Caveolin-1 regulation of store-operated Ca(2+) influx in human airway smooth muscle

Caveolae, plasma membrane invaginations with constitutive caveolin proteins, harbour proteins involved in intracellular calcium ([Ca(2+)](i)) regulation. In human airway smooth muscle (ASM), store-operated Ca(2+) entry (SOCE) is a key component of [Ca(2+)](i) regulation, and contributes to increased [Ca(2+)](i) in inflammation. SOCE involves proteins Orai1 and stromal interaction molecule (STIM)1. We investigated the link between caveolae, SOCE and inflammation in ASM. [Ca(2+)](i) was measured in human ASM cells using fura-2. Small interference RNA (siRNA) or overexpression vectors were used to alter expression of caveolin-1 (Cav-1), Orai1 or STIM1. Tumour necrosis factor (TNF)-α was used as a representative pro-inflammatory cytokine. TNF-α increased SOCE following sarcoplasmic reticulum Ca(2+) depletion, and increased whole-cell and caveolar Orai1 (but only intracellular STIM1). Cav-1 siRNA decreased caveolar and whole-cell Orai1 (but not STIM1) expression, and blunted SOCE, even in the presence of TNF-α. STIM1 overexpression substantially enhanced SOCE: an effect only partially reversed by Cav-1 siRNA. In contrast, Orai1 siRNA substantially blunted SOCE even in the presence of TNF-α. Cav-1 overexpression significantly increased Orai1 expression and SOCE, especially in the presence of TNF-α. These results demonstrate that caveolar expression and regulation of proteins such as Orai1 are important for [Ca(2+)](i) regulation in human ASM cells and its modulation during inflammation.     

Sodium chloride or plasmalyte-148 for patients presenting to emergency departments with diabetic ketoacidosis: A nested cohort study within a multicentre,cluster, crossover,randomised, controlled trial

Objective

To test the hypothesis that fluid resuscitation in the ED with plasmalyte-148 (PL) compared with 0.9% sodium chloride (SC) would result in a lower proportion of patients with diabetic ketoacidosis (DKA) requiring intensive care unit (ICU) admission.

Methods

We performed a prespecified nested cohort study at two hospitals within a cluster, crossover, open label, randomised, controlled trial comparing the effects of PL versus SC as fluid therapy for patients who presented to the ED with DKA. All patients presenting within a fixed recruitment period were included. The primary outcome was the proportion of patients admitted to ICU.

Results

Eighty-four patients were enrolled (SC n = 38, PL n = 46). The SC group had a lower median pH on admission (SC: 7.09 [interquartile range (IQR) 7.01–7.21], PL: 7.17 [IQR 6.99–7.26]). The median volume of intravenous fluids administered in ED was 2150 mL (IQR 2000–3200 mL; SC) and 2200 mL (IQR 2000–3450; PL); respectively. A higher proportion of patients in the SC group, 19 (50%), was admitted to ICU compared with PL group, 18 (39.1%); however, after adjustment for pH at presentation and diabetes type in a multivariable logistic regression model, the PL group did not have a significantly different rate of ICU admission compared with the SC group (odds ratio for ICU admission 0.73, 95% confidence interval 0.13–3.97, P = 0.71).

Conclusion

Patients with DKA treated with PL compared with SC in the EDs had similar rates of requiring ICU admission.     

Proliferation and cytogenetic analysis of hairy cell leukemia upon stimulation via the CD40 antigen

Using the CD40 system, in vitro proliferation of hairy cell leukemia (HCL) was examined in 43 patients. In this culture system, cells were stimulated by interleukin-4 (IL-4) and anti-CD40 monoclonal antibodies (MoAbs) that were added in soluble form or were cross-linked via their Fc part using Fc gamma RII-transfected mouse fibroblast cells. Proliferation was induced and confirmed by 3H-thymidine incorporation in 14 cases and by the presence of metaphases in 42 cases. 3H-thymidine incorporation showed a heterogeneous pattern: cross-linking of anti- CD40 gave the highest proliferation in 8 cases; in 11 cases, stimulation with anti-CD40 MoAbs alone, without cross-linking also resulted in proliferation; the addition of IL-4 further enhanced 3H- thymidine incorporation in 5 cases, but suppressed this phenomenon in 5 other cases. The CD40 system proved to be very effective in obtaining cytogenetic data. With a success rate of 42 of 43 patients tested, we found clonal abnormalities in 8 cases (19%) and nonclonal abnormalities with involvement of one or two abnormal metaphases in another 7 cases. The chromosomes most frequently involved in the abnormal karyotypes, both structurally and numerically, were chromosomes 5, 7, and 14. By fluorescence-activated cell-sorting analysis of the cultured cells, and by immunophenotypic analysis of metaphase spreads, T-cell growth could be excluded and the HCL-lineage confirmed. Stimulation via the CD40 antigen is an excellent tool for growing hairy cell leukemia cells.     

Effects of monoclonal antibody therapy in patients with chronic lymphocytic leukemia

A phase I clinical trial was initiated to treat patients with stage IV B-derived chronic lymphocytic leukemia (CLL) with the IgG2a murine monoclonal antibody T101. This antibody binds to a 65,000-mol wt (T65) antigen found on normal T lymphocytes, malignant T lymphocytes, and B- derived CLL cells. All of the patients had a histologically confirmed diagnosis of advanced B-derived CLL and were refractory to standard therapy, and more than 50% of their leukemia cells reacted with the T101 antibody in vitro. The patients received T101 antibody two times per week, over two to 50 hours by intravenous administration in 100 mL of normal saline containing 5% human albumin. Twelve patients were treated with a fixed dosage of 1, 10, 50, or 100 mg, and one patient was treated with 140 mg of antibody. It was demonstrated that patients given two-hour infusions of 50 mg developed pulmonary toxicity, with shortness of breath and chest tightness. This toxicity was eliminated when infusions of 50 or 100 mg of T101 were prolonged to 50 hours. All dose levels caused a rapid but transient decrease in circulating leukemia cell counts. In vivo binding to circulating and bone marrow leukemia cells was demonstrated at all dose levels with increased binding at higher dosages. Antimurine antibody responses were not demonstrated in any patients at any time during treatment. Circulating free murine antibody was demonstrated in the serum of only the two patients treated with 100 mg of antibody as a 50-hour infusion and the patient treated with 140 mg of antibody over 30 hours. Antigenic modulation was demonstrated in patients treated at all dose levels but was particularly apparent in patients treated with prolonged infusions of 50 and 100 mg of antibody. We were also able to demonstrate antigenic modulation in lymph node cells, which strongly suggests in vivo labeling of these cells. Overall, T101 antibody alone appears to have a very limited therapeutic value for patients with CLL. The observations of in vivo labeling of tumor cells, antigenic modulation, antibody pharmacokinetics, toxicity, and antimurine antibody formation may be used in the future for more effective therapy when drugs or toxins are conjugated to the antibody.     

Changes in the proteins coating monosodium urate crystals during active and subsiding inflammation. immunogold studies of synovial fluid from patients with gout and of fluid obtained using the rat subcutaneous air pouch model

Objective. In this in vivo study, we investigated changes in the proteins that coat monosodium urate (MSU) crystals in human synovial fluid samples and rat air pouch fluid samples obtained sequentially during periods of active and resolving inflammation, in order to evaluate whether in vivo findings are consistent with hypotheses on roles of protein coating based on in vitro findings. Methods. Crystals from patients with gout were isolated from joint fluids with acute inflammation, and subsequently from the same joints at the time inflammation was resolving. Crystals were also obtained using the rat subcutaneous air pouch model. Immunogold was used to label proteins coating MSU crystals, for light microscopy (LM) and transmission electron microscopy (TEM) studies. Results. Dense immunogold–silver labeling for IgG was observed under LM on crystals from fluid with acute inflammation, whereas other proteins (apolipoproteins [Apo], fibronectin, fibrinogen, albumin) were not labeled significantly. Apo B became strongly positive on crystals as the inflammation subsided, whereas other proteins were only weakly positive and IgG became absent or weakly positive. Quantitative TEM evaluation confirmed the LM observations. Conclusion. This study provides the first in vivo evidence supporting the notion derived from previous in vitro studies that proteins coating MSU crystals change as inflammation evolves. Protein coatings may play an important role in the self-limited nature of gouty inflammation. IgG coating MSU crystals may enhance the inflammation. As the inflammation subsides, Apo B could displace the IgG by competitively coating sites on crystals and could contribute in part to the resolution of the acute gouty arthritis.     

Light and electron microscopic studies on the synovial membrane in reiter's syndrome.

Studies by light microscopy on synovium obtained from 11 patients with Reiter's syndrome during the first month of an episode showed proliferation of synovial lining cells, polymorphonuclear neutrophils among the synovial lining cells, increased surface fibrin, and vascular congestion. Biopsy specimens taken later showed vascular congestion and still proliferated synovial lining cells, fewer polymorphonuclear neutrophils in some, and a tendency toward increased infiltration with lymphocytes and plasma cells. Electron microscopy of samples from 8 patients during the first month of disease activity showed occlusion of vessels by platelets in 4, and fibrin or dense granular material in the vessel walls in 4. Five of the patients with arthritis of less than 4 weeks duration had unidentified intracellular and extracellular particles; some of these were highly suggestive of Chlamydia. No such particles were noted in samples from patients with more chronic cases. Using an antibody to Chlamydia trachomatis and the peroxidase-antiperoxidase technique, immunocytochemistry showed reaction product in synovial macrophages in 2 patients with arthritis of less than 4 weeks duration, but not in the 1 patient studied who had more chronic disease. These studies provide support for dramatic synovial vascular injury consistent with that caused by endotoxin and the presence of chlamydial antigen in synovial macrophages, at least in the early phases of synovitis.     

Mechanisms underlying greater sensitivity of neonatal cardiac muscle to volatile anesthetics

BACKGROUND: In neonatal heart, plasma membrane Na+-Ca2+ exchange (NCX) and Ca2+ influx channels play greater roles in intracellular Ca2+ concentration [Ca2+]i regulation compared with the sarcoplasmic reticulum (SR). In neonatal (aged 0-3 days) and adult (aged 84 days) rat cardiac myocytes, we determined the mechanisms underlying greater sensitivity of the neonatal myocardium to inhibition by volatile anesthetics. METHODS: The effects of 1 and 2 minimum alveolar concentration halothane and sevoflurane on Ca2+ influx during electrical stimulation in the presence or blockade of NCX and the Ca2+ channel agonist BayK8644 were examined. [Ca2+]i responses to caffeine were used to examine anesthetic effects on SR Ca2+ release (via ryanodine receptor channels) and reuptake (via SR Ca2+ adenosine triphosphatase). Ca2+ influx via NCX was examined during rapid activation in the presence of the reversible SR Ca2+ adenosine triphosphatase inhibitor cyclopiazonic acid and ryanodine to inhibit the SR. Efflux mode NCX was examined during activation by extracellular Na+ in the absence of SR reuptake. RESULTS: Intracellular Ca2+ concentration transients during electrical stimulation were inhibited to a greater extent in neonates by halothane (80%) and sevoflurane (50%). Potentiation of [Ca2+]i responses by BayK8644 (160 and 120% control in neonates and adults, respectively) was also blunted by anesthetics to a greater extent in neonates. [Ca2+]i responses to caffeine in neonates ( approximately 30% adult responses) were inhibited to a lesser extent compared with adults (35 vs. 60% by halothane). Both anesthetics inhibited Ca2+ reuptake at 2 minimum alveolar concentration, again to a greater extent in adults. Reduction in NCX-mediated influx was more pronounced in neonates (90%) compared with adults (65%) but was comparable between anesthetics. Both anesthetics also reduced NCX-mediated efflux to a greater extent in neonates. Potentiation of NCX-mediated Ca2+ efflux by extracellular Na+ and NCX-mediated Ca2+ influx by intracellular Na+ were both prevented by halothane, especially in neonates. CONCLUSIONS: These data indicate that greater myocardial depression in neonates induced by volatile anesthetics may be mediated by inhibition of NCX and Ca2+ influx channels rather than inhibition of SR Ca2+ release.     

Effects of halothane on sarcoplasmic reticulum calcium release channels in porcine airway smooth muscle cells

BACKGROUND: Volatile anesthetics relax airway smooth muscle (ASM) by altering intracellular Ca2+ concentration ([Ca2+]i). The authors hypothesized that relaxation is produced by decreasing sarcoplasmic reticulum Ca2+ content via increased Ca2+ "leak" through both inositol trisphosphate (IP3) and ryanodine receptor channels. METHODS: Enzymatically dissociated porcine ASM cells were exposed to acetylcholine in the presence or absence of 2 minimum alveolar concentration (MAC) halothane, and IP3 levels were measured using radioimmunoreceptor assay. Other cells were loaded with the Ca2+ indicator fluo-3 and imaged using real-time confocal microscopy. RESULTS: Halothane increased IP3 concentrations in the presence and absence of acetylcholine. Inhibition of phospholipase C blunted the IP3 response to halothane. Exposure to 2 MAC halothane induced a transient [Ca2+]i response, suggesting depletion of sarcoplasmic reticulum Ca2+. Exposure to 20 microM Xestospongin D, a cell-permeant IP3 receptor antagonist, resulted in a 45+/-13% decrease in the [Ca2+]i response to halothane compared with halothane exposure alone. In permeabilized cells, Xestospongin D or 0.5 mg/ml heparin decreased the [Ca2+]i response to halothane by 65+/-13% and 68+/-22%, respectively, compared with halothane alone. In both intact and permeabilized cells, 20 microM ryanodine blunted the [Ca2+]i response to halothane by 32+/-13% and 39+/-21%, respectively, compared with halothane alone. Simultaneous exposure to Xestospongin D and ryanodine completely inhibited the [Ca2+]i response to halothane. CONCLUSIONS: The authors conclude that halothane reduces sarcoplasmic reticulum Ca2+ content in ASM cells via increased Ca2+ leak through both IP3 receptor and ryanodine receptor channels. Effects on IP3 receptor channels are both direct and indirect via elevation of IP3 levels.