A case of donepezil-related torsades de pointes.

An 80-year-old woman with Alzheimer's dementia presented with diarrhoea, vomiting and worsening confusion following an increase in donepezil dose from 5 to 10 mg. The ECG revealed prolongation of QTc interval. Soon after admission, she became unresponsive with polymorphic ventricular tachycardia (VT). Cardiopulmonary resuscitation with a 200 J shock was successful in establishing cardiac output. Following the discontinuation of donepezil, the QTc interval normalised and no further arrhythmias were recorded. Treatment with anticholinesterase inhibitors may result in life-threatening VT. Vigilance is required for the identification of this condition in patients presenting with presyncope, syncope or seizures.

B cells from patients with chronic GVHD are activated and primed for survival via BAFF-mediated pathways.

Recent data reveal an important role for B cells in the pathogenesis of chronic GVHD (cGVHD). Patients with cGVHD have delayed B-cell reconstitution and elevated BAFF to B-cell ratios compared to patients without cGVHD. The mechanisms promoting and sustaining B-cell activation in this disease, however, remain unknown. As BAFF increases murine B-cell metabolism and survival and maintains autoreactive B-cell clones, we performed ex vivo analyses of peripheral B cells from 51 patients who either had or did not have active cGVHD and were greater than 1 year from the time of allogeneic hematopoietic stem cell transplantation. We found that B cells from patients with active cGVHD were in a heightened metabolic state and were resistant to apoptosis. Exogenous BAFF treatment amplified cell size and survival in B cells from these patients. We found significantly increased signaling through ERK and AKT that associated with decreased levels of proapoptotic Bim, suggesting a mechanistic link between elevated BAFF levels and aberrant B-cell survival. Thus, we identify a role for BAFF in the pathogenesis of cGVHD and define B-cell activation and survival pathways suitable for novel therapeutic development in cGVHD.

Chemomobilization with Etoposide is Highly Effective in Patients with Multiple Myeloma and Overcomes the Effects of Age and Prior Therapy.

The optimal mobilization strategy prior to autologous stem cell transplantation for patients with multiple myeloma remains unclear. Mobilization with cytokines alone appears to yield suboptimal results in older patients as well as patients who have received prior lenalidomide. To avoid the marked cytopenias and risks of hemorrhagic cystitis associated with the administration of cyclophosphamide, we investigated the efficacy and safety of chemomobilization with an intermediate dose etoposide (VP-16; 375 mg/m(2) on days +1 and +2) and granulocyte-colony stimulating factor (G-CSF) (5 µg/kg twice daily from day +3 through the final day of collection). We reviewed our institutional experience with 152 myeloma patients mobilized with this regimen. The addition of VP-16 to G-CSF resulted in successful mobilization in 100% of patients, including 143 (94%) who collected successfully in a single day. A total of 99% of patients, including those with prior XRT and/or prior lenalidomide or thalidomide therapy, collected at least 5 × 10(6) cells/kg in 1 or 2 days of apheresis, and the median total number of CD34(+) cells collected in the entire population was 12 × 10(6) cells/kg. Collection was predictable, with 61% of patients collecting on day +11, and the rest between days +7 and +13. There were no variables, including age, prior imid exposure, radiation therapy, or total amount of prior therapy that were associated with suboptimal mobilization. Adverse effects of the regimen included supportive transfusions required in 31 (20%) patients, and fevers requiring hospitalization or intravenous antibiotics in 26 (17%) patients. VP-16 and G-CSF appears to be a safe and effective mobilization regimen for patients with multiple myeloma undergoing autologous stem cell transplantation, producing excellent stem cell yield with the majority of patients requiring 1 day of apheresis.

Alteration of thioredoxin reductase 1 levels in elucidating cancer etiology.

Thioredoxin reductase 1 (TR1) is a major antioxidant and redox regulator in mammalian cells and appears to function as a double-edged sword in that it has roles in preventing and promoting/sustaining cancer. TR1 is overexpressed in many cancer cells and targeting its removal often leads to a reversal in numerous malignant characteristics which has marked this selenoenzyme as a prime target for cancer therapy. Since alterations in TR1 activity may lead to a better understanding of the etiology of cancer and new avenues for providing better therapeutic procedures, we have described herein techniques for removing and reexpressing TR1 employing RNAi technology and for assessing the catalytic activity of this enzyme.

Role of selenium-containing proteins in T-cell and macrophage function.

Selenium (Se) has been known for many years to have played a role in boosting the immune function, but the manner in which this element acts at the molecular level in host defence and inflammatory diseases is poorly understood. To elucidate the role of Se-containing proteins in the immune function, we knocked out the expression of this protein class in T-cells or macrophages of mice by targeting the removal of the selenocysteine tRNA gene using loxP-Cre technology. Mice with selenoprotein-less T-cells manifested reduced pools of mature and functional T-cells in lymphoid tissues and an impairment in T-cell-dependent antibody responses. Furthermore, selenoprotein deficiency in T-cells led to an inability of these cells to suppress reactive oxygen species production, which in turn affected their ability to proliferate in response to T-cell receptor stimulation. Selenoprotein-less macrophages, on the other hand, manifested mostly normal inflammatory responses, but this deficiency resulted in an altered regulation in extracellular matrix-related gene expression and a diminished migration of macrophages in a protein gel matrix. These observations provided novel insights into the role of selenoproteins in the immune function and tissue homeostasis.

Deficiency in the 15-kDa selenoprotein inhibits tumorigenicity and metastasis of colon cancer cells.

Selenium has cancer-preventive activity that is mediated, in part, through selenoproteins. The role of the 15-kDa selenoprotein (Sep15) in colon cancer was assessed by preparing and using mouse colon CT26 cells stably transfected with short hairpin RNA constructs targeting Sep15. Metabolic (75)Se labeling and Northern and Western blot analyses revealed that >90% of Sep15 was downregulated. Growth of the resulting Sep15-deficient CT26 cells was reduced (P < 0.01), and cells formed significantly (P < 0.001) fewer colonies in soft agar compared with control CT26 cells. Whereas most (14 of 15) BALB/c mice injected with control cells developed tumors, few (3 of 30) mice injected with Sep15-deficient cells developed tumors (P < 0.0001). The ability to form pulmonary metastases had similar results. Mice injected with the plasmid-transfected control cells had >250 lung metastases per mouse; however, mice injected with cells with downregulation of Sep15 only had 7.8 +/- 5.4 metastases. To investigate molecular targets affected by Sep15 status, gene expression patterns between control and knockdown CT26 cells were compared. Ingenuity Pathways Analysis was used to analyze the 1,045 genes that were significantly (P < 0.001) affected by Sep15 deficiency. The highest-scored biological functions were cancer and cellular growth and proliferation. Consistent with these observations, subsequent analyses revealed a G(2)-M cell cycle arrest in cells with targeted downregulation of Sep15. In contrast to CT26 cells, Sep15-targeted downregulation in Lewis lung carcinoma (LLC1) cells did not affect anchorage-dependent or anchorage-independent cell growth. These data suggest tissue specificity in the cancer-protective effects of Sep15 downregulation, which are mediated, at least in part, by influencing the cell cycle.

Selenoproteins regulate macrophage invasiveness and extracellular matrix-related gene expression.

BACKGROUND: Selenium, a micronutrient whose deficiency in diet causes immune dysfunction and inflammatory disorders, is thought to exert its physiological effects mostly in the form of selenium-containing proteins (selenoproteins). Incorporation of selenium into the amino acid selenocysteine (Sec), and subsequently into selenoproteins is mediated by Sec tRNA([Ser]Sec). RESULTS: To define macrophage-specific selenoprotein functions, we generated mice with the Sec tRNA([Ser]Sec) gene specifically deleted in myeloid cells. These mutant mice were devoid of the "selenoproteome" in macrophages, yet exhibited largely normal inflammatory responses. However, selenoprotein deficiency led to aberrant expression of extracellular matrix-related genes, and diminished migration of macrophages in a protein gel matrix. CONCLUSION: Selenium status may affect immune defense and tissue homeostasis through its effect on selenoprotein expression and the trafficking of tissue macrophages.

Selenoproteins mediate T cell immunity through an antioxidant mechanism.

Selenium is an essential dietary element with antioxidant roles in immune regulation, but there is little understanding of how this element acts at the molecular level in host defense and inflammatory disease. Selenium is incorporated into the amino acid selenocysteine (Sec), which in turn is inserted into selenoproteins in a manner dependent on Sec tRNA([Ser]Sec). To investigate the molecular mechanism that links selenium to T cell immunity, we generated mice with selenoprotein-less T cells by cell type-specific ablation of the Sec tRNA([Ser]Sec) gene (trsp). Herein, we show that these mutant mice exhibit decreased pools of mature T cells and a defect in T cell-dependent antibody responses. We also demonstrate that selenoprotein deficiency leads to oxidant hyperproduction in T cells and thereby suppresses T cell proliferation in response to T cell receptor stimulation. These findings offer novel insights into immune function of selenium and physiological antioxidants.

Ethnic variations in the experiences of mental health service users in England: results of a national patient survey programme.

BACKGROUND: Minority ethnic groups in the UK are reported to have a poor experience of mental health services, but comparative information is scarce. AIMS: To examine ethnic differences in patients' experience of community mental health services. METHOD: Trusts providing mental health services in England conducted surveys in 2004 and 2005 of users of community mental health services. Multiple regression was used to examine ethnic differences in responses. RESULTS: About 27 000 patients responded to each of the surveys, of whom 10% were of minority ethnic origin. In the 2004 survey, age, living alone, the 2004 survey, age, living alone, detention and hospital admissions were stronger predictors of patient experience than ethnicity. Self-reported mental health status had the strongest explanatory effect. In the 2005 survey, the main negative differences relative to the White British were for Asians. CONCLUSIONS: Ethnicity had a smaller effect on patient experience than other variables. Relative to the White British, the Black group did not report negative experiences whereas the Asian group were most likely to respond negatively. However, there is a need for improvements in services for minority ethnic groups, including access to talking therapies and better recording of ethnicity.

Selenophosphate synthetase 2 is essential for selenoprotein biosynthesis.

Selenophosphate synthetase (SelD) generates the selenium donor for selenocysteine biosynthesis in eubacteria. One homologue of SelD in eukaryotes is SPS1 (selenophosphate synthetase 1) and a second one, SPS2, was identified as a selenoprotein in mammals. Earlier in vitro studies showed SPS2, but not SPS1, synthesized selenophosphate from selenide, whereas SPS1 may utilize a different substrate. The roles of these enzymes in selenoprotein synthesis in vivo remain unknown. To address their function in vivo, we knocked down SPS2 in NIH3T3 cells using small interfering RNA and found that selenoprotein biosynthesis was severely impaired, whereas knockdown of SPS1 had no effect. Transfection of SPS2 into SPS2 knockdown cells restored selenoprotein biosynthesis, but SPS1 did not, indicating that SPS1 cannot complement SPS2 function. These in vivo studies indicate that SPS2 is essential for generating the selenium donor for selenocysteine biosynthesis in mammals, whereas SPS1 probably has a more specialized, non-essential role in selenoprotein metabolism.

Both selenoproteins and low molecular weight selenocompounds reduce colon cancer risk in mice with genetically impaired selenoprotein expression.

Selenium has cancer protective effects in a variety of experimental systems. Currently, it is not known whether selenoproteins or low molecular weight selenocompounds are responsible for this activity. To evaluate the contribution of selenoproteins to the cancer protective effects of selenium, we used transgenic mice that carry a mutant selenocysteine transfer RNA gene, which causes reduced selenoprotein synthesis. Selenium homeostasis was characterized in liver and colon of wild-type and transgenic mice fed selenium-deficient diets supplemented with 0, 0.1, or 2.0 microg selenium (as selenite)/g diet. (75)Se-labeling, Western blot analysis, and enzymatic activities revealed that transgenic mice have reduced (P < 0.05) liver and colon glutathione peroxidase expression, but conserved thioredoxin reductase expression compared with wild-type mice, regardless of selenium status. Transgenic mice had more (P < 0.05) selenium in the nonprotein fraction of the liver and colon than wild-type mice, indicating a greater amount of low molecular weight selenocompounds. Compared with wild-type mice, transgenic mice had more (P < 0.05) azoxymethane-induced aberrant crypt formation (a preneoplastic lesion for colon cancer). Supplemental selenium decreased (P < 0.05) the number of aberrant crypts and aberrant crypt foci in both wild-type and transgenic mice. These results provide evidence that a lack of selenoprotein activity increases colon cancer susceptibility. Furthermore, low molecular weight selenocompounds reduced preneoplastic lesions independent of the selenoprotein genotype. These results are, to our knowledge, the first to provide evidence that both selenoproteins and low molecular weight selenocompounds are important for the cancer-protective effects of selenium.

n-3 PUFA fail to affect in vivo, antigen-driven CD8+T-cell proliferation in the spleen of naïve mice.

One of the most frequently reported immunomodulatory actions of n-3 PUFA is their ability to diminish in vitro lymphocyte proliferation. The purpose of this study was to determine if n-3 PUFA intake affects the kinetics or magnitude of the antigen-driven expansion of CD8(+)T-lymphocytes in vivo. In this study we utilized a well-characterized model of T-cell immunity (i.e. infection with the intracellular bacterium, Listeria monocytogenes). Weanling BALB/c mice were fed one of two experimental diets that differed solely in fat source. Our control diet contained lard (180 g/kg) and was devoid of long-chain n-3 PUFA. The experimental diet contained 150 g/kg menhaden fish oil and 30 g/kg corn oil, thus providing approximately 8 % of energy from long-chain n-3 PUFA. After 4 weeks, mice were infected intravenously with 10(6) colony-forming units of actA-deficient L. monocytogenes. Clonal expansion of antigen-specific CD8(+)T-cells in the spleen was measured at 5, 7, 9 and 14 d post-challenge using a class I MHC tetramer loaded with the immunodominant peptide from this pathogen (i.e. K(d):LLO91-99). We report that feeding mice a diet rich in n-3 fatty acids did not significantly impact either the kinetics or magnitude of in vivo, antigen-driven expansion of CD8(+)T-cells. Furthermore, contraction of this T-cell population was not affected by n-3 PUFA treatment. To our knowledge this is the first time MHC tetramers have been used to investigate the influence of n-3 PUFA on in vivo CD8(+)T-cell proliferation.

Omega-3 polyunsaturated fatty acid impairment of early host resistance against Listeria monocytogenes infection is independent of neutrophil infiltration and function.

The primary objective of this study was to determine whether the n-3 PUFA-mediated changes in host response to a Listeria monocytogenes infection (e.g., cytokine production and bacterial clearance) were dependent upon neutrophils. Balb/c mice were fed one of two semi-purified diets that contained either 0 or 41 g of n-3 PUFA/kg. After 4 week, mice were injected with a neutrophil-depleting (RB6-8C5) or isotype-control antibody 24 h prior to infection. Bacterial clearance from the liver and spleen at 3 days post-challenge was measured and the concentration of five pro-inflammatory cytokines in sera 24 h post-infection were determined using a novel protein multiplexing kit. We found that neutrophil depletion impaired bacterial clearance independent of the effect of n-3 PUFA. Interestingly, we observed a rather complex interaction between neutrophil-depletion and n-3 PUFA intake on in vivo pro-inflammatory cytokine production. For example, neutrophil depletion elevated circulating IL-6 and MCP-1 (2- to 5-fold; p<0.05) in n-3 PUFA-fed mice, but less so or not at all in mice fed the control diet. In summary, our data suggest that n-3 PUFA-mediated reduction of host resistance to L. monocytogenes is independent of neutrophil activity.

Dietary (n-3) polyunsaturated fatty acids do not affect the in vivo development and function of Listeria-specific CD4+ and CD8+ effector and memory/effector T cells in mice.

We previously reported that in a mouse model, a diet high in (n-3) PUFA diminishes host survival following an infection from Listeria monocytogenes, a gram-positive bacterial pathogen. In this study we investigated the impact of (n-3) PUFA on the adaptive immune response to L. monocytogenes. BALB/c mice were fed experimental diets either devoid of or rich in (n-3) PUFA from fish oil for 4 wk and then infected with 10(6) actA-deficient L. monocytogenes. At 7 and 35 d postchallenge, effector and memory/effector T cells in the spleen were enumerated by flow cytometry. Surprisingly, the number of Listeria-specific CD4(+) and CD8(+) effector and memory/effector T cells in the spleen was not affected by (n-3) PUFA. Also, the effector cells derived from mice fed either diet were equally capable of conferring protective immunity upon adoptive transfer to naive recipients. Despite our previous data, which demonstrated that (n-3) PUFA profoundly impaired host resistance to L. monocytogenes, pathogen-specific T cell responses were not substantially affected by dietary (n-3) PUFA.

Omega-3 polyunsaturated fatty acids impair in vivo interferon- gamma responsiveness via diminished receptor signaling.

BACKGROUND: A high intake of omega-3 polyunsaturated fatty acids (n-3 PUFAs) in mice causes impaired host resistance to Listeria monocytogenes. We wished to determine the role of interferon (IFN)-gamma signaling in this increased disease susceptibility. METHODS: A feeding trial was conducted with mice unable to produce IFN-gamma (IFN-gamma KO); we provided exogenous recombinant IFN-gamma during L. monocytogenes challenge. The experimental diets were nutritionally complete and differed only in fat source: lard (devoid of n-3 PUFAs) or menhaden fish oil (rich in n-3 PUFAs). RESULTS: The administration of IFN-gamma significantly enhanced bacterial clearance in IFN-gamma KO mice fed a diet devoid of n-3 PUFAs but had no effect in mice fed a diet rich in n-3 PUFAs. Ex vivo analysis of immune cells showed that n-3 PUFAs did not affect IFN-gamma receptor expression on immune cells. However, on IFN-gamma treatment, the phosphorylation of signal transducer and activator of transcription 1 was significantly reduced in peritoneal macrophages isolated from mice fed n-3 PUFAs. CONCLUSIONS: These data suggest that diminished IFN-gamma signaling in murine macrophages is one mechanism by which n-3 PUFAs impair host resistance to L. monocytogenes. To our knowledge, this is the first report of a nutrient affecting IFN-gamma signaling and in vivo responsiveness to this cytokine.

Dietary fish oil impairs primary host resistance against Listeria monocytogenes more than the immunological memory response.

The primary objective of this study was to determine whether dietary (n-3) polyunsaturated fatty acids (PUFA) impair the ability of mice to generate an immunological memory response against the bacterial pathogen, Listeria monocytogenes. Weanling BALB/c female mice were fed for 28 d one of two semipurified high fat diets containing either lard or refined menhaden fish oil, rich in long-chain (n-3) PUFA. Mice were immunized with 10(4) or 10(3) colony forming units (cfu) bacteria. Thirty-five days later, these immune mice and age-matched naïve (i.e., unimmunized) mice were challenged with 10(5) cfu bacteria. Three days postchallenge, bacterial clearance was determined. Compared with lard-fed mice, naïve mice in the fish oil treatment group had higher bacterial loads in their liver and spleen (P < 0.001). When mice were immunized with 10(4) cfu bacteria before rechallenge with 10-fold more bacteria, both lard- and fish oil-fed mice had significantly lower bacterial loads in their liver and spleen (e.g., approximately 2 log(10); P < 0.001) compared with their naïve counterparts. However, when the immunization dose was reduced to 10(3) bacteria, a modest diet treatment effect was observed, such that compared with immune lard-fed mice, immune fish oil-fed mice had significantly greater bacterial loads in their liver and spleen (i.e., approximately 0.5 log(10); P < 0.01). These data demonstrate for the first time that although dietary (n-3) PUFA can significantly impair host resistance to a primary as well as a secondary L. monocytogenes infection, the impairment of the immunological memory response is much less severe.