Costs of non-small cell lung cancer in the Netherlands.

OBJECTIVES: Real-world resource use and cost data on non-small cell lung cancer (NSCLC) are scarce. This data is needed to inform health-economic modelling to assess the impact of new diagnostic and/or treatment technologies. This study provides detailed insight into real-world medical resource use and costs of stage I-IV NSCLC in the Netherlands. MATERIALS AND METHODS: A random sample of patients newly diagnosed with NSCLC (2009-2011) was selected from four Dutch hospitals. Data was retrospectively collected from patient charts. This data included patient characteristics, tumour characteristics, treatment details, adverse events, survival and resource use. Resource use was multiplied by Dutch unit costs expressed in EUR 2012. Total mean costs were corrected for censoring using the Bang and Tsiatis weighted complete-case estimator. Furthermore, costs of adverse events, costs per phase of NSCLC management and costs of second opinions are presented. RESULTS: Data was collected on 1067 patients. Total mean costs for NSCLC diagnosis, treatment and follow-up are €28,468 during the study period and €33,143 when corrected for censoring. Adverse events were recorded in the patient charts for 369 patients (41%) and 82 patients (9%) experienced an adverse event of grade III or higher. For these patients, adverse event-related hospital admissions cost on average €2,091. Mean total costs are €1,725 for the diagnostic period, €17,296 for first treatment line, and €13,236 for each later treatment line. Costs of providing a second opinion are €2,580 per patient. CONCLUSIONS: Total mean hospital costs per NSCLC patient are €33,143 for the total duration of the disease. Ignoring censoring in our data underestimates these costs by 14%. Main limitations of the study relate to the short follow-up time, staging difficulties and missing data. Its main strength is that it provides highly detailed, real-world data on the costs of NSCLC.

DNA hypermethylation analysis in sputum for the diagnosis of lung cancer: training validation set approach.

BACKGROUND: Lung cancer has the highest mortality of all cancers. The aim of this study was to examine DNA hypermethylation in sputum and validate its diagnostic accuracy for lung cancer. METHODS: DNA hypermethylation of RASSF1A, APC, cytoglobin, 3OST2, PRDM14, FAM19A4 and PHACTR3 was analysed in sputum samples from symptomatic lung cancer patients and controls (learning set: 73 cases, 86 controls; validation set: 159 cases, 154 controls) by quantitative methylation-specific PCR. Three statistical models were used: (i) cutoff based on Youden's J index, (ii) cutoff based on fixed specificity per marker of 96% and (iii) risk classification of post-test probabilities. RESULTS: In the learning set, approach (i) showed that RASSF1A was best able to distinguish cases from controls (sensitivity 42.5%, specificity 96.5%). RASSF1A, 3OST2 and PRDM14 combined demonstrated a sensitivity of 82.2% with a specificity of 66.3%. Approach (ii) yielded a combination rule of RASSF1A, 3OST2 and PHACTR3 (sensitivity 67.1%, specificity 89.5%). The risk model (approach iii) distributed the cases over all risk categories. All methods displayed similar and consistent results in the validation set. CONCLUSIONS: Our findings underscore the impact of DNA methylation markers in symptomatic lung cancer diagnosis. RASSF1A is validated as diagnostic marker in lung cancer.

Solute transport in intervertebral disc: experiments and finite element modeling.

Loss of nutrient supply to the human intervertebral disc (IVD) cells is thought to be a major cause of disc degeneration in humans. To address this issue, transport of molecules of different size have been analyzed by a combination of experimental and modeling studies. Solute transport has been compared for steady-state and transient diffusion of several different solutes with molecular masses in the range 3-70 kDa, injected into parts of the disc where degeneration is thought most likely to occur first and into the blood supply to the disc. Diffusion coefficients of fluorescently tagged dextran molecules of different molecular weights have been measured in vitro using the concentration gradient technique in thin specimens of disc outer annulus and nucleus pulposus. Diffusion coefficients were found to decrease with molecular weight following a nonlinear relationship. Diffusion coefficients changed more rapidly for solutes with molecular masses less than 10 kDa. Although unrealistic or painful, solutes injected directly into the disc achieve the largest disc coverage with concentrations that would be high enough to be of practical use. Although more practical, solutes injected into the blood supply do not penetrate to the central regions of the disc and their concentrations dissipate more rapidly. Injection into the disc would be the best method to get drugs or growth factors to regions of degeneration in IVDs quickly; else concentrations of solute must be kept at a high value for several hours in the blood supply to the discs.

Sexual dysfunction in psychiatric inpatients the role of antipsychotic medication.

INTRODUCTION: Sexual dysfunction is a common side effect of antipsychotic medication. Although increased prolactin levels caused by antipsychotic agents are believed to play a major role with regard to sexual side effects, the underlying mechanism of antipsychotic agent-induced sexual dysfunction remains poorly understood. METHODS: In a multicentric study 587 psychiatric inpatients were assessed by means of a self-rating sexual questionnaire. Focussing on antipsychotic treatment three subgroups were drawn from the original sample. One group was treated with prolactin-increasing antipsychotics (n=119), the other with prolactin-neutral medication (n=109) and the third patient group was comprised of non-medicated clinical controls (n=105). RESULTS: The majority of all patients (50-75%) reported at least minor sexual dysfunction. On comparison of the subgroups, only female patients treated with prolactin-increasing medication reported more severe sexual dysfunction. However, multiple regression analysis did not confirm an association between the type of treatment and sexual impairment. DISCUSSION: Sexual dysfunction frequently occurs in psychiatric inpatients treated with antipsychotics. Our findings only partly support the assumptions concerning a major role of prolactin-increasing neuroleptics for medication-induced sexual impairment.

Sexual impairment in psychiatric inpatients: focus on depression.

INTRODUCTION: Although sexual side effects are a common reason for noncompliance with medication, information on impairment of sexuality in psychiatric inpatients is scarce. METHODS: In the present multi-center study, data on several aspects of sexual functioning were collected in psychiatric inpatients using a previously validated questionnaire. RESULTS: A high overall prevalence of sexual dysfunction was reported by participants and was highest in depressed subjects. Patients receiving antidepressants suffered from more frequent and more severe impairment of sexuality than did subjects receiving neither antidepressants nor antipsychotics or opioids. DISCUSSION: Judging from this data, sexual impairment appears to be a frequent and underestimated problem in psychiatric inpatients with a high prevalence across all diagnostic groups, particularly in depressed subjects. Female patients attribute this impairment mainly to their mental illness, whereas male patients tend to assign their impairments primarily to their medication.

Analysis of calcium channels by conditional mutagenesis.

Ca2+ influx through various ion channels is an important determinant of the cytosolic Ca2+ concentration, which plays a pivotal role in countless cellular processes. The cardiac L-type Ca2+ channel, Ca(v)1.2, represents a major pathway for Ca2+ entry and is in many cells expressed together with other high- and low-voltage-activated Ca2+ channels. This article will focus on the use of conditional transgenic mouse models to clarify the roles of Ca2+ channels in several biological systems. The phenotypes of conditional Ca2+ channel transgenic mice have provided novel, and often unexpected, insights into the in vivo function of L-type and T-type Ca2+ channels as mediators of signaling between cell membrane and intracellular processes in blood pressure regulation, smooth muscle contractility, insulin secretion, cardiac function, sleep, learning, and memory.

Inhibition of L-type Cav1.2 Ca2+ channels by 2,(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) and 2-[1-(3-dimethyl-aminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl) maleimide (Go6983).

Phosphatidylinositol 3-kinase (PI3-K) is involved in physiological processes of cellular proliferation and inflammation and, as postulated recently, in the regulation of L-type Ca(2+) channels. The latter conclusion arose in part from the inhibitory action of the compound 2,(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), which has been established as a selective PI3-K inhibitor (IC(50) = 1.4 microM). Herein we show, however, that LY294002 and an inhibitor of protein kinase C (PKC), 2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl) maleimide (Go6983), act as direct Ca(2+)-channel inhibitors, with IC(50) values of approximately 20 and 10 microM, respectively. Because both drugs are commonly used at concentrations of approximately 10 microM or higher, the interpretation of such experiments is questionable with respect to a regulatory action of PI3-K or PKC on L-type Ca(2+) channels.

Functional embryonic cardiomyocytes after disruption of the L-type alpha1C (Cav1.2) calcium channel gene in the mouse.

The L-type alpha(1C) (Ca(v)1.2) calcium channel is the major calcium entry pathway in cardiac and smooth muscle. We inactivated the Ca(v)1.2 gene in two independent mouse lines that had indistinguishable phenotypes. Homozygous knockout embryos (Ca(v)1. 2-/-) died before day 14.5 postcoitum (p.c.). At day 12.5 p.c., the embryonic heart contracted with identical frequency in wild type (+/+), heterozygous (+/-), and homozygous (-/-) Ca(v)1.2 embryos. Beating of isolated embryonic cardiomyocytes depended on extracellular calcium and was blocked by 1 microm nisoldipine. In (+/+), (+/-), and (-/-) cardiomyocytes, an L-type Ba(2+) inward current (I(Ba)) was present that was stimulated by Bay K 8644 in all genotypes. At a holding potential of -80 mV, nisoldipine blocked I(Ba) of day 12.5 p.c. (+/+) and (+/-) cells with two IC(50) values of approximately 0.1 and approximately 1 microm. Inhibition of I(Ba) of (-/-) cardiomyocytes was monophasic with an IC(50) of approximately 1 microm. The low affinity I(Ba) was also present in cardiomyocytes of homozygous alpha(1D) (Ca(v)1.3) knockout embryos at day 12.5 p.c. These results indicate that, up to day 14 p.c., contraction of murine embryonic hearts requires an unidentified, low affinity L-type like calcium channel.

Alternatively spliced IS6 segments of the alpha 1C gene determine the tissue-specific dihydropyridine sensitivity of cardiac and vascular smooth muscle L-type Ca2+ channels.

Dihydropyridines (DHPs) block the vascular smooth muscle L-type Ca2+ channel at lower concentrations than the cardiac Ca2+ channel, although their alpha 1 subunit, which binds the DHPs, is derived from the same gene. This alpha 1C gene gives rise to several splice variants, among which the alpha 1C-b variant is affected by lower concentrations of nisoldipine than the alpha 1C-a variant. Functional expression of chimeras of alpha 1C-a and alpha 1C-b subunits demonstrated that the transmembrane segment IS6 is responsible for the different dihydropyridine sensitivity. Northern blot analysis showed that transcripts coding for the IS6 segment of the alpha 1C-a subunit were expressed in heart but not in aorta, whereas the IS6 segment of the alpha 1C-b subunit was expressed predominantly in vascular smooth muscle. In situ hybridization of rat heart sections confirmed this expression pattern of IS6 alpha 1C-a and IS6 alpha 1C-b in ventricular and smooth muscle myocytes, respectively. These results suggest that the different dihydropyridine sensitivities of cardiac and vascular L-type Ca2+ channels are caused at least partially by the tissue-specific expression of alternatively spliced IS6 segments of the alpha 1C gene.

Modified plant plasma membrane H(+)-ATPase with improved transport coupling efficiency identified by mutant selection in yeast.

Transport across the plasma membrane is driven by an electrochemical gradient of H+ ions generated by the plasma membrane proton pump (H(+)-ATPase). Random mutants of Arabidopsis H(+)-ATPase AHA1 were isolated by phenotypic selection of growth of transformed yeast cells in the absence of endogenous yeast H(+)-ATPase (PMA1). A Trp-874-Leu substitution as well as a Trp-874 to Lys-935 deletion in the hydrophilic C-terminal domain of AHA1 conferred growth of yeast cells devoid of PMA1. A Trp-874-Phe substitution in AHA1 was produced gy site-directed mutagenesis. The modified enzymes hydrolyzed ATP at 200-500% of wild-type level, had a sixfold increase in affinity for ATP (from 1.2 to 0.2 mM; pH 7.0), and had the acidic pH optimum shifted towards neutral pH. AHA1 did not contribute significantly to H+ extrusion by transformed yeast cells. The different specifies of aha1, however, displayed marked differences in initial rates of net H+ extrusion and in their ability to sustain an electrochemical H+ gradient. These results provide evidence that Trp-874 plays an important role in auto-inhibition of the plant H(+)-ATPase and may be involved in controlling the degree of coupling between ATP hydrolysis and H+ pumping. Finally, these results demonstrate the usefulness of yeast as a generalized screening tool for isolating regulatory mutants of plant transporters.

Two stable cell lines for screening of calcium channel blockers.

Stable cell lines are potentially excellent tools for large-scale screening of new compounds. Two carboxyterminal-deleted constructs of the two splice variants a and b of the calcium channel class C alpha 1 subunit were expressed stably in HEK 293 cells. Each cell line produced regular L-type calcium currents. The opening and closing of the calcium channel elicited by potassium depolarization was followed by Fura-2 transients. These transients were blocked by the calcium channel blocker mibefradil with a concentration for 50% inhibition of 1.7 microM. The cell lines expressing the truncated cardiac alpha 1C-a or smooth muscle alpha 1C-b calcium channel were both blocked by nisoldipine under patch clamp conditions. Nisoldipine interacted with higher affinity with the alpha 1C-b channel than with the alpha 1C-a channel. These results indicate that the two cell lines retain the differential dihydropyridine sensitivity of smooth muscle and cardiac calcium channels and may be potential tools for the screening of L-type calcium channel blockers.

Interaction of Ro 40-5967 and verapamil with the stably expressed alpha 1-subunit of the cardiac L-type calcium channel.

The interaction of the nondihydropyridine calcium channel antagonist Ro 40-5967 with the stably expressed class C alpha 1-subunit of the cardiac L-type calcium channel was investigated and compared with that of verapamil by using the whole cell patch clamp configuration. Both compounds blocked the Ba++ inward current. The IC50 values at a holding potential of -80 or -40 mV were 4.9 and 1.4 microM for Ro 40-5967 and 250 and 15.5 microM for verapamil. Both Ro 40-5967 and verapamil induced a partial tonic block at a holding potential of -80 mV. The block increased with high depolarization rates. Both Ro 40-5967 and verapamil shifted the steady-state inactivation curve by more than 20 mV to hyperpolarized membrane potentials and decreased the inactivation rate constant. The effect of Ro 40-5967, but not that of verapamil, was attenuated by intracellular dialysis with GTP gamma S. The affinity for verapamil was not affected by replacing Ba++ by Ca++, but was increased by the coexpression of the beta 3-subunit. These results indicate that both compounds interact with high affinity with the inactivated channel state, but may interact additionally with the open channel.

On the regulation of the expressed L-type calcium channel by cAMP-dependent phosphorylation.

The Ca2+ channel subunits alpha 1C-a and alpha 1C-b were stably expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cells. The peak Ba2+ current (IBa) of these cells was not affected significantly by internal dialysis with 0.1 mM cAMP-dependent protein kinase inhibitor peptide (mPKI), 25 microM cAMP-dependent protein kinase catalytic subunit (PKA), or a combination of 25 microM PKA and 1 microM okadaic acid. The activity of the alpha 1C-b channel subunit expressed stably in HEK 293 cells was depressed by 1 microM H 89 and was not increased by superfusion with 5 microM forskolin plus 20 microM isobutyl-methylxanthine (IBMX). The alpha 1C-a.beta 2.alpha 2/delta complex was transiently expressed in HEK 293 cells; it was inhibited by internal dialysis of the cells with 1 microM H 89, but was not affected by internal dialysis with mPKI, PKA or microcystin. Internal dialysis of cells expressing the alpha 1C-a.beta 2.alpha 2/delta channel with 10 microM PKA did not induce facilitation after a 150-ms prepulse to +50 mV. The Ca2+ current (ICa) of cardiac myocytes increased threefold during internal dialysis with 5 microM PKA or 25 microM microcystin and during external superfusion with 0.1 microM isoproterenol or 5 microM forskolin plus 50 microM IBMX. These results indicate that the L-type Ca2+ channel expressed is not modulated by cAMP-dependent phosphorylation to the same extent as in native cardiac myocytes.

Expression of the L-type calcium channel with two different beta subunits and its modulation by Ro 40-5967.

The smooth muscle alpha 1Cb subunit of the L-type calcium channel was expressed alone (CHO alpha 1 cell) or together with the skeletal beta 1 (CHO alpha 1 beta 1 cell) subunit or smooth muscle beta 3 (CHO alpha 1 beta 3 cell) subunit in Chinese hamster ovary (CHO) cells. The interaction of the expressed calcium channel with the non-dihydropyridine calcium channel blocker Ro 40-5967 was studied. Ro 40-5967 decreased isradipine binding by an apparent allosteric interaction and blocked the barium inward currents (IBa) in a voltage- and use-dependent manner in all cells. The steady-state inactivation curves were shifted to hyperpolarizing potentials in the presence of Ro 40-5967. The rate of channel inactivation was increased in CHO alpha 1 and CHO alpha 1 beta 3 cells. The shift in the steady-state inactivation curve and the increase in channel inactivation were less pronounced in CHO alpha 1 beta 1 cells than in the other cell lines. Low concentrations of Ro 40-5967 increased IBa by up to 198% in 33% of the CHO alpha 1 beta 1 cells. In addition, higher concentrations of Ro 40-5967 were required to inhibit IBa in 60% of the CHO alpha 1 beta 3 cells. These results suggest that the beta subunits modify the interaction of the non-dihydropyridine Ro 40-5967 with the expressed calcium channel alpha 1 subunit.

Stable co-expression of calcium channel alpha 1, beta and alpha 2/delta subunits in a somatic cell line.

1. The high-voltage-activated L-type calcium channel is a multi-protein complex of alpha 1, alpha 2/delta, beta and gamma subunits. The alpha 1 subunit contains the voltage-dependent calcium-conducting pore. Chinese hamster ovary (CHO) cells were stably transfected with the complementary DNA of the alpha 1, beta and alpha 2/delta subunits. These subunits were not detected in wild-type CHO cells. 2. The alpha 1 (CaCh2b) subunit itself directed the expression of functional calcium channels which bound calcium channel blockers and showed voltage-dependent activation and inactivation. 3. The co-expression of the alpha 1 subunit with the beta subunit (CaB1 gene) enhanced the density of the dihydropyridine binding sites 2- to 3-fold and increased dihydropyridine-sensitive barium inward currents (IBa) up to 3.5-fold from -13.3 microA/cm2 (alpha 1 subunit) to -46.7 microA/cm2 (alpha 1 and beta subunits). 4. Co-expression of the beta subunit did not change the sensitivity of IBa towards dihydropyridines, but accelerated current activation and inactivation and shifted the half-maximal steady-state activation and inactivation to slightly more hyperpolarizing potentials. 5. The co-expression of the alpha 2/delta subunit together with alpha 1 and beta subunits accelerated the inactivation kinetics of the channel without a major effect on the other parameters. 6. These results indicate that the beta and alpha 2/delta subunit interact with the alpha 1 subunit and modulate thereby the properties of the alpha 1 subunit-dependent inward current.

Subunit-dependent modulation of recombinant L-type calcium channels. Molecular basis for dihydropyridine tissue selectivity.

At least four calcium channel subtypes (P, T, N, and L) have now been classified on the basis of their biophysical and/or pharmacological properties. L-type channels, a channel family particularly important to physiological function of the cardiovascular system, are identified by their slow voltage- and calcium-dependent inactivation as well as their sensitivity to dihydropyridine (DHP) calcium channel antagonists. In this study, we report the results of experiments in which we have measured the DHP modulation of recombinant calcium channel activity in cells transfected with alpha 1 subunits of cardiac and smooth muscle L-type calcium channels. We find subunit-dependent differences in the voltage and concentration dependence of channel modulation. Our results provide evidence for a molecular basis for DHP sensitivity of heart and smooth muscle calcium channels and, additionally, indicate that, even within one family of calcium channels, slight differences in channel structure can cause marked differences in channel pharmacology.

Stable and functional expression of the calcium channel alpha 1 subunit from smooth muscle in somatic cell lines.

Voltage-activated calcium channels are membrane spanning proteins that allow the controlled entry of Ca2+ into the cytoplasm of cells. The principal channel forming subunit of an L-type calcium channel is the alpha 1 subunit. Transfection of Chinese hamster ovary (CHO) cells with complementary DNA encoding the calcium channel alpha 1 subunit from smooth muscle led to the expression of functional calcium channels which bind calcium channel blockers and show the voltage-dependent activation and slow inactivation and unitary current conductance characteristic of calcium channels in smooth muscle. The currents mediated by these channels are sensitive towards dihydropyridine-type blockers and agonists indicating that the calcium channel blocker receptor sites were present in functional form. The smooth muscle alpha 1 subunit cDNA alone is sufficient for stable expression of functional calcium channels with the expected kinetic and pharmacological properties in mammalian somatic cells.

Hormonal regulation of calcium current in freshly isolated airway smooth muscle cells.

Single freshly isolated smooth muscle cells of adult bovine trachea were voltage clamped, and the calcium inward current was separated from K+ currents by blocking the large outward currents with intra- and extracellular Cs+ and extracellular tetraethylammonium chloride. Isoproterenol stimulated peak calcium current (ICa) in a dose-dependent manner through the beta-adrenergic receptor. The isoproterenol effect was not mediated or caused by the stimulation of a K+ or Na+ current, a decrease in the intracellular concentrations of Ca2+ or H+, the stimulation of the Na(+)-H+ or the Na(+)-Ca2+ exchanger. Neither basal nor isoproterenol-stimulated ICa was affected by internal dialysis of the cell with adenosine 3',5'-cyclic monophosphate (cAMP), cAMP analogues, or the catalytic subunit of cAMP-kinase. Internal dialysis of the cells with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) blocked the stimulation of isoproterenol whereas dialysis with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) induced an isoproterenol-like maximal increase of ICa. These results show that the beta-adrenergic receptor stimulates the L-type calcium current of isolated tracheal smooth muscle cells independent of cAMP and cAMP-kinase through a GTP/GDP regulated protein.

Cyclic GMP-dependent protein kinase and smooth muscle relaxation.

Cyclic guanosine monophosphate (cGMP)-dependent protein kinase has been cloned from bovine trachea. The isozymes I alpha and I beta, which differ only in their amino-terminal domains were expressed transiently in COS-7 cells. Both isozymes were activated by cGMP and cyclic adenosine monophosphate (cAMP). However, approximately 10-fold higher concentrations of cyclic nucleotides were needed to activate the I beta enzyme than the I alpha enzyme. The KA values for cAMP were 9.1 and greater than 20 microM for the I alpha and I beta isozymes, respectively. It is therefore unlikely that an unmodified I beta enzyme that occurs in high concentrations in vascular smooth muscle can be activated in vivo by cAMP.