Acceleration by ceramide of calcium-dependent translocation of phospholipase A2 from cytosol to membranes in platelets

The effect of ceramide on Ca2+-dependent translocation of cytosolic phospholipase A2 (cPLA2) to membranes was studied. Pretreatment of platelets with sphingomyelinase or C6-ceramide (N-hexanoylsphingosine) led to apparent enhancement of Ca2+-ionophore A23187-stimulated arachidonic acid release but did not affect the cytosolic phospholipase A2 (cPLA2) activity. Under these conditions, the cPLA2 proteins in membranes increased significantly, compared with those by A23187 alone. Sphingomyelinase and C6-ceramide, but not C6-dihydroceramide, a control analog of C6-ceramide, also facilitated the Ca2+-dependent increase in the cPLA2 protein, as well as the activity, in membranes induced by addition of Ca2+ into platelet lysate. Protein kinase Calpha, which possesses a Ca2+-dependent lipid binding domain, was increased in membranes in a Ca2+-dependent manner, but the increase was not accelerated by sphingomyelinase or C6-ceramide. These findings suggest that ceramide in membranes potentiates Ca2+-dependent cPLA2 translocation from cytosol to membranes, probably through modification of membrane phospholipid organization.     

Dietary Patterns and Clinical Outcomes in Hemodialysis Patients in Japan: A Cohort Study

Background & Objectives

Little is known about actual dietary patterns and their associations with clinical outcomes in hemodialysis patients. We identified dietary patterns in hemodialysis patients in Japan and examined associations between dietary patterns and clinical outcomes.

Design, setting, participants, measurements

We used data from 3,080 general-population participants in the Hisayama study (year 2007), and data from 1,355 hemodialysis patients in the Japan Dialysis Outcomes and Practice Patterns Study (JDOPPS: years 2005–2007). Food intake was measured using a brief self-administered diet-history questionnaire (BDHQ). To identify food groups with the Hisayama population data, we used principal components analysis with Promax rotation. We adjusted the resulting food groups for total daily energy intake, and then we used those adjusted food-group scores to identify dietary patterns in the JDOPPS patients by cluster analysis (Ward’s method). We then used Cox regression to examine the association between dietary patterns and a composite of adverse clinical outcomes: hospitalization due to cardiovascular disease or death due to any cause.

Results

We identified three food groups: meat, fish, and vegetables. Using those groups we then identified three dietary patterns: well-balanced, unbalanced, and other. After adjusting for potential confounders, we found an association between an unbalanced diet and important clinical events (hazard ratio 1.90, 95% C.I. 1.19–3.04).

Conclusions

Hemodialysis patients whose diet was unbalanced were more likely to have adverse clinical outcomes. Thus hemodialysis patients might benefit not only from portion control, but also from a diet that is well-balanced diet with regard to the food groups identified here as meat, fish, and vegetables.     

Combined blockade of TIM-3 and TIM-4 augments cancer vaccine efficacy against established melanomas

Cancer vaccines have been developed to instruct the endogenous immune responses to autologous tumors and to generate durable clinical responses. However, the therapeutic benefits of cancer vaccines remain insufficient due to the multiple immunosuppressive signals delivered by tumors. Thus, to improve the clinical efficacy of cancer immunotherapy, it is important to develop new modalities to overcome immunosuppressive tumor microenvironments and elicit effective antitumor immune responses. In this study, we show that novel monoclonal antibodies (mAbs) specifically targeting either T cell immunoglobulin mucin protein-3 (TIM-3) or T cell immunoglobulin mucin protein-4 (TIM-4) enhance the therapeutic effects of vaccination against established B16 murine melanomas. This is true for vaccination with irradiated B16 melanoma cells engineered to express the flt3 ligand gene (FVAX). More importantly, combining anti-TIM-3 and anti-TIM-4 mAbs markedly increased vaccine-induced antitumor responses against established B16 melanoma. TIM-3 blockade mainly stimulated antitumor effector activities via natural killer cell-dependent mechanisms, while CD8⁺ T cells served as the main effectors induced by anti-TIM-4 mAb. Our findings reveal that therapeutic manipulation of TIM-3 and TIM-4 may provide a novel strategy for improving the clinical efficacy of cancer immunotherapy.     

Amelioration of age‐related brain function decline by Bruton's tyrosine kinase inhibition

One of the hallmarks of aging is the progressive accumulation of senescent cells in organisms, which has been proposed to be a contributing factor to age‐dependent organ dysfunction. We recently reported that Bruton's tyrosine kinase (BTK) is an upstream component of the p53 responses to DNA damage. BTK binds to and phosphorylates p53 and MDM2, which results in increased p53 activity. Consistent with this, blocking BTK impairs p53‐induced senescence. This suggests that sustained BTK inhibition could have an effect on organismal aging by reducing the presence of senescent cells in tissues. Here, we show that ibrutinib, a clinically approved covalent inhibitor of BTK, prolonged the maximum lifespan of a Zmpste24^?/? progeroid mice, which also showed a reduction in general age‐related fitness loss. Importantly, we found that certain brain functions were preserved, as seen by reduced anxiety‐like behaviour and better long‐term spatial memory. This was concomitant to a decrease in the expression of specific markers of senescence in the brain, which confirms a lower accumulation of senescent cells after BTK inhibition. Our data show that blocking BTK has a modest increase in lifespan in Zmpste24^?/? mice and protects them from a decline in brain performance. This suggests that specific inhibitors could be used in humans to treat progeroid syndromes and prevent the age‐related degeneration of organs such as the brain.     

RECQL1 DNA Repair Helicase: A Potential Therapeutic Target and a Proliferative Marker against Ovarian Cancer

Objective

This study analyzed the clinicopathological correlation between ovarian cancer (OC) and RECQL1 DNA helicase to assess its therapeutic potential.

Methods

Surgically resected OC from 118 retrospective cases, for which paraffin blocks and all clinical data were complete, were used in this study. RECQL1 and Ki-67 immunostaining were performed on sections to correlate RECQL1 staining with subtype and patient survival. Ten OC and two normal cell lines were then examined for RECQL1 expression and were treated with siRNA against RECQL1 to assess its effect on cell proliferation.

Results

Of the 118 cases of adenocarcinoma (50, serous; 26, endometrioid; 21, clear cell; 15, mucinous; 6, other histology), 104 (90%) showed varying levels of RECQL1 expression in the nuclei of OC cells. The Cox hazards model confirmed that diffuse and strong staining of RECQL1 was correlated with histological type. However, RECQL1 expression did not correlate with overall patient survival or FIGO stage. In vitro, RECQL1 expression was exceptionally high in rapidly growing OC cell lines, as compared with normal cells. Using a time-course analysis of RECQL1-siRNA transfection, we observed a significant inhibition in cell proliferation.

Conclusions

RECQL1 DNA helicase is a marker of highly proliferative cells. RECQL1-siRNA may offer a new therapeutic strategy against various subtypes of OC, including platinum-resistant cancers, or in recurrent cancers that gain platinum resistance.     

Duodenal brush border intestinal alkaline phosphatase activity affects bicarbonate secretion in rats

We hypothesized that duodenal HCO(3)(-) secretion alkalinizes the microclimate surrounding intestinal alkaline phosphatase (IAP), increasing its activity. We measured AP activity in rat duodenum in situ in frozen sections with the fluorogenic substrate ELF-97 phosphate and measured duodenal HCO(3)(-) secretion with a pH-stat in perfused duodenal loops. We examined the effects of the IAP inhibitors L-cysteine or L-phenylalanine (0.1-10 mM) or the tissue nonspecific AP inhibitor levamisole (0.1-10 mM) on AP activity in vitro and on acid-induced duodenal HCO(3)(-) secretion in vivo. AP activity was the highest in the duodenal brush border, decreasing longitudinally to the large intestine with no activity in stomach. Villous surface AP activity measured in vivo was enhanced by PGE(2) intravenously and inhibited by luminal L-cysteine. Furthermore, incubation with a pH 2.2 solution reduced AP activity in vivo, whereas pretreatment with the cystic fibrosis transmembrane regulator (CFTR) inhibitor CFTR(inh)-172 abolished AP activity at pH 2.2. L-Cysteine and L-phenylalanine enhanced acid-augmented duodenal HCO(3)(-) secretion. The nonselective P2 receptor antagonist suramin (1 mM) reduced acid-induced HCO(3)(-) secretion. Moreover, L-cysteine or the competitive AP inhibitor glycerol phosphate (10 mM) increased HCO(3)(-) secretion, inhibited by suramin. In conclusion, enhancement of the duodenal HCO(3)(-) secretory rate increased AP activity, whereas inhibition of AP activity increased the HCO(3)(-) secretory rate. These data support our hypothesis that HCO(3)(-) secretion increases AP activity by increasing local pH at its catalytic site and that AP hydrolyzes endogenous luminal phosphates, presumably ATP, which increases HCO(3)(-) secretion via activation of P2 receptors.