Impact of Implementing the Paris System for Reporting Urine Cytology in the Performance of Urine Cytology: A Correlative Study of 124 Cases.

OBJECTIVES: We assessed the performance of urine cytology using the Paris System for Reporting Urine Cytology (PSRUC) in comparison to our current system. METHODS: In total, 124 specimens with histologic correlation were reviewed and assigned to the PSRUC categories: benign, atypical urothelial cells (AUCs), suspicious for high-grade urothelial carcinoma (SHGUC), and high-grade urothelial carcinoma (HGUC). Original cytological diagnoses were recorded. RESULTS: Fewer cases were given an AUC diagnosis using the PSRUC in comparison to the original diagnoses (26% vs 39%), while the association of AUCs with subsequent HGUC increased from 33% to 53% with the PSRUC. Using the PSRUC resulted in a higher number of low-grade carcinomas assigned to the benign (40%) rather than the AUC (22%) category. The performance of SHGUC/HGUC diagnoses was similar in both systems (predictive value = 94%). CONCLUSIONS: The PSRUC seems to improve the performance of urine cytology by limiting the AUC category to cases that are more strongly associated with HGUC.

Histopathogenesis of non-HPV-related differentiated oral squamous intraepithelial neoplasia.

INTRODUCTION: A study of immunohistopathologic and cytohistopathologic changes of the parabasal/basal layers in the differentiated squamous intraepithelial neoplasia (DSIN) may elucidate the histopathogenesis and reveal changes aiding early diagnosis and grading of the lesion. MATERIALS AND METHODS: A total of 55 consecutive resection specimens of nonbasaloid squamous cell carcinoma of the anterior oral cavity and 8 biopsies before resections displaying DSIN in the overlying squamous epithelium were examined. RESULTS: Squamous epithelium that is continuous/immediately adjacent to invasive squamous cell carcinoma (type 1) and the more peripheral (type 2) epithelium of resection specimens displayed consistent changes in the parabasal/basal layers: (A) cytologic atypia with proliferation of parabasal cells with downward expansion causing reactive proliferation of the basal cell layer in the early stage, invading the basal layer in the late stage; (B) disordered nuclear/cytoplasmic arrangement; (C) "Cobblestone" appearance. Immunoreactivity for TP53 and Ki67 was helpful in the diagnosis. The epithelial spectrum of changes decreased as one moved from type 1 to type 2 lesions. Five out of 8 biopsies showed type 1 lesions (followed by resection in a period of 11±6 mo) and 3 showed type 2 lesions (followed by resection in a period of 55±20 mo). In addition, resections were margin positive for type 2 lesions in 5 cases associated with recurrence at the site of resection during a period of 69±9 months. CONCLUSIONS: DSIN is characterized by a proliferation of neoplastic parabasal cells with dyskeratosis, downward expansion/pushing of the basal layer with elongation of rete ridges. We proposed grading of DSIN based on the changes of the parabasal/basal layers.

Treatment with a sphingosine-1-phosphate analog inhibits airway remodeling following repeated allergen exposure.

Sphingosine-1-phosphate (S1P) is an immunomodulatory lipid mediator that plays an important role in lymphocyte trafficking. Elevated levels of S1P are found in bronchoalveolar lavage (BAL) fluid of patients with asthma; however, its role in disease is not known. FTY720, a synthetic analog of S1P, has been shown to abrogate allergic inflammation and airway hyperresponsiveness following acute allergen challenge. However, its effects on asthmatic airway remodeling induced by repeated allergen exposure are unknown. Ovalbumin (OVA)-sensitized rats were challenged on days 14, 19, and 24 after sensitization. FTY720 or vehicle (PBS) therapy was administered 1 h prior to each challenge. BAL fluid and quantitative histological analysis were performed 48 h after the last challenge. FTY720 inhibited OVA-induced features of airway remodeling including increased airway smooth muscle mass and bronchial neovascularization, without affecting lymphocyte numbers in secondary lymphoid organs. Furthermore, CD3+ cells adjacent to airway smooth muscle bundles were increased in OVA-challenged rats but the increase was inhibited by FTY720. There was an expansion of bronchus-associated lymphoid tissue following FTY720 treatment of OVA-challenged animals. Real-time quantitative PCR revealed that Th2-associated transcription factors were inhibited following FTY720 therapy. Airway remodeling is a cardinal feature of severe asthma. These results demonstrate that allergen-driven airway remodeling can be inhibited by FTY720, offering potential new therapies for the treatment of severe asthma.

LTD4 induces HB-EGF-dependent CXCL8 release through EGFR activation in human bronchial epithelial cells.

Airway epithelial cells release proinflammatory mediators that may contribute to airway remodeling and leukocyte recruitment. We explored the hypothesis that leukotriene D4 (LTD4) may trigger the release of proremodeling factors through activation of the EGF receptor (EGFR). We particularly focused on the effects of LTD4 on release of heparin-binding EGF-like factor (HB-EGF) and IL-8 (CXCL8), a potent neutrophil chemoattractant that may be released downstream of EGFR activation. To address this hypothesis, both primary (NHBE) and transformed bronchial human epithelial cells (BEAS-2B) were grown on an air-liquid interface and stimulated with LTD4. HB-EGF and CXCL8 were evaluated by ELISA in cell culture supernatants. To explore the EGFR signaling pathway, we used a broad-spectrum matrix metalloproteinase (MMP) inhibitor, GM-6001, two selective EGFR tyrosine kinase inhibitors, AG-1478 and PD-153035, an HB-EGF neutralizing antibody, and a specific small interfering RNA (siRNA) against the EGFR. Expression of the CysLT1 cysteinyl leukotriene receptor was demonstrated by RT-PCR and immunocytochemistry in both BEAS-2B and NHBE cells. Four hours after stimulation with LTD4, HB-EGF and CXCL8 were significantly increased in cell culture supernatant. GM-6001 and montelukast, a specific CysLT1 receptor antagonist, blocked the LTD4-induced increase in HB-EGF. All inhibitors/antagonists decreased LTD4-induced CXCL8 release. siRNA against EGFR abrogated CXCL8 release following stimulation with LTD4 and exogenous HB-EGF. These findings suggest LTD4 induced EGFR transactivation through the release of HB-EGF in human bronchial epithelial cells with downstream release of CXCL8. These effects may contribute to epithelial-mediated airway remodeling in asthma and other conditions associated with cysteinyl leukotriene release.

Airway smooth muscle remodeling is a dynamic process in severe long-standing asthma.

BACKGROUND: The origin of the excess airway smooth muscle in asthma and when in the course of the disease it is acquired are uncertain. OBJECTIVES: We examined the relative sensitivities of 2 markers of proliferation, proliferating cell nuclear antigen (PCNA) and Ki 67, in airway smooth muscle in vivo and in vitro. We then studied whether muscle remodeling is a dynamic process in asthma by quantifying proliferation rate and area. Finally we examined heparin-binding epidermal growth factor as a biomarker of remodeling. METHODS: We obtained bronchoscopic biopsies from subjects with moderate or severe asthma and healthy controls (n = 9/group). For in vitro studies, airway smooth muscle cells were cultured from tracheas of transplant donors. The proliferation rate was quantified from PCNA and Ki 67, co-localized to smooth muscle-specific alpha-actin cells in vivo. Muscle area was assessed morphometrically. We examined the expression of heparin-binding epidermal growth factor on tissues by in situ hybridization and by immunohistochemistry and in cells in culture by RT-PCR. RESULTS: Proliferating cell nuclear antigen and Ki 67 were highly correlated, but PCNA was a significantly more sensitive marker both in vivo and in vitro. Muscle area was 3.4-fold greater and the fraction of PCNA(+) nuclei in muscle was 5-fold greater in severe asthma than in healthy subjects. Heparin-binding epidermal growth factor was upregulated in proliferating muscle cells in culture and in airway smooth muscle in severe asthmatic tissues. CONCLUSION: Proliferating cell nuclear antigen is a highly sensitive marker of proliferation and heparin-binding epidermal growth factor is a potential biomarker during active remodeling of ASM in severe asthma.

The effects of repeated allergen challenge on airway smooth muscle structural and molecular remodeling in a rat model of allergic asthma.

The effects of remodeling of airway smooth muscle (SM) by hyperplasia on airway SM contractility in vivo are poorly explored. The aim of this study was to investigate the relationship between allergen-induced airway SM hyperplasia and its contractile phenotype. Brown Norway rats were sensitized with ovalbumin (OVA) or saline on day 0 and then either OVA-challenged once on day 14 and killed 24 h later or OVA-challenged 3 times (on days 14, 19, and 24) and killed 2 or 7 days later. Changes in SM mass, expression of total myosin, SM myosin heavy chain fast isoform (SM-B) and myosin light chain kinase (MLCK), tracheal contractions ex vivo, and airway responsiveness to methacholine (MCh) in vivo were assessed. One day after a single OVA challenge, the number of SM cells positive for PCNA was greater than for control animals, whereas the SM mass, contractile phenotype, and tracheal contractility were unchanged. Two days after three challenges, SM mass and PCNA immunoreactive cells were increased (3- and 10-fold, respectively; P < 0.05), but airway responsiveness to MCh was unaffected. Lower expression in total myosin, SM-B, and MLCK was observed at the mRNA level (P < 0.05), and total myosin and MLCK expression were lower at the protein level (P < 0.05) after normalization for SM mass. Normalized tracheal SM force generation was also significantly lower 2 days after repeated challenges (P < 0.05). Seven days after repeated challenges, features of remodeling were restored toward control levels. Allergen-induced hyperplasia of SM cells was associated with a loss of contractile phenotype, which was offset by the increase in mass.

Role of the cystic fibrosis transmembrane conductance channel in human airway smooth muscle.

Patients with cystic fibrosis (CF) suffer from asthma-like symptoms and gastrointestinal cramps, attributed to a mutation in the CF transmembrane conductance regulator (CFTR) gene present in a variety of cells. Pulmonary manifestations of the disease include the production of thickened mucus and symptoms of asthma, such as cough and wheezing. A possible alteration in airway smooth muscle (ASM) cell function of patients with CF has not been investigated. The aim of this study was to determine whether the (CFTR) channel is present and affects function of human ASM cells. Cell cultures were obtained from the main or lobar bronchi of patients with and without CF, and the presence of the CFTR channel detected by immunofluorescence. Cytosolic Ca(2+) was measured using Fura-2 and dual-wavelength microfluorimetry. The results show that CFTR is expressed in airway bronchial tissue and in cultured ASM cells. Peak Ca(2+) release in response to histamine was significantly decreased in CF cells compared with non-CF ASM cells (357 +/- 53 nM versus 558 +/- 20 nM; P < 0.001). The CFTR pharmacological blockers, glibenclamide and N-phenyl anthranilic acid, significantly reduced histamine-induced Ca(2+) release in non-CF cells, and similar results were obtained when CFTR expression was varied using antisense oligonucleotides. In conclusion, these data show that the CFTR channel is present in ASM cells, and that it modulates the release of Ca(2+) in response to contractile agents. In patients with CF, a dysfunctional CFTR channel could contribute to the asthma diathesis and gastrointestinal problems experienced by these patients.

Chronic asthma-induced airway remodeling is prevented by toll-like receptor-7/8 ligand S28463.

RATIONALE: Allergic asthma is a heterogeneous disease, the pathology of which is a result of improper immune responses to innocuous antigens. We and others have previously shown that one of the Toll-like receptor (TLR)-7/8 ligands, the synthetic compound S28463 (resiquimod, R-848), is able to inhibit acute allergic asthma in mice. OBJECTIVES: Given that the efficiency of this pharmacologic compound against the smooth muscle mass increase and goblet cell hyperplasia that are characteristic of chronic allergic asthma has not been previously assessed, we investigated the ability of this compound to prevent these aspects of chronic airway remodeling. METHODS: The impact of S28463 treatment was assessed in a Brown Norway rat model of chronic asthma by histologic, morphometric, and molecular techniques. MEASUREMENTS AND MAIN RESULTS: We demonstrate that treatment with S28463 is able to prevent the development of goblet cell hyperplasia and increases in airway smooth muscle mass, and that this effect is at least partially mediated by inhibiting proliferation of goblet and smooth muscle cells, respectively. Furthermore, we show that the abrogation of airway remodeling is preceded by inhibition of the inflammatory reaction normally occurring in response to allergen challenge in sensitized animals. This inhibition was associated with a reduction of both helper T cell type 1 and type 2 cytokine protein expression in the lungs, demonstrating the potent antiinflammatory effect of this pharmaceutical compound in the context of allergic reactions. CONCLUSIONS: Taken together, our results indicate great potential for the use of S28463 as an antiinflammatory therapeutic agent for the management of chronic asthma.

Immune responses to viral infections: relevance for asthma.

Severe respiratory viral infections in childhood are associated with the development of asthma later in life. Rhinovirus, respiratory syncytial virus and metapneumovirus are of particular importance as triggers of asthma. Effects of virus infection on dendritic cell function in the airways may predispose children to allergic sensitization. Asthmatic subjects may have impaired interferon responses to viral infection that also predispose to allergic sensitization. Difference in Toll-like receptor expression on airway epithelial cells is a potential mechanism for the altered immune responses of asthmatic children.