Smaller Cortisol Awakening Responses Are Associated with Greater Visual Dependence in Postural Control.

There are known links between the hypothalamic-pituitary-adrenal (HPA) axis and systems responsible for regulating posture. Our aim was to explore directly, for the first time, whether an aspect of circadian HPA axis activity (the cortisol awakening response: CAR) was associated with greater visual dependency in postural control. For measurement of the CAR, electronically monitored saliva samples were collected by participants following morning awakening in their home environment. On the afternoons of the same days, postural sway was measured in the laboratory by exposing participants to static (control) and moving visual stimuli whilst standing still and upright on a force platform. Visual dependence was assessed as the increase in postural sway (path length) during exposure to the moving compared with the static condition. The 44 measurement days were derived from four days for each of eleven healthy participants (mean ± SD age: 51.18 ± 3.3 years). As expected, postural sway was greater when exposed to moving versus static cues. Mixed regression modelling showed that participants with smaller four day average CARs had greater deterioration in postural sway when presented with moving stimuli. These data are the first to document associations between the CAR and visual dependency in postural sway.

CYP3A4 mediates growth of estrogen receptor-positive breast cancer cells in part by inducing nuclear translocation of phospho-Stat3 through biosynthesis of (±)-14,15-epoxyeicosatrienoic acid (EET).

CYP3A4 expression in breast cancer correlates with decreased overall survival, but the mechanisms are unknown. Cytochrome P450 gene profiling by RNAi silencing demonstrates that CYP3A or 2C8 gene expression is specifically required for growth of the breast cancer lines MCF7, T47D, and MDA-MB-231. CYP3A4 silencing blocks the cell cycle at the G(2)/M checkpoint and induces apoptosis in the MCF7 line, thereby inhibiting anchorage-dependent growth and survival. CYP3A4 was profiled for NADPH-dependent arachidonic acid (AA) metabolism and synthesized AA epoxygenase products (±)-8,9-, (±)-11,12-, and (±)-14,15-epoxyeicosatrienoic acid (EET) (total turnover of ∼2 pmol/pmol CYP3A4/min) but not hydroxylase products (±)-15-, (±)-19-, or 20-hydroxyeicosatetraenoic acid. Furthermore, eicosanoid profiling revealed that MCF7 cells synthesize EETs in a CYP3A4-dependent manner. The (±)-14,15-EET regioisomer selectively rescues breast cancer cells from CYP3A4 silencing in a concentration-dependent fashion and promotes mitogenesis and anchorage-dependent cloning. Stat3 (Tyr-705) phosphorylation was inhibited by CYP3A4 silencing, providing a potential mechanism for CYP3A4 involvement in breast cancer cell growth. Silencing Stat3 blocks breast cancer cell growth and abrogates (±)-14,15-EET-induced proliferation, indicating a Stat3 requirement for (±)-14,15-EET-mediated cell growth. Although silencing of CYP3A4 reduces nuclear Tyr(P)-705-Stat3, (±)-14,15-EET restores this signaling process and promotes Tyr(P)-705-Stat3 translocation to the nucleus, suggesting that (±)-14,15-EET may be involved in an autocrine/paracrine pathway driving cell growth. These studies indicate that CYP3A4 is a highly active AA epoxygenase that promotes Stat3-mediated breast cancer cell growth in part through (±)-14,15-EET biosynthesis. Furthermore, these studies indicate an essential role for Stat3 as a mediator of epoxygenase activity in breast cancer.

The human immunodeficiency virus protease inhibitor ritonavir inhibits lung cancer cells, in part, by inhibition of survivin.

INTRODUCTION: Ritonavir is a potential therapeutic agent in lung cancer, but its targets in lung adenocarcinoma are unknown, as are candidate biomarkers for its activity. METHODS: RNAi was used to identify genes whose expression affects ritonavir sensitivity. Synergy between ritonavir, gemcitabine, and cisplatin was tested by isobologram analysis. RESULTS: Ritonavir inhibits growth of K-ras mutant lung adenocarcinoma lines A549, H522, H23, and K-ras wild-type line H838. Ritonavir causes G0/G1 arrest and apoptosis. Associated with G0/G1 arrest, ritonavir down-regulates cyclin-dependent kinases, cyclin D1, and retinoblastoma protein phosphorylation. Associated with induction of apoptosis, ritonavir reduces survivin messenger RNA and protein levels more than twofold. Ritonavir inhibits phosphorylation of c-Src and signal transducer and activator of transcription protein 3, which are important events for survivin gene expression and cell growth, and induces cleavage of PARP1. Although knock down of survivin, c-Src, or signal transducer and activator of transcription protein 3 inhibits cell growth, only survivin knock down enhances ritonavir inhibition of growth and survivin overexpression promotes ritonavir resistance. Ritonavir was tested in combination with gemcitabine or cisplatin, exhibiting synergistic and additive effects, respectively. The combination of ritonavir/gemcitabine/cisplatin is synergistic in the A549 line and additive in the H522 line, at clinically feasible ritonavir concentrations (<10 µM). CONCLUSIONS: Ritonavir is of interest for lung adenocarcinoma therapeutics, and survivin is an important target and potential biomarker for its sensitivity. Ritonavir cooperation with gemcitabine/cisplatin might be explained by involvement of PARP1 in repair of cisplatin-mediated DNA damage and survivin in repair of gemcitabine-mediated double-stranded DNA breaks.

Prediction of postoperative recurrence-free survival in non-small cell lung cancer by using an internationally validated gene expression model.

PURPOSE: This study was performed to discover prognostic genomic markers associated with postoperative outcome of stage I to III non-small cell lung cancer (NSCLC) that are reproducible between geographically distant and demographically distinct patient populations. EXPERIMENTAL DESIGN: American patients (n = 27) were stratified on the basis of recurrence and microarray profiling of their tumors was performed to derive a training set of 44 genes. A larger Korean patient validation cohort (n = 138) was also stratified by recurrence and screened for these genes. Four reproducible genes were identified and used to construct genomic and clinicogenomic Cox models for both cohorts. RESULTS: Four genomic markers, DBN1 (drebrin 1), CACNB3 (calcium channel beta 3), FLAD1 (PP591; flavin adenine dinucleotide synthetase), and CCND2 (cyclin D2), exhibited highly significant differential expression in recurrent tumors in the training set (P < 0.001). In the validation set, DBN1, FLAD1 (PP591), and CACNB3 were significant by Cox univariate analysis (P ≤ 0.035), whereas only DBN1 was significant by multivariate analysis. Genomic and clinicogenomic models for recurrence-free survival (RFS) were equally effective for risk stratification of stage I to II or I to III patients (all models P < 0.0001). For stage I to II or I to III patients, 5-year RFS of the low- and high-risk patients was approximately 70% versus 30% for both models. The genomic model for overall survival of stage I to III patients was improved by addition of pT and pN stage (P < 0.0013 vs. 0.010). CONCLUSION: A 4-gene prognostic model incorporating the multivariate marker DBN1 exhibits potential clinical utility for risk stratification of stage I to III NSCLC patients.

Monitoreo de DDI en la provincia de La Rioja (2006) / Monitoring of DDI in the province of La Rioja (2006)

Un total de 712 alumnos de escolaridad primaria, de ambos sexos, fue estudiado en este monitoreo de bocio endémico en dos localidades de la provincia de La Rioja: Ciudad de La Rioja (442 niños) y Chilecito (270 niños). La edad de los escolares osciló entre 5 y 16 años. La palpación tiroidea fue hecha por el conjunto de los médicos participantes. Sin embargo, con la finalidad de aunar criterios con lo realizado previamente (1-24), se tomó como única referencia la palpación de H.N., que se llevó a cabo en la totalidad de los niños estudiados. La definición del grado de bocio fue similar a la utilizada en los otros relevamientos (1). Se determinó la yoduria en muestras casuales de orina emitidas por los niños una vez que fueron palpados (125 de La Rioja y 128 de Chilecito). Se recolectaron 251 muestras de sal de consumo hogareño de La Rioja y 141 de Chilecito, para medir su contenido en yodo. El examen palpatorio de los niños reveló la existencia de bocio grado 1 solamente. La prevalencia de bocio encontrada fue de 1,8% en La Rioja y de 2,6% en Chilecito. Los niveles de yoduria alcanzaron, en La Rioja, una media de 226 ± 181 (DS) μg/L y una mediana de 186μg/L, al tiempo que en Chilecito la media fue de 217 ± 161μg/L y la mediana de 185μg/L. El contenido de yodo de las sales, que aportaron los alumnos desde sus hogares, fue adecuado para casi todas las marcas. De esta manera, observamos que en la ciudad de La Rioja el promedio de yodo en la sal, tomada en conjunto, fue de 33,1 ± 12,5 mg/Kg, mientras que para Chilecito fue de 28,1 ± 8,2 mg/Kg. Al analizar las concentraciones de yodo <15mg/Kg, observamos que fue del 10,7% en La Rioja y del 9,5% en Chilecito. Teniendo en cuenta la línea de corte del 10% que fija el ICCIDD (25) como valor óptimo, podemos observar que la situación fue prácticamente satisfactoria en ambas poblaciones. Concluímos que en estas dos poblaciones de la provincia de La Rioja no existe, actualmente, bocio endémico por deficiencia de yodo. Estos resultados indican que la profilaxis con sal yodada fue óptima en esta provincia, dado que en el pasado solía ser una típica zona yodo-deficiente con un muy alto porcentaje de bocio endémico.

Anastrozole Use in Early Stage Breast Cancer of Post-Menopausal Women.

The majority of breast cancers express the estrogen receptor and depend on estradiol (E2) for their growth. Hormonal therapy aims at depriving estrogen signaling either by using selective estrogen receptor modulators (SERM)-that interfere with the binding of E2 to its receptor (ER)-or aromatase inhibitors (AI)-that block the aromatase-dependent synthesis of E2. While SERMs are recommended for both pre- and post-menopausal patients, AIs are indicated only for post-menopausal patients. For the past 20 years, the SERM tamoxifen has been considered the "gold standard" for the treatment of hormone receptor positive breast cancers. However, tamoxifen's role is now challenged by third generation AIs, such as anastrozole, which exhibit greater efficacy in the adjuvant setting in several recently reported trials. This review will focus on anastrozole's mechanism of action, dosing, pharmacology, pharmacokinetics, and clinical applications. It will briefly discuss the clinical trials that determined anastrozole's efficacy in the treatment of advanced breast cancer (ABC) and in the neoadjuvant setting. Finally, it will present the clinical trials that established anastrozole as a frontline agent in the treatment of post-menopausal women with hormone receptor positive early breast cancer.

Epitope specificity of anti-factor VIII antibodies from inhibitor positive acquired and congenital haemophilia A patients using synthetic peptides spanning A and C domains.

Development of antibodies (Ab) that either block the function of coagulation factor VIII (FVIII) (inhibitors) or clear it from circulation, seriously complicate the treatment of haemophilia A patients with FVIII products. Autoantibodies which develop in subjects without congenital FVIII defects, cause acquired haemophilia, a rare but life-threatening coagulopathy. Identification of the FVIII epitopes to which inhibitor Abs bind will help understanding the mechanisms of inhibitor activity, and perhaps development of new therapies. Here, we examined the FVIII peptide sequence regions recognised by anti-FVIII Ab in the plasma of six congenital and one acquired haemophilia patients with high inhibitor titers (24.4-2000 BU/ml). We used indirect ELISA and overlapping synthetic peptides, 20 residues long, spanning the sequence of the A and C FVIII domains. None of the plasma samples reacted with A1, A3 or C1 domain peptides. Six plasmas reacted with A2 and/or C2 peptides. Peptides spanning residues A2-521-690 and C2-2251-2332 were recognised most frequently and strongly. They include residues that contribute to the binding sites for activated factor IX and phosphatidyl serine/von Willebrand factor. These results suggest that anti-FVIII Abs share a pattern of antigen specificity in our panel of patients, and that exposed regions of the FVIII molecule that form functionally important binding sites elicit an intense Ab response.

CD4+ T cells and cytokines in the pathogenesis of acquired myasthenia gravis.

Although human and experimental acquired myasthenia gravis (MG) are prototypic antibody (Ab)-mediated autoimmune diseases, synthesis of the pathogenic anti-acetylcholine receptor (AChR) Abs, which are high affinity IgG, requires intervention of CD4(+) T helper cells and their cytokines. Moreover, cytokine signaling is crucial for development, modulation, and downregulation of immune responses, and therefore influences the initiation and evolution of the anti-AChR response in acquired MG. Cytokines are involved in the growth and differentiation of CD4(+) T cells, and are secreted by activated CD4(+) T cells as effectors of their functions: differentiated CD4(+) T cells are classified into subtypes based on the cytokines they synthesize and secrete. Because cytokines are synthesized by and act on a variety of cells and because they may influence the synthesis and/or the activity of other cytokines, the effects of their signaling, in both normal and autoimmune responses, are complex and sometimes even contrasting, depending on the circumstances when the cytokine action took place. In this chapter, we will review studies on the effects on the development of acquired MG symptoms of several cytokines secreted by activated CD4(+) T cells or influencing the activation of particular CD4(+) T cell subsets.

C57BL/6 mice genetically deficient in IL-12/IL-23 and IFN-gamma are susceptible to experimental autoimmune myasthenia gravis, suggesting a pathogenic role of non-Th1 cells.

Immunization with Torpedo acetylcholine receptor (TAChR) induces experimental autoimmune myasthenia gravis (EAMG) in C57BL/6 (B6) mice. EAMG development needs IL-12, which drives differentiation of Th1 cells. The role of IFN-gamma, an important Th1 effector, is not clear and that of IL-17, a proinflammatory cytokine produced by Th17 cells, is unknown. In this study, we examined the effect of simultaneous absence of IL-12 and IFN-gamma on EAMG susceptibility, using null mutant B6 mice for the genes of both the IL-12/IL-23 p40 subunit and IFN-gamma (dKO mice). Wild-type (WT) B6 mice served as control for EAMG induction. All mice were immunized with TAChR in Freund's adjuvant. dKO mice developed weaker anti-TAChR CD4(+)T cells and Ab responses than WT mice. Yet, they developed EAMG symptoms, anti-mouse acetylcholine receptor (AChR) Ab, and CD4(+) T cell responses against mouse AChR sequences similar to those of WT mice. dKO and WT mice had similarly reduced AChR content in their muscles, and IgG and complement at the neuromuscular junction. Naive dKO mice had significantly fewer NK, NKT, and CD4(+)CD25(+)Foxp3(+) T regulatory (Treg) cells than naive WT mice. Treg cells from TAChR-immunized dKO mice had significantly less suppressive activity in vitro than Treg cells from TAChR-immunized WT mice. In contrast, TAChR-specific CD4(+) T cells from TAChR-immunized dKO and WT mice secreted comparable amounts of IL-17 after stimulation in vitro with TAChR. The susceptibility of dKO mice to EAMG may be due to reduced Treg function, in the presence of a normal function of pathogenic Th17 cells.

Myasthenia gravis: past, present, and future.

Myasthenia gravis (MG) is an autoimmune syndrome caused by the failure of neuromuscular transmission, which results from the binding of autoantibodies to proteins involved in signaling at the neuromuscular junction (NMJ). These proteins include the nicotinic AChR or, less frequently, a muscle-specific tyrosine kinase (MuSK) involved in AChR clustering. Much is known about the mechanisms that maintain self tolerance and modulate anti-AChR Ab synthesis, AChR clustering, and AChR function as well as those that cause neuromuscular transmission failure upon Ab binding. This insight has led to the development of improved diagnostic methods and to the design of specific immunosuppressive or immunomodulatory treatments.

CD4+ T and B cells cooperate in the immunoregulation of Experimental Autoimmune Myasthenia Gravis.

C57Bl6 mice (B6 mice) immunized with Torpedo acetylcholine receptor (TAChR) in Freund's adjuvants (FA) develop Experimental Autoimmune Myasthenia Gravis (EAMG). In mouse EAMG Th2 cytokines may be protective. Aluminum hydroxide (Alum) was used to immunize B6 mice to the TAChR and prime CD4+ T and B cells secreting Th2 cytokines. Mice immunized with TAChR/Alum developed anti-AChR CD4+ T cells response, but minimal antibody levels and symptoms. TAChR/Alum treatments prior immunization with TAChR/FA protected mice from EAMG. Cell transfer experiments demonstrated that B and CD4+ T cells mediated the protective effect by causing intense reduction of complement-fixing anti-TAChR IgG subclasses.

Breakdown of tolerance to a self-peptide of acetylcholine receptor alpha-subunit induces experimental myasthenia gravis in rats.

Experimental autoimmune myasthenia gravis (EAMG), a model for human myasthenia (MG), is routinely induced in susceptible rat strains by a single immunization with Torpedo acetylcholine receptor (TAChR). TAChR immunization induces anti-AChR Abs that cross-react with self AChR, activate the complement cascade, and promote degradation of the postsynaptic membrane of the neuromuscular junction. In parallel, TAChR-specific T cells are induced, and their specific immunodominant epitope has been mapped to the sequence 97-116 of the AChR alpha subunit. A proliferative T cell response against the corresponding rat sequence (R97-116) was also found in TAChR-immunized rats. To test whether the rat (self) sequence can be pathogenic, we immunized Lewis rats with R97-116 or T97-116 peptides and evaluated clinical, neurophysiological, and immunological parameters. Clinical signs of the disease were noted only in R97-116-immunized animals and were confirmed by electrophysiological signs of impaired neuromuscular transmission. All animals produced Abs against the immunizing peptide, but anti-rat AChR Abs were observed only in animals immunized with the rat peptide. These findings suggested that EAMG in rats can be induced by a single peptide of the self AChR, that this sequence is recognized by T cells and Abs, and that breakdown of tolerance to a self epitope might be an initiating event in the pathogenesis of rat EAMG and MG.

The susceptibility to experimental myasthenia gravis of STAT6-/- and STAT4-/- BALB/c mice suggests a pathogenic role of Th1 cells.

Autoantibodies to the muscle acetylcholine receptor (AChR) cause the symptoms of human and experimental myasthenia gravis (EMG). AChR-specific CD4+ T cells permit development of these diseases, but the role(s) of the Th1 and Th2 subsets is unclear. The STAT4 and STAT6 proteins, which mediate intracellular cytokine signaling, are important for differentiation of Th1 and Th2 cells, respectively. Wild-type (WT) BALB/c mice, which are prone to develop Th2 rather than Th1 responses to Ag, are resistant to EMG. We have examined the role of Th1 and Th2 cells in EMG using STAT4 (STAT4-/-)- or STAT6 (STAT6-/-)-deficient BALB/c mice. After AChR immunization, STAT6-/- mice were susceptible to EMG: they developed more serum anti-AChR Ab, and had more complement-fixing anti-AChR IgG2a and 2b and less IgG1 than WT or STAT4-/- mice. The susceptibility to EMG of STAT6-/- mice is most likely related to the Th1 cell-induced synthesis of anti-AChR Ab, which trigger complement-mediated destruction of the neuromuscular junction. CD4+ T cells of the STAT6-/- mice had proliferative responses to the AChR comparable to those of WT and STAT4-/- mice, and recognized similar AChR epitopes. STAT6-/- mice had abundant AChR-specific Th1 cells, which were nearly absent in WT and STAT4-/- mice. Spleen and lymph nodes from STAT6-/- mice contained cells that secreted IL-4 when cultured with AChR: these are most likely STAT6-independent cells, stimulated in a non-Ag-specific manner by the cytokines secreted by AChR-specific Th1 cells.

T cells and cytokines in the pathogenesis of acquired myasthenia gravis.

Although the symptoms of myasthenia gravis (MG) and experimental MG (EAMG) are caused by autoantibodies, CD4(+) T cells specific for the target antigen, the nicotinic acetylcholine receptor, and the cytokines they secrete, have an important role in these diseases. CD4(+) T cells have a pathogenic role, by permitting and facilitating the synthesis of high-affinity anti-AChR antibodies. Th1 CD4(+) cells are especially important because they drive the synthesis of anti-AChR complement-fixing IgG subclasses. Binding of those antibodies to the muscle AChR at the neuromuscular junction will trigger the complement-mediated destruction of the postsynaptic membrane. Thus, IL-12, a crucial cytokine for differentiation of Th1 cells, is necessary for development of EAMG. Th2 cells secrete different cytokines, with different effects on the pathogenesis of EAMG. Among them, IL-10, which is a potent growth and differentiation factor for B cells, facilitates the development of EAMG. In contrast, IL-4 appears to be involved in the differentiation of AChR-specific regulatory CD4(+) T cells, which can prevent the development of EAMG and its progression to a self-maintaining, chronic autoimmune disease. Studies on the AChR-specific CD4(+) cells commonly present in the blood of MG patients support a crucial role of CD4(+) T cells in the development of MG. Circumstantial evidence supports a pathogenic role of IL-10 also in human MG. On the other hand, there is no direct or circumstantial evidence yet indicating a role of IL-4 in the modulatory or immunosuppressive circuits in MG.

Absence of IL-4 facilitates the development of chronic autoimmune myasthenia gravis in C57BL/6 mice.

Myasthenia gravis (MG) is a T cell-dependent, Ab-mediated autoimmune disease. Ab against muscle acetylcholine receptor (AChR) cause the muscular weakness that characterizes MG and its animal model, experimental MG (EMG). EMG is induced in C57BL6 (B6) mice by three injections of Torpedo AChR (TAChR) in adjuvant. B6 mice develop anti-TAChR Ab that cross-react with mouse muscle AChR, but their CD4+ T cells do not cross-react with mouse AChR sequences. Moreover, murine EMG is not self-maintaining as is human MG, and it has limited duration. Several studies suggest that IL-4 has a protecting function in EMG. Here we show that B6 mice genetically deficient in IL-4 (IL-4-/-) develop long-lasting muscle weakness after a single immunization with TAChR. They develop chronic self-reactive Ab, and their CD4+ T cells respond not only to the TAChR and TAChR subunit peptides, but also to several mouse AChR subunit peptides. These results suggest that in B6 mice, regulatory mechanisms that involve IL-4 contribute to preventing the development of a chronic Ab-mediated autoimmune response to the AChR.

Adoptive protection from experimental myasthenia gravis with T cells from mice treated nasally with acetylcholine receptor epitopes.

Nasal administration of synthetic CD4(+) epitopes of the acetylcholine receptor (AChR) prevents experimental myasthenia gravis (EMG) in C57Bl/6 mice, but not in IL4-deficient C57Bl/6 (IL4(-/-)) mice. Here we verify that nasal tolerance requires IL4, by showing that CD4(+) cells from C57Bl/6 mice treated nasally with a pool of AChR CD4(+) epitopes protected IL4(-/-) mice from EMG and caused a reduced production of anti-AChR antibody. CD4(+) cells from C57Bl/6 mice treated with unrelated peptides or sham-treated did not induce protection. CD4(+) cells from C57Bl/6 mice treated with just one AChR peptide protected IL4(-/-) mice from EMG without affecting antibody synthesis.