Deletions in DNAL1 Cause Primary Ciliary Dyskinesia Across North American Indigenous Populations.

We report 4 cases of primary ciliary dyskinesia in unrelated indigenous North American children caused by identical, homozygous, likely pathogenic deletions in the DNAL1 gene. These shared DNAL1 deletions among dispersed indigenous populations suggest that primary ciliary dyskinesia accounts for more lung disease with bronchiectasis than previously recognized in indigenous North Americans.

Metabolomic profiling of asthma and chronic obstructive pulmonary disease: A pilot study differentiating diseases.

BACKGROUND: Differentiating asthma from other causes of chronic airflow limitation, such as chronic obstructive pulmonary disease (COPD), can be difficult in a typical outpatient setting. The inflammation of asthma typically is different than that of COPD, and the degree of inflammation and cellular damage varies with asthma severity. Metabolomics is the study of molecules created by cellular metabolic pathways. OBJECTIVES: We hypothesized that the metabolic activity of adults with asthma would differ from that of adults with COPD. Furthermore, we hypothesized that nuclear magnetic resonance spectroscopy (NMR) would measure such differences in urine samples. METHODS: Clinical and urine-based NMR data were collected on adults meeting the criteria of asthma and COPD before and after an exacerbation (n = 133 and 38, respectively) and from patients with stable asthma or COPD (n = 54 and 23, respectively). Partial least-squares discriminant analysis was performed on the NMR data to create models of separation (86 metabolites were measured per urine sample). Some subjects' metabolomic data were withheld from modeling to be run blindly to determine diagnostic accuracy. RESULTS: Partial least-squares discriminant analysis of the urine NMR data found unique differences in select metabolites between patients with asthma and those with COPD seen in the emergency department and even in follow-up after exacerbation. By using these select metabolomic profiles, the model could correctly diagnose blinded asthma and COPD with greater than 90% accuracy. CONCLUSION: This is the first report showing that metabolomic analysis of human urine samples could become a useful clinical tool to differentiate asthma from COPD.

Eosinophils in human oral squamous carcinoma; role of prostaglandin D2.

Eosinophils are often predominant inflammatory leukocytes infiltrating oral squamous carcinoma (OSC) sites. Prostaglandins are secreted by oral carcinomas and may be involved in eosinophil infiltration. The objective of this study was to determine the factors contributing to eosinophil migration and potential anti-neoplastic effects on OSC. Eosinophil degranulation was evaluated by measuring release of eosinophil peroxidase (EPO). Eosinophil chemotaxis towards OSC cells was assessed using artificial basement membrane. Eosinophil infiltration was prominent within the tissue surrounding the OSC tumor mass. We observed growth inhibition of the OSC cell line, SCC-9, during co-culture with human eosinophils, in vitro, which correlated with EPO activity that possesses growth inhibitory activity. The PGD2 synthase inhibitor, HQL-79, abrogated migration towards SCC-9. Our data suggest that OSC-derived PGD2 may play an important role via CRTH2 (the PGD2 receptor on eosinophils) in eosinophil recruitment and subsequent anti-tumor activity through the action of eosinophil cationic proteins.

Rhinovirus has the unique ability to directly activate human T cells in vitro.

BACKGROUND: Rhinovirus infection is a leading cause of exacerbation of airway diseases. We hypothesize that airway viruses activate inflammatory cells, inducing airway dysfunction. We have previously shown that airway viruses can induce eosinophil degranulation when cocultured with T cells and monocyte-derived dendritic cells (moDCs). These findings suggested that antigen presentation was important for T-cell activation. OBJECTIVE: Given the clinical importance of rhinovirus, we sought to determine whether it had any unique abilities to activate inflammatory cells compared with another common virus, such as respiratory syncytial virus (RSV). METHODS: We cocultured combinations of human leukocytes (T cells, moDCs, and eosinophils) with each virus. Using assays of BrdU incorporation, flow cytometry, and ELISA, we measured T-cell activation, rhinovirus expression, T-cell death, and eosinophil cysteinyl leukotriene release. RESULTS: In contrast to RSV, rhinovirus induced T-cell activation without the involvement of moDCs. Without moDCs, rhinovirus induced T-cell proliferation of both CD4 and CD8(+) cells, cytokine production, and ultimately, eosinophil stimulation. Although chloroquine inhibited RSV-induced activation of T cells through moDCs, rhinovirus was not inhibited; UV inactivation did block the rhinovirus effect. We also found that T cells could be infected by rhinovirus in vitro and within human nasal explant tissue. Although Toll-like receptors did not appear to be involved in T-cell activation, antagonists of Jun N-terminal kinase and nuclear factor κB did inhibit T-cell responses to rhinovirus. CONCLUSION: Rhinovirus has the unique ability to bypass antigen presentation and directly infect and activate human T cells. This could explain the strong association of rhinovirus with exacerbation of airway diseases.

Human dendritic cells promote an antiviral immune response when stimulated by CVT-E002.

OBJECTIVES: There is interest in developing new compounds to enhance the immune response to airway virus infections. CVT-E002 is a patented ginseng extract shown to decrease symptoms of virus infection in clinical trials. We hypothesized that the mechanism for this antiviral effect could be through modulation of dendritic cells leading to enhanced T-cell activation. METHODS: Human monocyte-derived dendritic cells (moDC) exposed to CVT-E002 (or not) were co-cultured with autologous T cells, with or without virus (respiratory syncytial virus or parainfluenza virus). Effects of CVT-E002 on cell function were determined through flow cytometry, 5-bromo-2'-deoxyuridine (BrdU) incorporation and ELISA. KEY FINDINGS: moDC cultured with CVT-E002 or virus induced greater activation of T cells, as measured by CD25 expression and BrdU incorporation, compared with untreated moDC. Responding T cells were CD4+CD45RO+. Co-cultures of CVT-E002 treated moDC with T cells responded with increased release of Th1-type cytokines (interferon-gamma, tumour necrosis factor and interleukin-12). CVT-E002-treated moDC showed increased expression of CD83, CD80 and CD86. Lipopolysaccharide levels were not detected in CVT-E002 and antagonists for Toll-like receptor-4 did not inhibit CVT-E002-induced moDC maturation. CONCLUSIONS: CVT-E002 induced moDC maturation, which caused increased memory T-cell activation and Th1-type cytokine response.

Human lymphocytes express the transcriptional regulator, Wilms tumor 1: the role of WT1 in mediating nitric oxide-dependent repression of lymphocyte proliferation.

The inhibitory roles of nitric oxide (NO) in T cell proliferation have been observed and studied extensively over the last two decades. Despite efforts, the fundamental pathway by which NO exerts its inhibitory actions remains to be elucidated although recent evidence suggests that the transcription factor Wilms tumor 1 (WT1) may be important. WT1 has been linked to numerous developmental pathways in particular nephrogenesis. Due to its roles in development and cell proliferation, polymorphisms within the WT1 gene can result in malignancies such as leukemia and Wilms tumor. WT1 functions as a transcriptional regulator and its activity is controlled through phosphorylation by protein kinase A (PKA). PKA-dependent WT1 phosphorylation results in translocation of WT1 from the nucleus to the cytosol, a process that interferes with WT1 transcriptional activities. In the current study we demonstrate that WT1 is expressed in human lymphocytes. Using the proliferative compound PHA we induced T cell proliferation and growth correlated with an increase in the expression of WT1 measured by RT-PCR, flow cytometry and immunoblot. Co-stimulation with the NO donor SNOG at concentrations of 0, 100, 300 and 600 microM reduced in a concentration dependent way the PHA-induced upregulation of WT1 that correlated with a reduction in T cell proliferation. We conclude that WT1 might be an important component of the NO-dependent regulation of T lymphocyte proliferation and potential function.

The induction of eosinophil peroxidase release: improved methods of measurement and stimulation.

The production and release of eosinophil peroxidase (EPO) has been associated with human pathology. Degranulation assays with eosinophils are typically very difficult to do, with very low release values. EPO is unique for its high cationic charge. As such, it adheres to most extracellular surfaces, rendering it more difficult to measure compared with other released cellular proteins. Based on the understanding of the sticky nature of EPO, we were concerned that EPO released in vitro cannot be reproducibly measured in the supernatants of stimulated cells. Instead, we suspected that much of the released EPO was left adherent to the tube walls. We chose to investigate the measurement of EPO activity using the peroxidase substrate, O-phenylenediamine (OPD). Unlike other peroxidase substrates, OPD is soluble in aqueous physiological solutions, which do not lyse cell membranes, thereby allowing us to add OPD directly to eosinophils and exclusively measure extracellular EPO. This novel approach would remove the concerns of incorrect EPO measurements due to its adhesive nature. In addition, we developed this method to quantify EPO release in terms of EPO concentration. Finally, using this technique, we have been able to demonstrate secretory IgA (s-IgA)-induced release of EPO. By using OPD, we have developed a more sensitive and specific method to analyze the release of extracellular EPO.