Citations

Abstract Thymic stromal lymphopoietin (TSLP) is a newly identified IL-7–like cytokine known to be expressed in airway biopsies of patients with asthma and chronic obstructive pulmonary disease. As both diseases may be induced or exacerbated by cigarette smoking, it is possible that TSLP represents an important link between cigarette smoke exposure and inflammatory signaling in the airways. In this regard, TSLP appears to also be expressed in airway smooth muscle (ASM); however, its role is unknown. In the current study, we examined TSLP and the TSLP receptor (TSLP-R) expression and function in human ASM cells under normal conditions and following exposure to cigarette smoke extract (CSE). Western blot analysis of human ASM cells showed significant expression of TSLP and TSLP-R, with increased expression of both by overnight exposure to 1 or 2% CSE. Furthermore, CSE increased TSLP release by ASM. In parallel experiments using enzymatically dissociated human ASM cells loaded with the Ca2+ indicator fura 2-AM and imaged using fluorescence microscopy, we evaluated the effects of CSE exposure on intracellular Ca2+ ([Ca2+]i) responses to agonist stimulation. [Ca2+]i responses to histamine were increased with overnight CSE exposure. Exposure to TSLP also resulted in elevated responses, which were blunted by TSLP and TSLP-R Abs. Importantly, the enhancing effects of CSE on [Ca2+]i responses were also blunted by these Abs. These effects were associated with CSE- and TSLP-induced changes in STAT5 phosphorylation. Overall, these novel data suggest that cigarette smoke, TSLP, and ASM are functionally linked and that cigarette smoke-induced increase in airway contractility may be mediated via ASM-derived increases in TSLP signaling.

Breast cancer is the most common type of cancer diagnosed in women. Anthracyclines and taxanes are the most active and widely used chemotherapeutic agents in the treatment of both early-stage and advanced breast cancer. In the past decade, novel formulations of these cytotoxic agents have been developed to improve efficacy and decrease toxicity. nab-paclitaxel is a solvent-free, albumin-bound 130-nm particle form of paclitaxel (Abraxane®, Abraxis Bioscience, CA, USA), which was developed to avoid toxicities associated with the Cremophor vehicle used in solvent-based paclitaxel. In a Phase III study, nab-paclitaxel demonstrated higher response rates, a better safety profile compared with conventional paclitaxel, and improved survival in patients receiving it as second-line therapy. Based on this pivotal trial, nab-paclitaxel is now approved in the USA for treatment of breast cancer after failure of combination chemotherapy for metastatic disease or relapse within 6 months of adjuvant therapy where prior therapy included an anthracycline unless clinically contraindicated. Recently, several Phase II studies have suggested a role for nab-paclitaxel as a single agent and in combination with other agents for first-line treatment of metastatic breast cancer. Studies are ongoing to explore the use of nab-paclitaxel in other solid tumors such as non-small-cell lung cancer, ovarian cancer and malignant melanoma.

The severity of asthma, a disease characterized by airway hyperresponsiveness and inflammation, is enhanced in some women during the menstrual cycle and during pregnancy but relieved in others. These clinical findings suggest that sex steroids modulate airway tone. Based on well-known relaxant effects of estrogens on vascular smooth muscle, we hypothesized that estrogens relax airway smooth muscle (ASM), thus facilitating bronchodilation. In ASM tissues from female patients, Western and immunocytochemical analyses confirmed the presence of both estrogen receptor (ER) isoforms, ERα and ERβ. In fura 2-loaded, dissociated ASM cells maintained in culture, acute exposure to physiological concentrations of 17β-estradiol (E2; 100 pM to 10 nM) decreased the intracellular Ca2+ ([Ca2+]i) response to 1 μM histamine, an effect reversed by the ER antagonist ICI-182,780. The ERα-selective agonist (R,R)-THC had a greater reducing effect on [Ca2+]i responses to histamine and 1 μM ACh compared with the ERβ-selective agonist (DPN). The effects of E2 on [Ca2+]i were mediated, at least in part, via decreased Ca2+ influx through l-type channels and store-operated Ca2+ entry but not via Ca2+-activated K+ channels, receptor-operated entry, or sarcoplasmic reticulum reuptake. Overall, these data support our hypothesis that estrogens relax ASM and suggest a potentially novel therapeutic target in airway hyperresponsiveness.

Rewarming patients after profound hypothermia may result in acute heart failure and high mortality (50–80%). However, the underlying pathophysiological mechanisms are largely unknown. We characterized cardiac contractile function in the temperature range of 15–30°C by measuring the intracellular Ca2+concentration ([Ca2+]i) and twitch force in intact left ventricular rat papillary muscles. Muscle preparations were loaded with fura-2 AM and electrically stimulated during cooling at 15°C for 1.5 h before being rewarmed to the baseline temperature of 30°C. After hypothermia/rewarming, peak twitch force decreased by 30–40%, but [Ca2+]iwas not significantly altered. In addition, we assessed the maximal Ca2+-activated force (Fmax) and Ca2+sensitivity of force in skinned papillary muscle fibers. Fmaxwas decreased by ∼30%, whereas the pCa required for 50% of Fmaxwas reduced by ∼0.14. In rewarmed papillary muscle, both total cardiac troponin I (cTnI) phosphorylation and PKA-mediated cTnI phosphorylation at Ser23/24 were significantly increased compared with controls. We conclude that after hypothermia/rewarming, myocardial contractility is significantly reduced, as evidenced by reduced twitch force and Fmax. The reduced myocardial contractility is attributed to decreased Ca2+sensitivity of force rather than [Ca2+]iitself, resulting from increased cTnI phosphorylation.

AbstractBackground: Parathyroid hormone (PTH) assays able to distinguish between full-length PTH (PTH1–84) and N-terminally truncated PTH (PTH7–84) are of increasing significance in the accurate diagnosis of endocrine and osteological diseases. We describe the discovery of new N-terminal and C-terminal PTH variants and the development of selected reaction monitoring (SRM)-based immunoassays specifically designed for the detection of full-length PTH [amino acid (aa)1–84] and 2 N-terminal variants, aa7–84 and aa34–84.Methods: Preparation of mass spectrometric immunoassay pipettor tips and MALDI-TOF mass spectrometric analysis were carried out as previously described. We used novel software to develop SRM assays on a triple-quadrupole mass spectrometer. Heavy isotope-labeled versions of target peptides were used as internal standards.Results: Top-down analysis of samples from healthy individuals and renal failure patients revealed numerous PTH variants, including previously unidentified aa28–84, aa48–84, aa34–77, aa37–77, and aa38–77. Quantitative SRM assays were developed for PTH1–84, PTH7–84, and variant aa34–84. Peptides exhibited linear responses (R2 = 0.90–0.99) relative to recombinant human PTH concentration limits of detection for intact PTH of 8 ng/L and limits of quantification of 16–31 ng/L depending on the peptide. Standard error of analysis for all triplicate measurements was 3%–12% for all peptides, with <5% chromatographic drift between replicates. The CVs of integrated areas under the curve for 54 separate measurements of heavy peptides were 5%–9%.Conclusions: Mass spectrometric immunoassays identified new clinical variants of PTH and provided a quantitative assay for these and previously identified forms of PTH.

Abstract There is increasing recognition that neurotrophin (NT) signaling occurs in non-neuronal tissues, including airway smooth muscle (ASM). We recently demonstrated that NTs, such as brain-derived neurotrophic factor (BDNF), enhance intracellular Ca2 + ([Ca2+]i) and force regulation in human ASM. Increased NT expression has been observed in airway diseases, such as asthma and allergy. In the present study, we tested the hypothesis that NTs contribute to inflammation-induced enhancement of ASM contractility. Using human ASM cells and real-time fluorescence [Ca2+]i imaging, we examined the contribution of the high-affinity tropomyosin-related kinase and low-affinity, pan-NT p75NTR receptors to [Ca2+]i regulation under control conditions and after exposure to the proinflammatory cytokine TNF-α (20 ng/ml). Exposure to TNF-α enhanced [Ca2+]i responses to agonist (acetylcholine, histamine). Exposure to 10 nM BDNF for even 30 minutes substantially and synergistically enhanced TNF-α effects on [Ca2+]i responses to agonist. Small interfering RNA suppression of tropomyosin-related kinase substantially blunted the effect of BDNF on [Ca2+]i responses to agonist (with greater effect on Ca2+ influx via store-operated Ca2+ entry compared with sarcoplasmic reticulum Ca2+ release) in both control and TNF-α–exposed cells. However, p75NTR suppression by small interfering RNA had no significant effect on [Ca2+]i responses in either cell group. These novel data demonstrate that NTs influence ASM contractility, and suggest a potential role for NTs in airway diseases.

Background: Although angina is a chronic disease, most clinical trials evaluating antiangi nal therapy are of a few weeks or months in duration. Methods and Results: To evaluate the effects of nicardipine, a second-generation dihydro pyridine calcium channel blocker, as long-term therapy, patients with chronic stable angina were treated for 1 year with open-label nicardipine. Anginal symptoms were controlled with 20 mg tid in 24%, 30 mg tid in 34%, and 40 mg tid in 42%. Of 72 patients completing the 1-year trial, only 14 (19%) had required the addition of long-acting nitrates for control of symptoms. The remaining 57 patients, who had anginal symptoms controlled with nicar dipine alone, were continued into the 3-week, double-blind period and were randomized to continue their usual dose of nicardipine or placebo. Throughout the 1-year, open-label treatment period, the number of anginal episodes and exercise parameters of angina were significantly reduced with nicardipine. Conclusions: During the double-blind period, the patients who continued on nicardipine had significantly greater exercise time and time to onset of angina than patients who were randomized to placebo. The exercise parameters in the patients randomized to placebo were improved over baseline after 1 year of therapy; however, the improvement with nicar dipine was significantly greater.

Background— Brugada syndrome is a rare, autosomal-dominant, male-predominant form of idiopathic ventricular fibrillation characterized by a right bundle-branch block and ST elevation in the right precordial leads of the surface ECG. Mutations in the cardiac Na + channel SCN5A on chromosome 3p21 cause ≈20% of the cases of Brugada syndrome; most mutations decrease inward Na + current, some by preventing trafficking of the channels to the surface membrane. We previously used positional cloning to identify a new locus on chromosome 3p24 in a large family with Brugada syndrome and excluded SCN5A as a candidate gene. Methods and Results— We used direct sequencing to identify a mutation (A280V) in a conserved amino acid of the glycerol-3-phosphate dehydrogenase 1–like ( GPD1-L ) gene. The mutation was present in all affected individuals and absent in >500 control subjects. GPD1-L RNA and protein are abundant in the heart. Compared with wild-type GPD1-L, coexpression of A280V GPD1-L with SCN5A in HEK cells reduced inward Na + currents by ≈50% ( P <0.005). Wild-type GPD1-L localized near the cell surface to a greater extent than A280V GPD1-L. Coexpression of A280V GPD1-L with SCN5A reduced SCN5A cell surface expression by 31±5% ( P =0.01). Conclusions— GPD1-L is a novel gene that may affect trafficking of the cardiac Na + channel to the cell surface. A GPD1-L mutation decreases SCN5A surface membrane expression, reduces inward Na + current, and causes Brugada syndrome.