IL-12 Can Target Human Lung Adenocarcinoma Cells and Normal Bronchial Epithelial Cells Surrounding Tumor Lesions

BACKGROUND: Non small cell lung cancer (NSCLC) is a leading cause of cancer death. We have shown previously that IL-12rb2 KO mice develop spontaneously lung adenocarcinomas or bronchioalveolar carcinomas. Aim of the study was to investigate i) IL-12Rbeta2 expression in human primary lung adenocarcinomas and in their counterparts, i.e. normal bronchial epithelial cells (NBEC), ii) the direct anti-tumor activity of IL-12 on lung adenocarcinoma cells in vitro and vivo, and the mechanisms involved, and iii) IL-12 activity on NBEC. METHODOLOGY/PRINCIPAL FINDINGS: Stage I lung adenocarcinomas showed significantly (P = 0.012) higher frequency of IL-12Rbeta2 expressing samples than stage II/III tumors. IL-12 treatment of IL-12R(+) neoplastic cells isolated from primary adenocarcinoma (n = 6) inhibited angiogenesis in vitro through down-regulation of different pro-angiogenic genes (e.g. IL-6, VEGF-C, VEGF-D, and laminin-5), as assessed by chorioallantoic membrane (CAM) assay and PCR array. In order to perform in vivo studies, the Calu6 NSCLC cell line was transfected with the IL-12RB2 containing plasmid (Calu6/beta2). Similar to that observed in primary tumors, IL-12 treatment of Calu6/beta2(+) cells inhibited angiogenesis in vitro. Tumors formed by Calu6/beta2 cells in SCID/NOD mice, inoculated subcutaneously or orthotopically, were significantly smaller following IL-12 vs PBS treatment due to inhibition of angiogenesis, and of IL-6 and VEGF-C production. Explanted tumors were studied by histology, immuno-histochemistry and PCR array. NBEC cells were isolated and cultured from lung specimens of non neoplastic origin. NBEC expressed IL-12R and released constitutively tumor promoting cytokines (e.g. IL-6 and CCL2). Treatment of NBEC with IL-12 down-regulated production of these cytokines. CONCLUSIONS: This study demonstrates that IL-12 inhibits directly the growth of human lung adenocarcinoma and targets the adjacent NBEC. These novel anti-tumor activities of IL-12 add to the well known immune-modulatory properties of the cytokine and may provide a rational basis for the development of a clinical trial

Genetic Inhibition of the Ubiquitin Ligase Rnf5 Attenuates Phenotypes Associated to F508del Cystic Fibrosis Mutation

Cystic fibrosis (CF) is caused by mutations in the CFTR chloride channel. Deletion of phenylalanine 508 (F508del), the most frequent CF mutation, impairs CFTR trafficking and gating. F508del-CFTR mistrafficking may be corrected by acting directly on mutant CFTR itself or by modulating expression/activity of CFTR-interacting proteins, that may thus represent potential drug targets. To evaluate possible candidates for F508del-CFTR rescue, we screened a siRNA library targeting known CFTR interactors. Our analysis identified RNF5 as a protein whose inhibition promoted significant F508del-CFTR rescue and displayed an additive effect with the investigational drug VX-809. Significantly, RNF5 loss in F508del-CFTR transgenic animals ameliorated intestinal malabsorption and concomitantly led to an increase in CFTR activity in intestinal epithelial cells. In addition, we found that RNF5 is differentially expressed in human bronchial epithelia from CF vs. control patients. Our results identify RNF5 as a target for therapeutic modalities to antagonize mutant CFTR proteins

The influence of personality disorders on desire for hastened death among terminally ill cancer patients

An ongoing debate exists regarding the legal, moral, and practical issues pertaining to physician-assisted suicide (PAS) in this country. Researchers have identified a construct called “desire for hastened death” which they believe accounts for interest in PAS and euthanasia as well as suicidality (Rosenfeld et al., 2000). Previous research on desire for hastened death has indicated that psychological and social factors play a significant role in terminally ill patients\u27 desire for death. This study tested the hypothesis that the presence of personality disorders would be associated with higher levels of desire for hastened death in a sample of terminally ill cancer patients living in a palliative care facility. In addition, it was hypothesized that personality psychopathology would moderate the relationships between the clinical variables of depression, hopelessness, spiritual well-being, social support, and symptom distress, and desire for hastened death. Personality psychopathology was assessed using the MCMI-III (Millon, 1994); desire for hastened death was assessed using the Schedule for Attitudes toward Hastened Death (Rosenfeld et al., 1999; 2000). The presence of personality disorders in general was not associated with desire for hastened death. Further, personality disorders did not appear to moderate the relationships between clinical variables and desire for hastened death. Several methodological limitations, most notably low power, may have contributed to lack of findings. Implications of supplemental analyses suggested that when grouped into clusters, the presence of certain disorders were predictive of increased desire for hastened death. Cluster A Personality Disorder significantly added to a multivariate model predicting desire for hastened death. Directions for future research in this area are presented

Handbook of Forensic Psychology

Abstract Given the consequences of infrequent visitation for incarcerated mothers an

Characterization of chloride and cationic channels in cultured human keratinocytes

Patch-clamp experiments on human cultured keratinocytes revealed the presence of three types of ion channel. The first type was a Cl(-)-selective channel, the current/voltage relationship of which showed outward rectification, the mean conductance at positive and negative membrane potentials being 66 pS and 16 pS respectively. The second type of channel showed almost equal permeability to alkali ions but was impermeable to Cl- and to the large organic cation N-methyl-D-glucamine. Its current/voltage relationship was linear with a mean unitary conductance of 18 pS in symmetrical 140 mmol/l NaCl. Finally, the third type was a large-conductance cation channel, which had in physiological ionic conditions a peculiar rectifying current/voltage relationship, the shape of which was strongly dependent on the concentration of divalent cations on both sides of the membrane. Lowering of Ca2+ and/or Mg2+ on either side of the patch led to a marked increase of the single-channel current. With identical solutions without Ca2+ Mg2+ on both sides of the patch the current/voltage relationship became ohmic and reached a conductance of 150-200 pS. In addition, channel activity was reversibly affected by changes of the external Ca2+ concentration. In particular, open-channel probability strongly increased at negative membrane potentials when the external Ca2+ was lowered from millimolar to micromolar values. Whole-cell experiments confirm the role of the extracellular Ca2+ as a modulator of the cation conductance

A volume-sensitive chloride conductance revealed in cultured human keratinocytes by 36Cl- efflux and whole-cell patch clamp recording

The Cl- transport mechanism responsible for the stimulation of 36Cl- efflux after exposure to hypotonic medium (210 mosmol/kg) was investigated in human keratinocytes. The involvement of the anion exchanger and of the Cl-/cation cotransporters was ruled out by the finding that replacement of extracellular Cl- by the poorly permeant anion gluconate, and the addition of bumetanide and furosemide, inhibitors of the Na+/K+/Cl- and K+/Cl- cotransporters, respectively, failed to significantly reduce the activation of Cl- efflux by hypotonic medium. 'Whole cell' configuration of the patch clamp technique directly revealed the presence of a macroscopic Cl- current, which was evoked by incubation with hypotonic medium and was reversed by elevation of the extracellular osmolality. Volume-sensitive current showed outward rectification of the current-voltage relationship and time-dependent inactivation at depolarizing voltages. This current was Cl- selective, because the zero-current reversal potential approached the Cl- equilibrium potential, when extracellular Cl- was replaced by gluconate. 0.1 mM 1,9-dideoxyforskolin significantly reduced either 36Cl- efflux and the Cl- current, suggesting that the Cl- efflux and the macroscopic current activated after exposure to hypotonic medium are mediated by the same pathway. Electronic cell sizing showed that in keratinocytes hypotonic swelling was not followed by a significant regulatory volume decrease response

Measuring Knowledge of the Insanity Defense: Scale Construction and Validation

Given the influence of social conformity and prejudice, defendants pleading not guilty by reason of insanity face the significant challenges of securing fair and impartial juries. Attitudes and knowledge of the insanity defense are factors that may influence levels of impartiality. In the light of this, we set out to develop a scale to examine knowledge levels of the insanity defense and their influence on decision-making. Two studies were conducted to construct a scale designed to assess laypersons\u27 knowledge of the insanity defense. Items measuring knowledge of the insanity defense were based on Perlin\u27s (1995) insanity defense myths. The first study identified particular items in need of revision and subscales that required the development of additional items in order to improve reliability and construct validity in the second study. The second study used the revised scale, demonstrating improved validity and reliability. The scale also had acceptable predictive validity with reference to insanity defense verdicts

Evaluation of Fused Pyrrolothiazole Systems as Correctors of Mutant CFTR Protein

Cystic fibrosis (CF) is a genetic disease caused by mutations that impair the function of the CFTR chloride channel. The most frequent mutation, F508del, causes misfolding and premature degradation of CFTR protein. This defect can be overcome with pharmacological agents named “correctors”. So far, at least three different classes of correctors have been identified based on the additive/synergistic effects that are obtained when compounds of different classes are combined together. The development of class 2 correctors has lagged behind that of compounds belonging to the other classes. It was shown that the efficacy of the prototypical class 2 corrector, the bithiazole corr-4a, could be improved by generating conformationally-locked bithiazoles. In the present study, we investigated the effect of tricyclic pyrrolothiazoles as analogues of constrained bithiazoles. Thirty-five compounds were tested using the functional assay based on the halide-sensitive yellow fluorescent protein (HS-YFP) that measured CFTR activity. One compound, having a six atom carbocyle central ring in the tricyclic pyrrolothiazole system and bearing a pivalamide group at the thiazole moiety and a 5-chloro-2-methoxyphenyl carboxamide at the pyrrole ring, significantly increased F508del-CFTR activity. This compound could lead to the synthesis of a novel class of CFTR correctors

Analysis of inhibitors of the anoctamin-1 chloride channel (transmembrane member 16A, TMEM16A) reveals indirect mechanisms involving alterations in calcium signalling

Background and purpose: Pharmacological inhibitors of TMEM16A (ANO1), a Ca2+ -activated Cl- channel, are important tools of research and possible therapeutic agents acting on smooth muscle, airway epithelia and cancer cells. We tested a panel of TMEM16A inhibitors, including CaCCinh -A01, niclosamide, MONNA, Ani9 and niflumic acid, to evaluate their possible effect on intracellular Ca2+ . Experimental approach: We recorded cytosolic Ca2+ increase elicited with UTP, ionomycin or IP3 uncaging. Key results: Unexpectedly, we found that all compounds, except for Ani9, markedly decreased intracellular Ca2+ elevation induced by stimuli acting on intracellular Ca2+ stores. These effects were similarly observed in cells with and without TMEM16A expression. We investigated in more detail the mechanism of action of niclosamide and CaCCinh -A01. Acute addition of niclosamide directly increased intracellular Ca2+ , an activity consistent with inhibition of the SERCA pump. In contrast to niclosamide, CaCCinh -A01 did not elevate intracellular Ca2+ , thus implying a different mechanism of action, possibly a block of inositol triphosphate receptors. Conclusions and implications: Most TMEM16A inhibitors are endowed with indirect effects mediated by alteration of intracellular Ca2+ handling, which may in part preclude their use as TMEM16A research tools