Inhaled nitric oxide improves systemic oxygenation in infants with persistent pulmonary hypertension and may reduce the need for more invasive treatments.

Objective. To assess the clinical effects of rituximab therapy in patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV).Methods. The study group comprised 11 patients who had active AAV despite receiving maximally tolerated doses of cyclophosphamide or had contraindications for cyclophosphamide use. All patients had ANCA reactive against proteinase 3. The patients received rituximab infusions and glucocorticoids to induce remission. Three patients also received plasma exchange. No other immunosuppressive agents were used. Glucocorticoids were tapered as soon as control of disease activity was achieved. Disease activity was monitored using the Birmingham Vasculitis Activity Score, modified for Wegener's granulomatosis.Results. Rituximab therapy was well tolerated by all patients, and adverse events were rare. Following the rituximab infusions, circulating B lymphocytes became undetectable, and ANCA titers decreased significantly. Remission was achieved in all patients and was maintained while B lymphocytes were absent.Conclusion. The ability to achieve stable remissions with rituximab in patients with AAV refractory to conventional therapy suggests that B lymphocyte depletion may be a safe, effective, mechanism-based treatment modality for treatment of patients with these conditions.

No abstract

We tested the hypothesis that prolonged serum deprivation would allow a subset of cultured airway myocytes to reacquire the abundant contractile protein content, marked shortening capacity, and elongated morphology characteristic of contractile cells within intact tissue. Passage 1 or 2 canine tracheal smooth muscle (SM) cells were grown to confluence, then serum deprived for up to 19 days. During serum deprivation, two differentiation pathways emerged. One-sixth of the cells developed an elongated morphology and aligned into bundles. Elongated myocytes contained cables of contractile myofilaments, dense bodies, gap junctions, and membrane caveoli, ultrastructural features of contractile SM in tissue. These cells immunostained intensely for SM α-actin, SM myosin heavy chain (MHC), and SM22 (an SM-specific actin-binding protein), and Western analysis of culture lysates disclosed 1.8 (SM α-actin)-, 7.7 (SM MHC)-, and 5.8 (SM22)-fold protein increases during serum deprivation. Immunoreactive M3 muscarinic receptors were present in dense foci distributed throughout elongated, SM MHC-positive myocytes. ACh (10−3 M) induced a marked shortening (59.7 ± 14.4% of original length) in 62% of elongated myocytes made semiadherent by gentle proteolytic digestion, and membrane bleb formation (a consequence of contraction) occurred in all stimulated cells that remained adherent and so did not shorten. Cultured airway myocytes that did not elongate during serum deprivation instead became short and flattened, lost immunoreactivity for contractile proteins, lacked the M3 muscarinic-receptor expression pattern seen in elongated cells, and exhibited no contractile response to ACh. Thus we demonstrate that prolonged serum deprivation induces distinct differentiation pathways in confluent cultured tracheal myocytes and that one subpopulation acquires an unequivocally functional contractile phenotype in which structure and function resemble contractile myocytes from intact tissue.

Previous studies have suggested that the proinflammatory cytokine, TNF-␣, contributes to airway hyperresponsivness by altering airway smooth muscle (ASM) Ca 2ϩ responses to agonist stimulation. The present study examined the effects of TNF-␣ on Ca 2ϩ influx pathways in cultured human ASM cells (HASMCs). Proteins encoded by the transient receptor potential (TRP) gene family function as channels through which receptor-operated and store-operated Ca 2ϩ entry (SOCE) occur. In the present study, the presence of TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6 mRNA and protein expression was confirmed in cultured HASMCs using RT-PCR and Western blot analysis. TNF-␣ treatment significantly increased TRPC3 mRNA and protein levels in HASMCs as well as SOCE. TNF-␣ treatment also increased both the peak and plateau intracellular Ca 2ϩ concentration responses in HASMCs elicited by acetylcholine and bradykinin. The effects of TNF-␣ treatment on SOCE and agonist-induced intracellular Ca 2ϩ concentration responses were attenuated using small interfering RNA transfection, which knocked down TRPC3 expression. Thus, in inflammatory airway diseases, TNF-␣ treatment may result in increased myocyte activation due to altered Ca 2ϩ influx pathways. These results suggest that TRPC3 may be an important therapeutic target in inflammatory airway diseases such as asthma and chronic obstructive pulmonary disease.

The introduction of continuous (24-hr) on-site presence by a staff academic critical care specialist was associated with improved processes of care and staff satisfaction and decreased intensive care unit complication rate and hospital length of stay.

Contractility of airway smooth muscle requires elevation of intracellular calcium concentration. Under resting conditions, airway smooth muscle cells maintain a relatively low intracellular calcium concentration, and activation of the surface receptors by contractile agonists results in an elevation of intracellular calcium, culminating in contraction of the cell. The pattern of elevation of intracellular calcium brought about by agonists is a dynamic process and involves the coordinated activities of ion channels located in the plasma membrane and the sarcoplasmic reticulum. Among the signaling molecules involved in this dynamic calcium regulation in airway smooth muscle cells are inositol 1,4,5-trisphosphate and cyclic ADPribose, which mobilize calcium from the sarcoplasmic reticulum by acting via the inositol 1,4,5-trisphosphate and ryanodine receptors, respectively. In addition, calcium influx from the extracellular space is critical for the repletion of the intracellular calcium stores during activation of the cells by agonists. Calcium influx can occur via voltage-and receptor-gated channels in the plasma membrane, as well as by influx that is triggered by depletion of the intracellular stores (i.e., store-operated calcium entry mechanism). Transient receptor potential proteins appear to mediate the calcium influx via receptor-and store-operated channels. Recent studies have shown that proinflammatory cytokines regulate the expression and activity of the pathways involved in intracellular calcium regulation, thereby contributing to airway smooth muscle cell hyperresponsiveness. In this review, we will discuss the specific roles of cyclic ADP-ribose/ryanodine receptor channels and transient receptor potential channels in the regulation of intracellular calcium in airway smooth muscle cells. DYNAMIC INTRACELLULAR CALCIUM REGULATION IN AIRWAY SMOOTH MUSCLE CELLSExposure of airway smooth muscle (ASM) cells to contractile agonists results in a biphasic elevation of intracellular calcium concentration ([Ca 21 ] i ) that is characterized by an initial rapid and transient rise in calcium, followed by a decline to a lower, sustained steady-state concentration above the basal level (1-3). This biphasic [Ca 21 ] i response results from calcium influx from the extracellular space and release of calcium from the intracellular stores (i.e., the sarcoplasmic reticulum [SR]). Earlier studies have attributed the initial rapid and transient phase of the [Ca 21 ] i response to release from the SR, while the sustained phase of the response was thought to be due to influx from the extracellular space (2-6). Recent investigations using the improved temporal and spatial resolution features of real-time confocal microscopy have shed light on the dynamics of the [Ca 21 ] i response in ASM cells. These studies have shown that the biphasic [Ca 21 ] i response of ASM cells elicited by contractile agonists in reality consists of propagating regenerative calcium oscillations that originate at a location within a cell and propagate...

(11,15,20,26,38,39,46) or by plasma membrane Ca 2ϩ influx.TNF␣, a potent proinflammatory cytokine found in bronchoalveolar lavage fluid (12) and sputum (50) from asthmatics, has been implicated as a mediator in the pathophysiology of asthma (45, 51, 52) and chronic obstructive pulmonary disease (8, 16). Indeed, TNF␣ has been shown to enhance ASM contractility (13,43,47 (11,20,46) as well as via ryanodine receptor (RyR) channels (15, 26), the latter resulting from increased levels of the novel second messenger cyclic ADP ribose (cADPR) (27,39). cADPR, in turn, is synthesized and degraded by the bifunctional ectoenzyme CD38 via ADP ribosyl cyclase and cADPR hydrolase activities, respectively (22). Indeed, the CD38/cADPR pathway has been implicated in [Ca 2ϩ ] i regulation in ASM (39, 57) and intestinal (30, 31), uterine (7, 53), and vascular (17, 25) smooth muscles. Recent studies suggest that the CD38/cADPR signaling pathway contributes to TNF␣-induced augmentation of [Ca 2ϩ ] i responses in ASM (14). Such an effect is thought to result from an increase in cADPRinduced Ca 2ϩ mobilization from the SR. However, this does not explain the effect of TNF␣ on both peak and plateau responses. While it is likely that SR Ca 2ϩ release is augmented by TNF␣, whether Ca 2ϩ influx is also increased remains to be determined.Ca 2ϩ influx in ASM can occur through voltage-gated (59), receptor-operated (33), and/or store-operated channels (6). In the last case, store-operated Ca 2ϩ entry (SOCE) is triggered by depletion of SR Ca 2ϩ stores (6,41,42,49,55,56). We and others have shown that different transient receptor potential channel (TRPC) isoforms are expressed in ASM (6,58). In a recent study using human ASM, we further demonstrated that TNF␣ treatment increases SOCE (58). TNF␣ treatment also increases CD38 expression. Studies in other cell types suggest that CD38 itself can also regulate SOCE (10, 21). Whether altered CD38 expression contributes to TNF␣-induced changes in Ca 2ϩ influx is not known. In the present study, we hypothesized that the effects of TNF␣ treatment on CD38 expression and SOCE in human ASM cells are linked. Accordingly, we examined the effects of CD38 overexpression vs. knockdown of CD38 expression [via specific small interfering RNA (siRNA)] on TNF␣-induced enhancement of SOCE. METHODSHuman ASM cells. Human bronchi were obtained from discarded surgical specimens in accordance with procedures reviewed and approved (as well as deemed exempt from Human Subjects classification under 45CFR 46) by the Mayo Clinic Institutional Review Board. The techniques for isolation of ASM cells from bronchi have