Addition of Rapamycin to Anti-CD3 Antibody Improves Long-Term Glycaemia Control in Diabetic NOD Mice

Aims/Hypothesis

Non-Fc-binding Anti CD3 antibody has proven successful in reverting diabetes in the non-obese diabetes mouse model of type 1 diabetes and limited efficacy has been observed in human clinical trials. We hypothesized that addition of rapamycin, an mTOR inhibitor capable of inducing operational tolerance in allogeneic bone marrow transplantation, would result in improved diabetes reversal rates and overall glycemia.

Methods

Seventy hyperglycemic non-obese diabetic mice were randomized to either a single injection of anti CD3 alone or a single injection of anti CD3 followed by 14 days of intra-peritoneal rapamycin. Mice were monitored for hyperglycemia and metabolic control.

Results

Mice treated with the combination of anti CD3 and rapamycin had similar rates of diabetes reversal compared to anti CD3 alone (25/35 vs. 22/35). Mice treated with anti CD3 plus rapamycin had a significant improvement in glycemia control as exhibited by lower blood glucose levels in response to an intra-peritoneal glucose challenge; average peak blood glucose levels 30 min post intra-peritoneal injection of 2 gr/kg glucose were 6.9 mmol/L in the anti CD3 plus rapamycin group vs. 10 mmo/L in the anti CD3 alone (P<0.05).

Conclusions/Interpretation

The addition of rapamycin to anti CD3 results in significant improvement in glycaemia control in diabetic NOD mice.     

2-Methoxyestradiol Induces Mitotic Arrest,Apoptosis, and Synergistic Cytotoxicity with Arsenic Trioxide in Human Urothelial Carcinoma Cells

2-Methoxyestradiol (2-ME), an endogenous derivative of 17β-estradiol, has been reported to elicit antiproliferative responses in various tumors. In this study, we investigated the effects of 2-ME on cell viability, proliferation, cell cycle, and apoptosis in human urothelial carcinoma (UC) cell lines. We used two high-grade human bladder UC cell lines (NTUB1 and T24). After treatment with 2-ME, the cell viability and apoptosis were measured by MTT assay and flow cytometry (fluorescence-activated cell sorting), with annexin V-FITC staining and propidium iodide (PI) labeling. DNA fragmentation was analyzed by agarose gel electrophoresis. Flow cytometry with PI labeling was used for the cell cycle analyses. The protein levels of caspase activations, poly (ADP-ribose) polymerase (PARP) cleavage, phospho-histone H2A.X, phospho-Bad, and cell cycle regulatory molecules were measured by Western blot. The effects of the drug combinations were analyzed using the computer software, CalcuSyn. We demonstrated that 2-ME effectively induces dose-dependent cytotoxicity and apoptosis in human UC cells after 24 h exposure. DNA fragmentation, PARP cleavage, and caspase-3, 7, 8, 9 activations can be observed with 2-ME-induced apoptosis. The decreased phospho-Bad (Ser136 and Ser155) and mitotic arrest of the cell cycle in the process of apoptosis after 2-ME treatment was remarkable. In response to mitotic arrest, the mitotic forms of cdc25C, phospho-cdc2, cyclin B1, and phospho-histone H3 (Ser10) were activated. In combination with arsenic trioxide (As₂O₃), 2-ME elicited synergistic cytotoxicity (combination index <1) in UC cells. We concluded that 2-ME significantly induces apoptosis through decreased phospho-Bad and arrests bladder UC cells at the mitotic phase. The synergistic antitumor effect with As₂O₃ provides a novel implication in clinical treatment of UC.     

Inverse Association of N-Terminal Pro-B-Type Natriuretic Peptide with Metabolic Syndrome in Patients with Congestive Heart Failure

Background

Metabolic syndrome has been shown to be associated with lower levels of plasma N-terminal pro-B-type natriuretic peptide (Nt-proBNP) in the general population. We sought to elucidate the relationship between Nt-proBNP and components of metabolic syndrome in patients with congestive heart failure (CHF).

Methods

Fasting blood samples were obtained from 93 patients in our institution. Plasma levels of Nt-proBNP and other biochemical data were measured. The New York Heart Association (NYHA) classification system (I-IV) was used to define the functional capacity of CHF. Metabolic syndrome and its components were defined using diagnostic criteria from the International Diabetes Federation.

Results

Forty-nine patients (52.7%) had CHF. There was a positive correlation between plasma Nt-proBNP levels and NYHA functional capacity in CHF patients. Plasma Nt-proBNP levels increased significantly with each increasing NYHA class of the disease. The prevalence of metabolic syndrome in CHF patients was higher than that in patients without CHF. Most importantly, we found that plasma Nt-proBNP levels were lower in CHF patients with metabolic syndrome attributable to inverse relationships between plasma Nt-proBNP and body mass index (β = −0.297), plasma triglyceride (β = −0.286) and homeostasis model assessment of insulin resistance (HOMA-IR; β = −0.346). Fasting glucose to insulin ratio (FGIR, an insulin sensitivity index) was positively associated with plasma Nt-proBNP levels (β = 0.491), and was the independent predictor of plasma Nt-proBNP levels in CHF patients.

Conclusions

Plasma Nt-proBNP levels are inversely associated with metabolic syndrome in CHF patients. Reduced plasma Nt-proBNP levels in CHF patients may lead to impaired lipolysis and metabolic function, and may contribute to the development of metabolic syndrome in CHF patients.     

Aliskiren Attenuates Steatohepatitis and Increases Turnover of Hepatic Fat in Mice Fed with a Methionine and Choline Deficient Diet

Background & Aims

Activation of the renin-angiotensin-system is known to play a role in nonalcoholic steatohepatitis. Renin knockout mice manifest decreased hepatic steatosis. Aliskiren is the first direct renin inhibitor to be approved for clinical use. Our study aims to evaluate the possible therapeutic effects and mechanism of the chronic administration of aliskiren in a dietary steatohepatitis murine model.

Methods

Male C57BL/6 mice were fed with a methionine and choline-deficient (MCD) diet to induce steatohepatitis. After 8 weeks of feeding, the injured mice were randomly assigned to receive aliskiren (50 mg_·kg^-1 per day) or vehicle administration for 4 weeks. Normal controls were also administered aliskiren (50 mg_·kg^-1 per day) or a vehicle for 4 weeks.

Results

In the MCD mice, aliskiren attenuated hepatic steatosis, inflammation and fibrosis. Aliskiren did not change expression of lipogenic genes but increase turnover of hepatic fat by up-regulating peroxisome proliferator-activated receptor α, carnitine palmitoyltransferase 1a, cytochrome P450-4A14 and phosphorylated AMP-activated protein kinase. Furthermore, aliskiren decreased the hepatic expression of angiotensin II and nuclear factor κB. The levels of oxidative stress, hepatocyte apoptosis, activation of Kupffer cells and hepatic stellate cells, and pro-fibrotic markers were also reduced in the livers of the MCD mice receiving aliskiren.

Conclusions

Aliskiren attenuates steatohepatitis and fibrosis in mice fed with a MCD diet. Thus, the noted therapeutic effects might come from not only the reduction of angiotensin II but also the up-regulation of fatty acid oxidation-related genes.     

BCRP/ABCG2 Inhibition Sensitizes Hepatocellular Carcinoma Cells to Sorafenib

The multikinase inhibitor, sorafenib (Nexavar®, BAY43-9006), which inhibits both the Raf/MEK/ERK pathway and several receptor tyrosine kinases (RTKs), has shown significantly therapeutic benefits in advanced hepatocellular carcinoma (HCC). However, not all HCC patients respond to sorafenib well and new therapeutic strategies to optimize the efficacy of sorafenib are urgently required. Overexpression of breast cancer resistance protein (BCRP/ABCG2) mediates the drug-efflux of several tyrosine kinase inhibitors (TKIs) to attenuate their efficacy. This study aimed to investigate the role of BCRP/ABCG2 in the sensitivity of HCC to sorafenib. Our data showed that BCRP/ABCG2 mediated the efflux of sorafenib. Co-treatment with a BCRP/ABCG2 inhibitor greatly augmented the cytotoxicity of sorafenib in HCC cells. Similar results were also achieved by the competitive inhibitor of BCRP/ABCG2, gefitinib, in combination with sorafenib. These results suggest not only that BCRP/ABCG2 is a potential predictor for the sorafenib sensitivity in HCC, but also that blockage of BCRP/ABCG2 may be a potential strategy to increase the response of HCC cells to sorafenib.     

Phytochemical Investigation of Annulohypoxylon ilanense,an Endophytic Fungus Derived from Cinnamomum Species

Cultivation of the fungal strain Annulohypoxylon ilanense, an endophytic fungus isolated from the wood of medicinal plant Cinnamomum species, resulted in the isolation of one new furanoid derivative, ilanefuranone ( 1 ), one new pyrrole alkaloid, ilanepyrrolal ( 2 ), and one new biarylpropanoid derivative, ilanenoid ( 3 ), together with 22 known compounds, of which one α‐tetralone analog, (?)‐(4R)‐3,4‐dihydro‐4,6‐dihydroxynaphthalen‐1(2H)‐one ( 4 ) was isolated for the first time from a natural source. The structures were elucidated on the basis of physicochemical evidence, in‐depth NMR spectroscopic analysis, and high‐resolution mass spectrometry, and the antimycobacterial activities were also evaluated.     

Proteomic analysis for Type I interferon antagonism of Japanese encephalitis virus NS5 protein

Japanese encephalitis virus (JEV) nonstructural protein 5 (NS5) exhibits a Type I interferon (IFN) antagonistic function. This study characterizes Type I IFN antagonism mechanism of NS5 protein, using proteomic approach. In human neuroblastoma cells, NS5 expression would suppress IFNβ‐induced responses, for example, expression of IFN‐stimulated genes PKR and OAS as well as STAT1 nuclear translocation and phosphorylation. Proteomic analysis showed JEV NS5 downregulating calreticulin, while upregulating cyclophilin A, HSP 60 and stress‐induced‐phosphoprotein 1. Gene silence of calreticulin raised intracellular Ca²⁺ levels while inhibiting nuclear translocalization of STAT1 and NFAT‐1 in response to IFNβ, thus, indicating calreticulin downregulation linked with Type I IFN antagonism of JEV NS5 via activation of Ca²⁺/calicineurin. Calcineurin inhibitor cyclosporin A attenuated NS5‐mediated inhibition of IFNβ‐induced responses, for example, IFN‐sensitive response element driven luciferase, STAT1‐dependent PKR mRNA expression, as well as phosphorylation and nuclear translocation of STAT1. Transfection with calcineurin (vs. control) siRNA enhanced nuclear translocalization of STAT1 and upregulated PKR expression in NS5‐expressing cells in response to IFNβ. Results prove Ca²⁺, calreticulin, and calcineurin involvement in STAT1‐mediated signaling as well as a key role of JEV NS5 in Type I IFN antagonism. This study offers insights into the molecular mechanism of Type I interferon antagonism by JEV NS5.     

Binding of HIV-1 gp120 to DC-SIGN Promotes ASK-1-Dependent Activation-Induced Apoptosis of Human Dendritic Cells

During disease progression to AIDS, HIV-1 infected individuals become increasingly immunosuppressed and susceptible to opportunistic infections. It has also been demonstrated that multiple subsets of dendritic cells (DC), including DC-SIGN(+) cells, become significantly depleted in the blood and lymphoid tissues of AIDS patients, which may contribute to the failure in initiating effective host immune responses. The mechanism for DC depletion, however, is unclear. It is also known that vast quantities of viral envelope protein gp120 are shed from maturing HIV-1 virions and form circulating immune complexes in the serum of HIV-1-infected individuals, but the pathological role of gp120 in HIV-1 pathogenesis remains elusive. Here we describe a previously unrecognized mechanism of DC death in chronic HIV-1 infection, in which ligation of DC-SIGN by gp120 sensitizes DC to undergo accelerated apoptosis in response to a variety of activation stimuli. The cultured monocyte-derived DC and also freshly-isolated DC-SIGN(+) blood DC that were exposed to either cross-linked recombinant gp120 or immune-complex gp120 in HIV(+) serum underwent considerable apoptosis after CD40 ligation or exposure to bacterial lipopolysaccharide (LPS) or pro-inflammatory cytokines such as TNFα and IL-1β. Furthermore, circulating DC-SIGN(+) DC that were isolated directly from HIV-1(+) individuals had actually been pre-sensitized by serum gp120 for activation-induced exorbitant apoptosis. In all cases the DC apoptosis was substantially inhibited by DC-SIGN blockade. Finally, we showed that accelerated DC apoptosis was a direct consequence of excessive activation of the pro-apoptotic molecule ASK-1 and transfection of siRNA against ASK-1 significantly prevented the activation-induced excessive DC death. Our study discloses a previously unknown mechanism of immune modulation by envelope protein gp120, provides new insights into HIV immunopathogenesis, and suggests potential therapeutic approaches to prevent DC depletion in chronic HIV infection.     

A novel prenylated C6–C3 compound with estrogen-like activity from the fruits of Illicium arborescens

A novel C₆–C₃ prenylated compound, illicarborene A (1), together with illioliganfunone D (2), 1-allyl-3,5-dimethoxy-4-(3-methylbut-2-enyloxy)benzene (3), (?)-illicinone A (4), (?)-illicinone B (5) and (?)-illicinone A derivative (6) was isolated and characterized from the fruits of Illicium arborescens Hayata. Compound 1 possesses a new class of tricyclic 6/6/5 ring system. The structure of 1 was determined by spectroscopic analysis such as ¹H–¹H COSY, HMQC, HMBC, and NOESY, and confirmed by chemical reaction to yield 7. Compounds 1–5 were found to increase proliferative activity in primary cell culture of osteoblast cells.