivo. Quiescent memory cells derived from a TH1-polarized effector population blocked the development of airway hyperreactivity in a model of allergic lung disease, in association with decreased induction of chemokines and eosinophil recruitment. Conversely, reactivation of quiescent TH2 cells after inhalation of antigen or virus infection was sufficient to lead to the development of airway hyperresponsiveness and allergic pulmonary inflammation in mice whose lungs were previously normal. Conclusion: These data provide evidence that dual-receptor memory T cells can regulate allergic disease susceptibility and suggest that they may play a role in mediating the influence of microbes on asthma pathogenesis. (J Allergy Clin Immunol 2004;114:1441-8.)

Background Respiratory syncytial virus (RSV) is the leading infectious cause of respiratory failure and wheezing in infants and young children. Prematurity is the greatest risk factor for severe RSV-induced disease, and recent studies suggest that premature children have lower levels of the type I IFNs (α/β), for which signal transducer and activator of transcription (STAT) 1 is a critical intracellular signaling molecule. Objective We hypothesized that RSV infection in STAT1 knockout ( STAT1 KO) mice would result in both increased airway resistance and airway hyperresponsiveness. Methods Wild-type (WT) and STAT1 KO mice on a BALB/c background were either RSV or mock infected. Phenotypic response to infection was assessed by means of plethysmography, immunohistochemistry, and lung cytokine measurement. Results We found that STAT1 KO mice infected with RSV ( STAT1 KO-RSV) had greater baseline lung resistance ( P =.05) and airway responsiveness ( P STAT1 KO ( STAT1 KO-MOCK), RSV-infected wild type (WT-RSV), and mock-infected wild type (WT-MOCK) mice. In addition, the STAT1 KO-RSV mice showed induction of mucus production and expression of gob-5 and Muc5ac, conditions not present in any of the other 3 groups. IL-17, a cytokine that regulates Muc5ac expression, was expressed in the lungs of the STAT1 KO-RSV mice, whereas lung levels of IL-17 were undetectable in the remaining groups. Expression of the IL-23–specific p19 subunit was also increased in the STAT1 KO-RSV mice but not in the WT-RSV mice. Conclusion These results show that STAT1 has an important regulatory role in RSV-induced alteration of airway function.

Severe respiratory syncytial virus (RSV) infection has been hypothesized to be a risk factor for the development of allergy and asthma, but epidemiologic studies in humans have been inconclusive. By use of a well-characterized murine model of RSV infection and allergic sensitization with ovalbumin, the effect of a preceding severe RSV infection on the development of the pulmonary allergic inflammatory response and airway hyperresponsiveness (AHR) was tested. The impact of prior allergic sensitization on RSV-induced illness, as measured by weight loss, also was evaluated. RSV infection before allergic sensitization decreased allergen-induced AHR, production of interleukin-13 in lung tissue, and lung eosinophilia. In contrast, allergic sensitization before RSV infection increased AHR and decreased RSV-related weight loss and lung levels of interferon-gamma but did not alter viral clearance. These data provide evidence that RSV-associated AHR occurs in hosts with allergic responses and that allergic inflammation is diminished when preceded by RSV infection.

PGE2 is an important cyclooxygenase product that modulates airway inflammatory and smooth muscle responses. Signal transduction is mediated by four EP receptor subtypes that cause distinct effects on cell metabolism. To determine the role of EP2 receptor activation, we produced a mouse lacking the EP2 receptor by targeted gene disruption. The effect of aerosolized PGE2 and other agonists was measured using barometric plethysmography and by measurements of lung resistance in mechanically ventilated mice. Inhalation of PGE2 inhibited methacholine responses in wild-type but not in mice lacking the EP2 receptor [EP2(−/−)]. After airway constriction was induced by methacholine aerosol, PGE2reduced the airway constriction enhanced pause in wild-type mice (from 0.88 ± 0.15 to 0.55 ± 0.06) but increased it in EP2(−/−) mice (from 0.73 ± 0.08 to 1.27 ± 0.19). Similar results were obtained in mechanically ventilated mice. These data indicate that the EP2 receptor mediates the bronchodilation effect of PGE2.

Cyclooxygenase (COX) inhibition during allergic sensitization and allergen airway challenge results in augmented allergic inflammation. We hypothesized that this increase in allergic inflammation was dependent on increased generation of leukotrienes that results from COX inhibition, as leukotrienes are important proinflammatory mediators of allergic disease. To test this hypothesis, we allergically sensitized and challenged mice deficient in 5-lipoxygenase (5-LO). We found that 5-LO knockout mice that were treated with a COX inhibitor during allergic sensitization and challenge had significantly increased airway hyperresponsiveness (AHR) (p < 0.01) and airway eosinophilia (p < 0.01) compared with 5-LO knockout mice that were treated with vehicle. The proinflammatory cytokines have also been hypothesized to be critical regulators of airway inflammation and AHR. We found that the increase in airway eosinophilia seen with COX inhibition is dependent on IL-5, whereas the increase in AHR is not dependent on this cytokine. In contrast, the COX inhibition-mediated increase in AHR is dependent on IL-13, but airway eosinophilia is not. These results elucidate the pathways by which COX inhibition exerts a critical effect of the pulmonary allergen-induced inflammatory response and confirm that COX products are important regulators of allergic inflammation.

Rationale The role of prostaglandins in allergic lung inflammation is still being defined. In vitro studies suggest that prostaglandin E 2 (PGE 2 ) has pro-allergic inflammatory effects, but in vivo studies in both humans and animals suggest that PGE 2 restrains allergic inflammation. In order to further clarify the immunomodulatory effect of PGE 2 in the allergic inflammatory response in the lung, we neutralized PGE 2 by administering a well characterized anti-PGE 2 antibody in a murine model of ovalbumin sensitization. Methods Female BALB/c mice underwent an allergic sensitization protocol which consisted of an intraperitoneal (IP) injection of ovalbumin formulated with alum on day 0, followed 14 days later by daily 1% ovalbumin aerosol treatments for 40 minutes on each of 4 consecutive days. Mice were either treated with anti-PGE 2 antibody on both the day prior to the intraperitoneal injection of ovalbumin/alum and the day prior to the first ovalbumin aerosol exposure, or control antibody treatment on the same days. Endpoints were measured on the day after the last ovalbumin aerosol exposure. Results The anti-PGE 2 treated mice had significantly greater levels of IL-5 and IL-13 levels in the lung supernatants compared to the mice treated with the control antibody. In addition, anti-PGE 2 treatment resulted in significantly increased bronchoalveolar lavage fluid eosinophilia and lymphocytosis. The anti-PGE 2 treated mice also had a significant increase in methaholine reactivity compared to the control antibody treated mice Conclusions These results suggest that PGE 2 restrains allergic inflammation and may be a therapeutic target in the treatment of allergic inflammation in the lung.

Effector and memory T lymphocytes differ significantly, and there is no experimental evidence that memory cells are sufficient to render an otherwise normal individual susceptible to localized allergic inflammation. Furthermore, nothing is known about the kinetics of memory responses after inhalation of antigen or interplay between an allergen-specific memory helper T (Th) cell Th2 population and uncommitted or competing Th1 cells. To study these processes, T cell receptor-transgenic CD4(+) effector cells were generated in vitro, transferred into naive recipients, and allowed to resume a quiescent state. Inhalation of protein antigen reactivated these Ag-specific Th2 donor cells, leading to allergic pulmonary inflammation and airway hyperreactivity. Susceptibility was correlated with the size of the input Th2 population, but Th1 cells neither enhanced nor reduced inflammation in this model. Importantly, the reactivation of these antigen-experienced cells by inhaled antigen did not skew the cytokine balance of recipient-derived T cells recruited to the lung nor did it inhibit the development of donor-derived Th1 cells from uncommitted antigen-experienced cells that form a normal part of immune responses. These data indicate that a quiescent memory Th2-cell population can create susceptibility to allergic pulmonary inflammation in a manner refractory to inhibition by Th1 cells or endogenous inhibitory mechanisms.