Rapid shedding of proinflammatory microparticles by human mononuclear cells exposed to cigarette smoke is dependent on Ca2+ mobilization

Objectives

Microparticles are membrane vesicles shed by cells upon activation and apoptosis. Agonists capable of inducing microparticle generation include cytokines, bacterial products, P-selectin, histamine. Cigarette smoke extract has also been recognized as an agonist involved in microparticle generation with an apoptosis-dependent mechanism. We investigated the possibility that cigarette smoke extract induces the rapid generation of proinflammatory microparticles by human mononuclear cells with a calcium-dependent mechanism.

Materials and methods

Human mononuclear cells were exposed to cigarette smoke extract. [Ca²⁺]_i mobilization was assessed with the fluorescent probe Fluo-4 NW. Microparticles were quantified with a prothrombinase assay and by flow cytometry. Normal human bronchial epithelial cells and A549 alveolar cells were incubated with cigarette smoke extract-induced microparticles and the generation of ICAM-1, IL-8, and MCP-1 was assessed by ELISA.

Results

Exposure to cigarette smoke extract induced a rapid increase in [Ca²⁺]_i mobilization. Microparticle generation was also increased. EGTA, verapamil and the calmodulin inhibitor, W-7, inhibited microparticle generation. Incubation of lung epithelial cells with cigarette smoke extract-induced microparticles increased the expression of proinflammatory mediators.

Conclusions

Exposure of mononuclear cells to cigarette smoke extract causes a rapid shedding of microparticles with a proinflammatory potential that might add to the mechanisms of disease from tobacco use.     

An experimental model to study the effects of a senna extract on the blood constituent labeling and biodistribution of a radiopharmaceutical in rats

ABSTRACT Cassia angustifolia Vahl (senna) is a natural product that contains sennosides, which are active components that affect the intestinal tract and induce diarrhea. Authors have shown that senna produces DNA (deoxyribonucleic acid) lesions in Escherichia coli cultures and can act as an antifungal agent. Natural drugs can alter the labeling of blood constituents with technetium-99m (^99mTc) and can affect the biodistribution of radiopharmaceuticals. In this work, we have evaluated the influence of a senna extract on the radiolabeling of blood constituents and on the biodistribution of the radiopharmaceutical sodium pertechnetate (Na^99mTcO₄) in Wistar rats. Twelve animals were treated with senna extract for 7 days. Blood samples were withdrawn from the animals and the radiolabeling procedure was carried out. The senna extract did not modify the radiolabeling of the blood constituents. A biodistributional assay was performed by administering Na^99mTcO₄ and determining its activity in different organs and in blood. The senna extract altered the biodistribution of Na^99mTcO₄ in the thyroid, liver, pancreas, lungs and blood. These results are associated with properties of the chemical substances present in the aqueous senna extract. Although these assays were performed in animals, our findings suggest that caution should be exercised when nuclear medicine examinations using Na^99mTcO₄ are conducted in patients who are using senna extract.     

A quantitative analysis of the Na+-dependence of\dot V_{max} of the fast action potential in mammalian ventricular myocardium

In microelectrode experiments on papillary muscles of guinea pigs, the quantitative dependence of \(\dot V_{{\text{max}}} \) of the fast action potential, taken as a measure of I_Na, on external Na⁺ concentration has been analyzed under different experimental conditions including the presence of antiarrhythmic drugs such as lidocaine, procaine and propafenone.

1. External Na⁺ concentration changes between 225 mmol/l and 45 mmol/l led to a non-linear response of \(\dot V_{{\text{max}}} \) in that the \(\dot V_{{\text{max}}} \) changes obtained experimentally were significantly smaller than predicted theoretically from a linear dependence of \(\dot V_{{\text{max}}} \) on [Na⁺] _o .
2. Each individual \(\dot V_{{\text{max}}} \) relationship exhibited saturation characteristics. In Lineweaver-Burk plots, \(\dot V_{{\text{max}}} \) correlated extremely well to 1/[Na⁺] _o with correlation coefficients between 0.995 and 0.999. In 16 experiments, the apparentK _m for Na⁺ varied within a range from 170 to 455 mmol/l. Values of 450 V/s–900 V/s were calculated for the saturated \(\dot V_{{\text{max}}} \) (at an infinitely large [Na⁺] _o ).
3. The apparentK _m for Na⁺ rose from 232.0±24.7 mmol/l to 544.0±50.2 mmol/l when the K⁺ concentration of the medium was increased from 5.4 to 10 mmol/l and, thus, resting potential declined from ?90.5±2.5 mV to ?76.1±1.8 mV.
4. Alkalization (pH 9.0) of the medium lowered the apparentK _m for Na⁺ and, simultaneously, reduced the saturated \(\dot V_{{\text{max}}} \) . This typical shift of the straight relating \(\dot V_{{\text{max}}} \) to 1/[Na⁺] _o in the Lineweaver-Burk plot excludes a competitive interaction between Na⁺ and H⁺ ions.
5. Lidocaine (5×10^?5–2×10^?4 mol/l), procaine (2×10^?4 mol/l) and propafenone (0.5–3×10^?5 mol/l) depressed \(\dot V_{{\text{max}}} \) the stronger the lower [Na⁺] _o was. The changes of the \(\dot V_{{\text{max}}} \) relationship induced by these drugs indicated neither a competitive nor a non-competitive interaction of these compounds with Na⁺.
6. As tested with propafenone, external Na⁺ changes modulated the tonic and the phasic block of \(\dot V_{{\text{max}}} \) . The Na⁺ sensitivity of both types of block differed considerably. In a Na⁺-poor (50 mmol/l) medium, the apparentK _m for the tonic block declined from 5×10^?5 to 1.4×10^?5 mol/l and the apparentK _m for the phasic block from 3.8×10^?5 to 2.3×10^?5 mol/l.
7. Na⁺ is not the sole cation that determines the strength of a drug-induced blockade of \(\dot V_{{\text{max}}} \) as it can be substituted by the permeant Li⁺.
8. In the presence of drugs like propafenone which are capable of shifting the steady state inactivation (h _∞) of I_Na to more negative potentials, the voltage-dependence ofh _∞ can be modified by external Na⁺ variations. Na⁺ withdrawal led to a considerable shift in the hyperpolarizing direction.

     

Perfusion scanning using 99mTc-HMPAO detects early cerebrovascular changes in the diabetic rat

Background

^99mTc-HMPAO is a well-established isotope useful in the detection of regional cerebral blood flow. Diabetes gives rise to arterial atherosclerotic changes that can lead to significant end organ dysfunction, prominently affecting perfusion to the heart, kidneys, eyes and brain. In the current study, we investigated the role of ^99mTc-HMPAO cerebral perfusion scans in detecting early vascular changes in the diabetic brain.

Methods

Cerebral perfusion studies were performed on both control and streptozotocin-(STZ) induced diabetic male Wistar rats. Rat brain imaging using a gamma camera was performed for each group 0.5, 2, 4, and 24 hours post ^99mTc-HMPAO injection. Data processing for each cerebral perfusion scan was performed by drawing a region of interest (ROI) circumferentially around the brain (B). Background (BKG) due to signal from the soft tissue of each rat was subtracted. Brain ^99mTc-HMPAO uptake minus background counts (net brain counts; NBC) were then compared between the two groups.

Results

The NBC (mean ± SD) for the STZ group were statistically significantly higher (p = 0.0004) than those of the control group at each of the time points studied.

Conclusion

^99mTc-HMPAO brain scan may be useful in the detection of early atherosclerotic changes in the diabetic rat brain.     

Hydrophilic and lipophilic radiopharmaceuticals as tracers in pharmaceutical development: In vitro – In vivo studies

Background

Scintigraphic studies have been performed to assess the release, both in vitro and in vivo, of radiotracers from tablet formulations. Four different tracers with differing physicochemical characteristics have been evaluated to assess their suitability as models for drug delivery.

Methods

In-vitro disintegration and dissolution studies have been performed at pH 1, 4 and 7. In-vivo studies have been performed by scintigraphic imaging in healthy volunteers. Two hydrophilic tracers, (^99mTc-DTPA) and (^99mTc-MDP), and two lipophilic tracers, (^99mTc-ECD) and (^99mTc-MIBI), were used as drug models.

Results

Dissolution and disintegration profiles, differed depending on the drug model chosen. In vitro dissolution velocity constants indicated a probable retention of the radiotracer in the formulation. In vivo disintegration velocity constants showed important variability for each radiopharmaceutical. Pearson statistical test showed no correlation between in vitro drug release, and in vivo behaviour, for ^99mTc-DTPA, ^99mTc-ECD and ^99mTc-MIBI. High correlation coefficients were found for ^99mTc-MDP not only for in vitro dissolution and disintegration studies but also for in vivo scintigraphic studies.

Conclusion

Scintigraphic studies have made a significant contribution to the development of drug delivery systems. It is essential, however, to choose the appropriate radiotracers as models of drug behaviour. This study has demonstrated significant differences in release patterns, depending on the model chosen. It is likely that each formulation would require the development of a specific model, rather than being able to use a generic drug model on the basis of its physicochemical characteristics.     

Estrogens regulate neuroinflammatory genes via estrogen receptors α and β in the frontal cortex of middle-aged female rats

Background

Increasing evidence links diverse forms of air pollution to neuroinflammation and neuropathology in both human and animal models, but the effects of long-term exposures are poorly understood.

Objective

We explored the central nervous system consequences of subchronic exposure to diesel exhaust (DE) and addressed the minimum levels necessary to elicit neuroinflammation and markers of early neuropathology.

Methods

Male Fischer 344 rats were exposed to DE (992, 311, 100, 35 and 0 μg PM/m³) by inhalation over 6 months.

Results

DE exposure resulted in elevated levels of TNFα at high concentrations in all regions tested, with the exception of the cerebellum. The midbrain region was the most sensitive, where exposures as low as 100 μg PM/m³ significantly increased brain TNFα levels. However, this sensitivity to DE was not conferred to all markers of neuroinflammation, as the midbrain showed no increase in IL-6 expression at any concentration tested, an increase in IL-1β at only high concentrations, and a decrease in MIP-1α expression, supporting that compensatory mechanisms may occur with subchronic exposure. Aβ42 levels were the highest in the frontal lobe of mice exposed to 992 μg PM/m³ and tau [pS199] levels were elevated at the higher DE concentrations (992 and 311 μg PM/m³) in both the temporal lobe and frontal lobe, indicating that proteins linked to preclinical Alzheimer's disease were affected. α Synuclein levels were elevated in the midbrain in response to the 992 μg PM/m³ exposure, supporting that air pollution may be associated with early Parkinson's disease-like pathology.

Conclusions

Together, the data support that the midbrain may be more sensitive to the neuroinflammatory effects of subchronic air pollution exposure. However, the DE-induced elevation of proteins associated with neurodegenerative diseases was limited to only the higher exposures, suggesting that air pollution-induced neuroinflammation may precede preclinical markers of neurodegenerative disease in the midbrain.     

Differential radio-sensitivities of human chromosomes 1 and 2 in one donor in interphase- and metaphase-spreads after 60Co γ-irradiation

Background

Skeletal uptake of ^99mTc labelled methylene diphosphonate (^99mTc-MDP) is used for producing images of pathological bone uptake due to its incorporation to the sites of active bone turnover. This study was done to validate bone turnover markers using total skeletal uptake (TSU) of ^99mTc-MDP.

Methods

22 postmenopausal women (52–80 years) volunteered to participate. Scintigraphy was performed by injecting 520 MBq of ^99mTc-MDP and taking whole body images after 3 minutes, and 5 hours. TSU was calculated from these two images by taking into account the urinary loss and soft tissue uptake. Bone turnover markers used were bone specific alkaline phosphatase (S-Bone ALP), three different assays for serum osteocalcin (OC), tartrate resistant acid phosphatase 5b (S-TRACP5b), serum C-terminal cross-linked telopeptides of type I collagen (S-CTX-I) and three assays for urinary osteocalcin (U-OC).

Results

The median TSU of ^99mTc-MDP was 23% of the administered activity. All bone turnover markers were significantly correlated with TSU with r-values from 0.52 (p = 0.013) to 0.90 (p < 0.001). The two resorption markers had numerically higher correlations (S-TRACP5b r = 0.90, S-CTX-I r = 0.80) than the formation markers (S-Total OC r = 0.72, S-Bone ALP r = 0.66), but the difference was not statistically significant. TSU did not correlate with age, weight, body mass index or bone mineral density.

Conclusion

In conclusion, bone turnover markers are strongly correlated with total skeletal uptake of ^99mTc-MDP. There were no significant differences in correlations for bone formation and resorption markers. This should be due to the coupling between formation and resorption.     

Lesion based diagnostic performance of dual phase <Superscript>99m</Superscript>Tc-MIBI SPECT/CT imaging and ultrasonography in patients with secondary hyperparathyroidism

Background

We aimed to evaluate the diagnostic performance of ^99mTc-MIBI SPECT/CT and ultrasonography in patients with secondary hyperparathyroidism (SHPT), and explored the factors that affect the diagnostic performance.

Methods

^99mTc-MIBI SPECT/CT and ultrasonography were performed in 50 patients with SHPT within 1 month before they underwent surgery. Imaging results were confirmed by the pathology. Pearson correlation analysis was used to determine the correlation of PTH level with clinical data. The optimal cutoff value for predicting positive ^99mTc-MIBI results was evaluated by ROC analysis in lesions diameter.

Results

Forty-nine patients had a positive ^99mTc-MIBI imaging results and 39 patients had positive ultrasonography results. The sensitivities of ^99mTc-MIBI and ultrasonography were 98.00% and 78.00%, respectively. A total of 199 lesions were resected in 50 patients. Among them, 183 lesions were proved to be parathyroid hyperplasia. On per-lesion basis analysis, the sensitivity and specificity of ^99mTc-MIBI and ultrasonography were 59.34% and 75.00% vs 46.24% and 80.00%, respectively. The Pearson correlation analysis showed that the serum AKP and PTH level had a significant linear association (r?=?0.699, P?<?0.001). The lesion diameter was a statistically significant predictive factor in predicting positive ^99mTc-MIBI SPECT/CT. The optimal cutoff value for predicting positive ^99mTc-MIBI results evaluated by ROC analysis in lesions diameter was 8.05 mm.

Conclusion

Dual phase ^99mTc-MIBI SPECT/CT imaging had a higher sensitivity in patients with SHPT than ultrasonography. Therefore, using ^99mTc-MIBI positioning the lesion could be an effective method pre-surgical in patients with SHPT.

     

Effects of the administration of a catalase inhibitor into the fourth cerebral ventricle on cardiovascular responses in spontaneously hypertensive rats exposed to sidestream cigarette smoke

OBJECTIVE:

Previous studies have demonstrated a relationship between brain oxidative stress and cardiovascular regulation. We evaluated the effects of central catalase inhibition on cardiovascular responses in spontaneously hypertensive rats exposed to sidestream cigarette smoke.

METHODS:

Male Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SH) (16 weeks old) were implanted with a stainless steel guide cannula leading into the fourth cerebral ventricle (4^th V). The femoral artery and vein were cannulated for arterial pressure and heart rate measurement and drug infusion, respectively. The rats were exposed to sidestream cigarette smoke for 180 minutes/day, 5 days/week for 3 weeks (CO: 100-300 ppm). The baroreflex was tested using a pressor dose of phenylephrine (8 μg/kg, bolus) and a depressor dose of sodium nitroprusside (50 μg/kg, bolus). Cardiovascular responses were evaluated before and 5, 15, 30 and 60 minutes after injection of a catalase inhibitor (3-amino-1,2,4-triazole, 0.001 g/100 μL) into the 4^th V.

RESULTS:

Vehicle administration into the 4^th V did not affect the cardiovascular response, whereas administration of the central catalase inhibitor increased the basal HR and attenuated the bradycardic peak (p<0.05) to a greater extent in WKY rats exposed to sidestream cigarette smoke than in WKY rats exposed to fresh air. However, in spontaneously hypertensive rats, the effect of the catalase inhibitor treatment was stronger in the fresh air condition (p<0.05).

CONCLUSION:

Administration of a catalase inhibitor into the 4^th V combined with exposure to sidestream cigarette smoke has a stronger effect in WKY rats than in SH rats.     

Different functional states of tetrodotoxin sensitive and tetrodotoxin resistant Na+ channels occur during the in vitro development of rat skeletal muscle

There are three stages of differentiation of voltage dependent Na⁺ channels during the in vitro development of rat skeletal muscle.

1. Myoblasts which are less than 60 h old in culture have Na⁺ channels which normally do not give rise to action potentials but do so after treatment of the cells with very low concentrations of sea anemone toxin. These Na⁺ channels revealed by sea anemone toxin are resistant to TTX.
2. Myoblasts prior to fusion are electrically excitable ( \(\dot V\) _max=10 V/s). Electrically activated Na⁺ channels are only blocked by high concentrations of TTX. Titration of TTX resistant Na⁺ channels with a tritiated derivative of TTX indicates a dissociation constant of the TTX-Na⁺ channel complex of 50 nM.
3. Myotubes have both high and low affinity binding sites for TTX (Frelin et al. 1983). Action potentials ( \(\dot V\) _max=100?200 V/s) are only inhibited at high concentrations of TTX. Experiments with rat myoballs indicate that only Na⁺ channels with a low affinity binding site for TTX are functional in voltage-clamp studies. The K_0.5 value for TTX inhibition of the peak Na⁺ current is observed at 70 nM. Spontaneous contractions of myotubes are blocked by TTX with a K_0.5 value of 100 nM, suggesting that TTX resistant Na⁺ channels are also the ones responsible for the spontaneous contractions in rat myotubes in culture.

²²Na⁺ flux studies after activation of the Na⁺ channel with neurotoxins have been carried out at the different stages of differentiation. Toxin activated Na⁺ channels have the same high affinity for sea anemone toxins at all stages of development; likewise, the sensitivity for TTX is the same. The in vitro differentiation of Na⁺ channels in rat and chick skeletal muscle cells is compared and discussed in relation to the action of neurotrophic influences.     

Tonic and phasicI Na blockade by antiarrhythmics

Drug-induced depression ofI _Na was studied in microelectrode experiments on papillary muscles of guinea pigs by taking \(\dot V_{\max } \) of the fast Na^ü action potential as an estimate forg _max thereby systematically discriminating between tonic and phasic blockade.

1. The antiarrhythmic compound propafenone and its derivatives butafenone and Sa 76, which are structurally related to local anesthetics, exerted a tonic and phasic \(\dot V_{\max } \) blockade. At a given drug concentration, the strength as well as the kinetics of development and removal of phasic block are functions of the interstimulus interval. The slope of the curve relating the strength to the interstimulus interval indicates the sensitivity of phasic block to changes in stimulation rate. It depends on the molecular structure of the drug and can be also affected by the resting potential. With increasingly shorter interstimulation intervals, phasic inhibition became more marked and developed faster. However, when the development kinetics are considered as an event-dependent process, the fraction of complete phasic block that is installed per single excitation decreases.
2. Each type of drug-induced \(\dot V_{\max } \) block possesses its own individual apparent dissociation constant and apparentn _H coefficient. In the case of propafenone, for instance, the apparentK _m's for tonic and phasic inhibition (at a frequency of 120/min) were 4.4×10^?5 mol/l and 1×10^?5 mol/l, respectively, and the apparentn _H's amounted to 1.76 and 0.84, respectively. The apparentK _m for the phasic block depends on the interstimulus interval and decreases with an increase in stimulation rate. The stoichiometry of the underlying drugreceptor interaction, however, remains virtually unaffected since the apparentn _H of the phasic \(\dot V_{\max } \) block proved insensitive to frequency changes.
3. Propafenone wash-out experiments revealed that tonic and phasic \(\dot V_{\max } \) block disappeared with individual time constants differing by a factor of 3–5 from each other.
4. TTX (5×10^?6 mol/l) enhanced the propafenone action and intensified tonic and phasic \(\dot V_{\max } \) blockade. Each block exhibited an individual quantitative response. Tonic blockade became stronger by a factor of 3.1±0.72, but phasic block by a factor of 1.9±0.1.
5. Chemical channel modification induced by formaldehyde increased the inhibitory efficacy of propafenone. Again, tonic \(\dot V_{\max } \) blockade responded more sensitive than phasic blockade and the former rose by a factor of 6.9 but the latter by a factor of only 1.5.
6. These results can be satisfactorily accounted for in terms of the modulated single receptor hypothesis (HHK model).

     

An Inhibitory Role for Sema4A in Antigen-Specific Allergic Asthma

Purpose

The class IV semaphorin Sema4A is critical for efficient T_H1 differentiation and Sema4a ^?/? mice exhibit impaired T_H1 immune responses. However, the role of Sema4A in T_H2 cell-mediated allergic diseases has not been fully studied. The aim of this study was to clarify the regulatory role possessed by Sema4A in mouse models of allergic diseases, particularly allergic asthma.

Methods

Sema4a ^?/? mice on a BALB/c background were examined for the development of allergic diseases. To induce experimental asthma, mice were sensitized with ovalbumin (OVA) followed by intranasal challenges with OVA. After challenge, airway hyperreactivity (AHR) and airway inflammation were evaluated. The role of Sema4A in asthma was examined using Sema4a ^?/? mice and Sema4A-Fc fusion proteins. The direct effects of Sema4A-Fc on antigen-specific effector CD4⁺ T cells were also examined.

Results

A fraction of Sema4a ^?/? BALB/c mice spontaneously developed skin lesions that resembled atopic dermatitis (AD) in humans. Furthermore, AHR, airway inflammation, and T_H2-type immune responses were enhanced in Sema4a ^?/? mice compared to wild type (WT) mice when immunized and challenged with OVA. In vivo systemic administration of Sema4A-Fc during the challenge period ameliorated AHR and lung inflammation and reduced the production of T_H2-type cytokines in WT mice. The inhibitory effects of Sema4A on airway inflammation were also observed in mice deficient in Tim-2, a Sema4A receptor. Finally, we showed that Sema4A-Fc directly inhibited IL-4-producing OVA-specific CD4⁺ T cells.

Conclusion

These results demonstrate that Sema4A plays an inhibitory role in T_H2-type allergic diseases, such as allergic asthma.     

A comparison of two methods for estimating odds ratios: Results from the National Health Survey

Background

The practice of dichotomizing a continuous outcome variable does not make use of within-category information. That means the loss of information. This study compared two approaches in the modelling of the association between sociodemographic and smoking with obesity in adult women in Iran.

Methods

We conducted a comparative study between two methods via an illustrative example, using data from the "National Health Survey in Iran (NHSI)" database. It included 14176 women aged 20–69 years. At first, body mass index(BMI) was treated as a continuous variable, OR_s and 95 per cent confidence intervals were calculated using the "without dichotomizing" method. Then subjects were classified into obese (BMI ≥ 30 kg/m²) and nonobese (BMI < 30 kg/m²) and logistic regression model was used to estimate OR_s and 95 per cent confidence intervals.

Results

The odds ratio estimates changed only slightly over the two methods. But the "without dichotomizing" method provided shorter confidence intervals on the odds ratio parameters than dichotomizing method. All relative confidence interval lengths were greater than 1.15.

Conclusion

If responses are continuous then the "without dichotomizing" method is certainly more useful than the "dichotomizing" method and leads to more precise estimation of odds ratios.     

Non-invasive imaging of atherosclerotic plaque macrophage in a rabbit model with F-18 FDG PET: a histopathological correlation

Background

Increased ^99mTc-sestamibi stress lung-to-heart ratio (sLHR) has been shown to predict cardiac outcomes similar to pulmonary uptake of thallium. Peak heart rate and use of pharmacologic stress affect the interpretation of lung thallium uptake. The current study was performed to determine whether ^99mTc-sestamibi sLHR measurements are affected by stress-related variables, and whether this in turn affects prognostic utility.

Methods

sLHR was determined in 718 patients undergoing ^99mTc-sestamibi SPECT stress imaging. sLHR was assessed in relation to demographics, hemodynamic variables and outcomes (mean follow up 5.6 ± 1.1 years).

Results

Mean sLHR was slightly greater in males than in females (P < 0.01) and also showed a weak negative correlation with age (P < 0.01) and systolic blood pressure (P < 0.01), but was unrelated to stress method or heart rate at the time of injection. In patients undergoing treadmill exercise, sLHR was also positively correlated with peak workload (P < 0.05) but inversely with double product (P < 0.05). The combined explanatory effect of sex, age and hemodynamic variables on sLHR was less than 10%. The risk of acute myocardial infarction (AMI) or death increased by a factor of 1.7–1.8 for each SD increase in unadjusted sLHR, and was unaffected by adjustment for sex, age and hemodynamic variables (hazard ratios 1.6–1.7). The area under the ROC curve for the unadjusted sLHR was 0.65 (95% CI 0.59–0.71, P < 0.0001) and was unchanged for the adjusted sLHR (0.65, 95% CI 0.61–0.72, P < 0.0001).

Conclusion

Stress-related variables have only a weak effect on measured sLHR. Unadjusted and adjusted sLHR provide equivalent prognostic information for prediction of AMI or death.     

Stimulus-secretion coupling in pancreatic acinar cells: influences of external sodium and calcium on responses to cholecystokinin-pancreozymin and ionophore A23187

1. The roles of Na and Ca ions in stimulus-secretion coupling were analysed in the isolated and perfused rat pancreas.2. Partial replacement of NaCl with LiCl produced a diminution in both amylase output and pancreatic juice flow which were induced by 5 m-u. CCK-PZ/ml., and almost normal responses were usually regained immediately after the reintroduction of a standard concentration of NaCl. Nearly total replacement of NaCl with LiCl caused an almost complete inhibition of the responses, although 25 mM-NaHCO₃ and 1 mM-NaH₂PO₄ were still present, and only partial recovery was obtained after the re-introduction of a standard concentration of NaCl.3. A quantitative relationship was found between the amount of amylase released by CCK-PZ and the [Na⁺]_o over the range 26-157 mM in the presence of 2·5 mM-Ca. A similar relationship was also observed when [Ca²⁺]_o was decreased to 1·0 mM, but the responses were reduced to about one half of those observed with 2·5 mM-Ca.4. The most satisfactory theory which explains the cellular mechanism of CCK-PZ-induced amylase output, and which fits the experimental data, requires the dominant activity of a complex composed of a carrier molecule bearing one Ca and four Na molecules, if there is no interaction between Na⁺ and Li⁺.5. A quantitative relationship was also found between the amount of pancreatic juice flow stimulated by CCK-PZ and [Na⁺]_o, over the range 26-157 mM, in the presence of 1·0 or 2·5 mM-Ca.6. A similar quantitative relationship was found between the amount of amylase released by Ca-ionophore A23187 and [Na⁺]_o in the presence of 2·5 or 5·0 mM-Ca. The most satisfactory theory which fits this experimental data also requires the dominant activity of a complex composed of a carrier molecule bearing one Ca and four Na molecules, if there is no interaction between Na⁺ and Li⁺.     

Critical role of interleukin-5 in the development of a mite antigen-induced chronic bronchial asthma model

Objective and design

Asthma is associated with eosinophilic airway inflammation and characterized by enhanced airway sensitivity. Interleukin (IL)-5 plays an important role in the pathogenesis of asthma. The involvement of IL-5 receptor-mediated cellular signals in the pathogenesis of a mite antigen-induced chronic asthma model was investigated.

Subjects

In this study, 48 female C57BL/6J (WT) mice and IL-5 receptor-deficient (IL-5RKO) mice were used.

Treatment

Mite antigen (50 μl) was intranasally administered 13 times to WT and IL-5RKO mice.

Methods

Airway hypersensitivity (Mch PC₂₀₀) and specific antigen exposure tests were performed, and lung tissue, bronchoalveolar lavage fluid (BALF), and blood were collected to investigate the asthma pathology and differences in the local pulmonary levels of cytokines and chemokines.

Results

Airway sensitivity was enhanced and antigen-specific airway resistance was increased in WT mice. In addition, the number of eosinophils and Th2 cytokine levels in the BALF were increased. In contrast, IL-5RKO mice did not acquire the asthma pathology, such as antigen-specific airway resistance and eosinophilic airway inflammation. Mch PC₂₀₀ was significantly correlated with cysteinyl leukotriene levels in WT mice.

Conclusion

These findings suggested that both IL-5 induced eosinophils and cysteinyl leukotrienes are involved in the pathology of this mite antigen-induced chronic asthma model.     

Urinary trypsin inhibitor suppresses excessive generation of superoxide anion radical, systemic inflammation, oxidative stress, and endothelial injury in endotoxemic rats

Objective and design

The protective effects of ulinastatin, a human urinary trypsin inhibitor (UTI), against superoxide radical (O ₂ ^?· ) generation, systemic inflammation, lipid peroxidation, and endothelial injury were investigated in endotoxemic rats.

Materials and treatment

Twenty-one Wistar rats were allocated to a control group, a UTI group, and a sham group. A bolus of lipopolysaccharide (LPS; 3 μg/g) was administered intravenously to the control group, a bolus of LPS and UTI (5 U/g) to the UTI group, and a bolus of saline to the sham group.

Methods

The O ₂ ^?· generated was measured as the current in the right atrium using an electrochemical O ₂ ^?· sensor. Plasma nitrite, high mobility group box 1 (HMGB1), tumor necrosis factor (TNF)-α, inteleukin (IL)-6, malondialdehyde, and soluble intercellular adhesion molecule-1 (sICAM-1) were measured 360 min after LPS administration.

Results

The O ₂ ^?· current increased in the control group and was significantly attenuated in the UTI group after 55 min (P < 0.05 at 55–60 min, P < 0.01 at 65–360 min). Plasma nitrite, HMGB1, TNF-α, IL-6, malondialdehyde, and sICAM-1 were attenuated in the UTI group.

Conclusions

UTI suppressed excessive O ₂ ^?· generation, systemic inflammation, lipid peroxidation, and endothelial injury in endotoxemic rats.     

Bone regeneration: current concepts and future directions

Background

Colon cancer is a public health problem worldwide. Adjuvant chemotherapy after surgical resection for stage III colon cancer has been shown to improve both progression-free and overall survival, and is currently recommended as standard therapy. However, its value for patients with stage II disease remains controversial. When this study was designed 5-fluorouracil (5FU) plus leucovorin (LV) was standard adjuvant treatment for colon cancer. Irinotecan (CPT-11) is a topoisomerase I inhibitor with activity in metastatic disease. In this multicenter adjuvant phase III trial, we evaluated the addition of irinotecan to weekly 5FU plus LV in patients with stage II or III colon cancer.

Methods

The study included 873 eligible patients. The treatment consisted of weekly administration of irinotecan 80 mg/m² intravenously (IV), LV 200 mg/m² and 5FU 450 mg/m² bolus (Arm A) versus LV 200 mg/m² and 5FU 500 mg/m² IV bolus (Arm B). In Arm A, treatments were administered weekly for four consecutive weeks, followed by a two-week rest, for a total of six cycles, while in Arm B treatments were administered weekly for six consecutive weeks, followed by a two-week rest, for a total of four cycles. The primary end-point was disease-free survival (DFS) at three years.

Results

The probability of overall survival (OS) at three years was 0.88 for patients in Arm A and 0.86 for those in Arm B, while the five-year OS probability was 0.78 and 0.76 for patients in Arm A and Arm B, respectively (P = 0.436). Furthermore, the probability of DFS at three years was 0.78 and 0.76 for patients in Arm A and Arm B, respectively (P = 0.334). With the exception of leucopenia and neutropenia, which were higher in patients in Arm A, there were no significant differences in Grades 3 and 4 toxicities between the two regimens. The most frequently recorded Grade 3/4 toxicity was diarrhea in both treatment arms.

Conclusions

Irinotecan added to weekly bolus 5FU plus LV did not result in improvement in disease-free or overall survival in stage II or III colon cancer, but did increase toxicity.

Trial registration

Australian New Zealand Clinical Trials Registry: ACTRN12610000148077     

Heterogeneity of external surface charges near sodium channels in the nodal membrane of frog nerve

1. The conductance γ and the numberN _o of Na channels in the nodal membrane of frog nerve fibres were determined from ensemble average values of the Na current and the variance of Na current fluctuations.
2. Replacement of extracellular Cl^? by NO ₃ ^? shifts the voltage dependencies of all Na gating parameters towards more negative voltages, reduces γ by a factor of 0.84 and hardly changes the numberN _o of channels not blocked by 8 nM TTX.
3. Adding 0.1 mM LaCl₃ to the extracellular solution shifts the voltage dependencies of all Na gating parameters towards more positive voltages, reduces γ by a factor of 0.75 and hardly changes the numberN _o of channels not blocked by 8 nM TTX.
4. It is concluded that changes of the external surface potential induced by Cl^?, NO ₃ ^? replacement do not alter the local Na⁺ concentration in the outer mouth of the Na channel and hardly affect the TTX binding to toxin receptors.
5. Surface potential changes by addition of LaCl₃ also have no clear effect on TTX binding. The reduction of γ in 0.1 mM LaCl₃ is probably due to a direct interaction of La³⁺ with Na channels.
6. Our results suggest a heterogeneous distribution of external fixed surface charges in the outer mouth of the Na channel, at the TTX binding site and near the Na channel gates.

     

Radiolabeled nano-peptides show specificity for an animal model of human PC3 prostate cancer cells

OBJECTIVES:

Cancer has been investigated using various pre‐targeting techniques or models focusing on radiobombesin analogues; however, both are not offered together. In this study, nano‐bombesin labeling by a pre‐targeting system was undertaken to develop an alternative approach for prostate tumor treatment.

METHODS:

A two‐step pre‐targeting system utilizing a combination of streptavidin (SA), biotinylated morpholino (B‐MORF), biotinylated BBN (B‐BBN) with two different spacers (β‐Ala and PEG), and a radiolabeled cMORF was evaluated in vitro and in vivo.

RESULTS:

Final conjugation conditions consisted of a 1:1:2 ratio of SA:B‐MORF:B‐BBN, followed by addition of ^99mTc‐cMORF to compensate for free MORF. In vitro binding experiments with prostate cancer cells (PC‐3) revealed that total binding was time‐dependent for the Ala spacer but not for the PEG spacer. The highest accumulation (5.06±1.98 %) was achieved with 1 hour of incubation, decreasing as time progressed. Specific binding fell to 1.05±0.35 %. The pre‐targeting biodistribution in healthy Swiss mice was measured at different time points, with the best responses observed for 7‐h and 15‐h incubations. The effector, ^99mTc‐MAG3‐cMORF, was administered 2 h later. Strong kidney excretion was always documented. The greatest tumor uptake was 2.58±0.59 %ID/g at 7 h for B‐βAla‐BBN, with a region of interest (ROI) value of 3.9 % during imaging. The tumor/blood ratio was low due to the slow blood clearance; however, the tumor/muscle ratio was 5.95.

CONCLUSIONS:

The pre‐targeting approach with a peptide was a viable concept. Further evaluation with modified sequences of MORF, including less cytosine, and additional test intervals could be worthwhile.