Epithelial Cell Adhesion Molecule in Primary Sjögren's Syndrome Patients: Characterization and Evaluation of a Potential Biomarker.

OBJECTIVE: To determine the subcellular localization of epithelial cell adhesion molecule (EpCAM) in labial salivary gland (LSG) and evaluate the diagnostic use of the extracellular domain of EpCAM (EpEX) and intracellular domain (EpICD) for primary Sjögren's syndrome (pSS). METHODS: Immunohistochemical (IHC) analysis was conducted using EpEX and EpICD domain-specific antibodies on labial salivary gland biopsy (LSGB) from participants. Chi-square or Fisher's exact analysis, Mann-Whitney U-test, and Kruskal-Wallis test compared differences among groups. Independent risk factors of pSS were determined by multiple logistic regression analysis. Receiver-operator characteristic curves (ROC) were carried out to estimate the diagnostic value. RESULTS: Compared to non-SS controls, loss of membranous EpEX and EpICD expression was observed in LSGB of pSS patients, which occurred in parallel with increased accumulation of cytoplastic and nuclear EpICD. The subcellular EpEX/EpICD expressions were associated with various features of pSS patients, especially histopathological grade of LSGB. Furthermore, high IHC scores of membranous EpEX were independent risk factors for pSS, even for the pSS patients at early stage. The IHC scores of subcellular EpEX/EpICD were of great diagnostic value for pSS with high sensitivity (70-80%) and specificity (85-95%). CONCLUSION: This study first found the aberrant expression pattern of EpCAM in LSG of pSS patients. The IHC scores of subcellular EpEX/EpICD were demonstrated to have the potential to act as diagnostic biomarkers for pSS.

Contrast-enhanced Ultrasonography for Monitoring Arterial Inflammation in Takayasu Arteritis.

OBJECTIVE: To evaluate the utility of contrast-enhanced ultrasound (CEUS) compared with 18F-fluorodeoxyglucose-positron emission tomography (FDG-PET) in assessing vessel inflammation of Takayasu arteritis (TA). METHODS: This is a retrospective analysis of 71 patients with TA who had undergone carotid CEUS. Twenty-two of 71 patients underwent FDG-PET after CEUS. Clinical disease activity was assessed by Kerr criteria and the Indian Takayasu Clinical Activity Score 2010 (ITAS2010). We investigated the correlation between carotid vascularization on CEUS and clinical data. The consistency of carotid CEUS and PET data has been analyzed for TA disease activity. RESULTS: There was a statistically significant correlation between the results of CEUS and ITAS2010 (p = 0.004) or Kerr criteria (p < 0.001). According to ITAS2010, thirty-four of 71 patients with TA were clinically inactive. Assessment of 34 TA patients with clinically inactive disease yielded 11 CEUS scans that showed active lesions (visual grade ≥ 2) in the left or right carotid artery. In 22 cases that underwent CEUS and FDG-PET, 12 were active and 10 were inactive on the basis of ITAS2010. Moreover, bilateral carotid CEUS vascularization score positively correlated with vascular FDG uptake in these patients with TA (p = 0.004). When vascular inflammation was defined as FDG uptake with visual grade ≥ 2, carotid CEUS showed sensitivity of 100% and specificity of 80%. CONCLUSION: For TA patients with clinically inactive disease, CEUS could help clinicians to identify active lesions in the carotid vascular region. Carotid CEUS may be a rapid and cost-effective imaging tool in the followup of patients with TA.

The role of the extracellular matrix protein mindin in airway response to environmental airways injury.

BACKGROUND: Our previous work demonstrated that the extracellular matrix protein mindin contributes to allergic airways disease. However, the role of mindin in nonallergic airways disease has not previously been explored. OBJECTIVES: We hypothesized that mindin would contribute to airways disease after inhalation of either lipopolysaccharide (LPS) or ozone. METHODS: We exposed C57BL/6J and mindin-deficient (-/-) mice to aerosolized LPS (0.9 µg/m3 for 2.5 hr), saline, ozone (1 ppm for 3 hr), or filtered air (FA). All mice were evaluated 4 hr after LPS/saline exposure or 24 hr after ozone/FA exposure. We characterized the physiological and biological responses by analysis of airway hyperresponsiveness (AHR) with a computer-controlled small-animal ventilator (FlexiVent), inflammatory cellular recruitment, total protein in bronchoalveolar lavage fluid (BALF), proinflammatory cytokine profiling, and ex vivo bronchial ring studies. RESULTS: After inhalation of LPS, mindin-/- mice demonstrated significantly reduced total cell and neutrophil recruitment into the airspace compared with their wild-type counterparts. Mindin-/- mice also exhibited reduced proinflammatory cytokine production and lower AHR to methacholine challenge by FlexiVent. After inhalation of ozone, mice had no detectible differences in cellular inflammation or total BALF protein dependent on mindin. However, mindin-/- mice were protected from increased proinflammatory cytokine production and AHR compared with their C57BL/6J counterparts. After ozone exposure, bronchial rings derived from mindin-/- mice demonstrated reduced constriction in response to carbachol. CONCLUSIONS: These data demonstrate that the extracellular matrix protein mindin modifies the airway response to both LPS and ozone. Our data support a conserved role of mindin in production of proinflammatory cytokines and the development of AHR in two divergent models of reactive airways disease, as well as a role of mindin in airway smooth muscle contractility after exposure to ozone.

Excitation-contraction coupling in airway smooth muscle.

Excitation-contraction (EC) coupling in striated muscles is mediated by the cardiac or skeletal muscle isoform of voltage-dependent L-type Ca(2+) channel (Ca(v)1.2 and Ca(v)1.1, respectively) that senses a depolarization of the cell membrane, and in response, activates its corresponding isoform of intracellular Ca(2+) release channel/ryanodine receptor (RyR) to release stored Ca(2+), thereby initiating muscle contraction. Specifically, in cardiac muscle following cell membrane depolarization, Ca(v)1.2 activates cardiac RyR (RyR2) through an influx of extracellular Ca(2+). In contrast, in skeletal muscle, Ca(v)1.1 activates skeletal muscle RyR (RyR1) through a direct physical coupling that negates the need for extracellular Ca(2+). Since airway smooth muscle (ASM) expresses Ca(v)1.2 and all three RyR isoforms, we examined whether a cardiac muscle type of EC coupling also mediates contraction in this tissue. We found that the sustained contractions of rat ASM preparations induced by depolarization with KCl were indeed partially reversed ( approximately 40%) by 200 mum ryanodine, thus indicating a functional coupling of L-type channels and RyRs in ASM. However, KCl still caused transient ASM contractions and stored Ca(2+) release in cultured ASM cells without extracellular Ca(2+). Further analyses of rat ASM indicated that this tissue expresses as many as four L-type channel isoforms, including Ca(v)1.1. Moreover, Ca(v)1.1 and RyR1 in rat ASM cells have a similar distribution near the cell membrane in rat ASM cells and thus may be directly coupled as in skeletal muscle. Collectively, our data implicate that EC-coupling mechanisms in striated muscles may also broadly transduce diverse smooth muscle functions.

Redox activation of intracellular calcium release channels (ryanodine receptors) in the sustained phase of hypoxia-induced pulmonary vasoconstriction.

Hypoxia-induced pulmonary vasoconstriction (HPV) is an important adaptive process that remains incompletely understood. In preconstricted rat pulmonary arteries (inner diameter, 250 to 400 microm), hypoxia (pO2 approximately 10 mm Hg) induces an initial transient phase and a more slowly developing sustained phase of vasoconstriction. Since the release of calcium ions (Ca2+) from intracellular stores by redox-sensitive intracellular Ca2+ release channels known as ryanodine receptors (RyRs) in pulmonary arterial smooth-muscle cells (PASMCs) may play a role in HPV, and considerable evidence now supports that levels of reactive oxygen species (ROS) are paradoxically increased in PASMC under hypoxia, we investigated whether redox activation of RyRs by ROS may transduce HPV. By reverse transcriptase-polymerase chain reaction, we found that all three RyR isoforms are expressed in rat pulmonary arteries and in PASMCs. The sustained phase, but not the transient phase, of HPV can be prevented by pretreating pulmonary arteries with RyR inhibitors ryanodine (200 micromol/L) or dantrolene (50 micromol/L). The addition of dantrolene, ryanodine or the thiol-reducing agent dithiothreitol (1 mmol/L) during the sustained phase of HPV reversed the hypoxic vasoconstriction. In contrast, the superoxide scavenger nitroblue tetrazolium (500 nmol/L) prevented further hypoxic pulmonary vasoconstriction during the sustained phase of HPV but did not reverse it. Taken together, our data suggest that redox activation of RyRs by ROS has an important role in transducing the sustained contraction of pulmonary arteries under hypoxia.

Ryanodine receptors in muscarinic receptor-mediated bronchoconstriction.

Ryanodine receptors (RyRs), intracellular calcium release channels essential for skeletal and cardiac muscle contraction, are also expressed in various types of smooth muscle cells. In particular, recent studies have suggested that in airway smooth muscle cells (ASMCs) provoked by spasmogens, stored calcium release by the cardiac isoform of RyR (RyR2) contributes to the calcium response that leads to airway constriction (bronchoconstriction). Here we report that mouse ASMCs also express the skeletal muscle and brain isoforms of RyRs (RyR1 and RyR3, respectively). In these cells, RyR1 is localized to the periphery near the cell membrane, whereas RyR3 is more centrally localized. Moreover, RyR1 and/or RyR3 in mouse airway smooth muscle also appear to mediate bronchoconstriction caused by the muscarinic receptor agonist carbachol. Inhibiting all RyR isoforms with > or = 200 microM ryanodine attenuated the graded carbachol-induced contractile responses of mouse bronchial rings and calcium responses of ASMCs throughout the range of carbachol used (50 nM to > or = 3 microM). In contrast, inhibiting only RyR1 and RyR3 with 25 microM dantrolene attenuated these responses caused by high (>500 nM) but not by low concentrations of carbachol. These data suggest that, as the stimulation of muscarinic receptor in the airway smooth muscle increases, RyR1 and/or RyR3 also mediate the calcium response and thus bronchoconstriction. Our findings provide new insights into the complex calcium signaling in ASMCs and suggest that RyRs are potential therapeutic targets in bronchospastic disorders such as asthma.