Expression pattern of sirtuins in innate immune cells and influence of Sirtuin 1/2 inhibition on innate immune responses to Toll-like receptor ligands and to infection

Purpose/Objective: The family of histone deacetylases comprises 18 members in mammals, among which seven sirtuins (SIRT1-7). Sirtuins are NADP-dependent enzymes that have been involved in the control of cell metabolism, proliferation and survival. The expression pattern of sirtuins and their influence on host response to microbial infection remain largely unknown. The aim of the study was to analyze the expression of SIRT1-7 and to address the effects of SIRT1/2 inhibition on innate immune responses in vitro and in vivo.. Materials and methods: in vitro: Bone marrow (BM), BM-derived macrophages (BMDMs) and dendritic cells (BMDCs) and RAW 264.7 and J774.1 macrophage cell lines were stimulated for 0, 2, 6 and 18 h with LPS, Pam3CSK4 and CpG ODN. SIRT1-7 mRNA was quantified by real time-PCR. TNF was measured by ELISA. In vivo: BALB/c mice were challenged with LPS (350 lg i.p.) with or without a SIRT1/2 inhibitor. Blood and organs were collected after 0, 1, 4, 8 and 24 h to quantify SIRT1-7 and TNF. Mortality was assessed daily. Results: Bone marrow, macrophages and DCs express, in order of abundance, SIRT2 > > SIRT1, SIRT3 and SIRT6 > SIRT4, SIRT5 and SIRT7. Microbial products decrease the expression of all sirtuins except SIRT6 in a time dependent manner in BMDMs (0_24 h). SIRT2 is the most expressed sirtuin also in the liver, kidney (together with SIRT3) and spleen. Upon LPS challenge, SIRT1, SIRT3, SIRT4 and SIRT7 mRNA levels decrease in the liver (from 4 h to 24 h), whereas SIRT1-7 mRNA levels decrease within 1 h in both kidney and spleen. Pharmacological inhibition of SIRT1/2 decreases TNF production by macrophages stimulated with LPS, Pam3CSK4 and CpG ODN (n = 6; P < 0.001). In agreement, prophylactic treatment with a SIRT1/2 inhibitor decreases TNF production (n = 8; P = 0.04) and increases survival (n = 13, P = 0.03) of mice challenged with LPS. Conclusions: Sirtuins are expressed in innate immune cells. Inhibition of SIRT1/2 activity decreases cytokine production by macrophages and protects from endotoxemia, suggesting that sirtuin inhibitors may represent novel adjunctive therapy for treating inflammatory disorders such as sepsis.

GLUT2 surface expression and intracellular transport via the constitutive pathway in pancreatic beta cells and insulinoma: evidence for a block in trans-Golgi network exit by brefeldin A.

The biosynthesis, intracellular transport, and surface expression of the beta cell glucose transporter GLUT2 was investigated in isolated islets and insulinoma cells. Using a trypsin sensitivity assay to measure cell surface expression, we determined that: (a) greater than 95% of GLUT2 was expressed on the plasma membrane; (b) GLUT2 did not recycle in intracellular vesicles; and (c) after trypsin treatment, reexpression of the intact transporter occurred with a t1/2 of approximately 7 h. Kinetics of intracellular transport of GLUT2 was investigated in pulse-labeling experiments combined with glycosidase treatment and the trypsin sensitivity assay. We determined that transport from the endoplasmic reticulum to the trans-Golgi network (TGN) occurred with a t1/2 of 15 min and that transport from the TGN to the plasma membrane required a similar half-time. When added at the start of a pulse-labeling experiment, brefeldin A prevented exit of GLUT2 from the endoplasmic reticulum. When the transporter was first accumulated in the TGN during a 15-min period of chase, but not following a low temperature (22 degrees C) incubation, addition of brefeldin A (BFA) prevented subsequent surface expression of the transporter. This indicated that brefeldin A prevented GLUT2 exit from the TGN by acting at a site proximal to the 22 degrees C block. Together, these data demonstrate that GLUT2 surface expression in beta cells is via the constitutive pathway, that transport can be blocked by BFA at two distinct steps and that once on the surface, GLUT2 does not recycle in intracellular vesicles.

The role of bile acid retention and a common polymorphism in the ABCB11 gene as host factors affecting antiviral treatment response in chronic hepatitis C.

Summary.  The outcome of hepatitis C virus (HCV) infection and the likelihood of a sustained virological response (SVR) to antiviral therapy depends on both viral and host characteristics. In vitro studies demonstrated that bile acids (BA) interfere with antiviral interferon effects. We investigate the influence of plasma BA concentrations and an ABCB11 polymorphism associated with lower transporter expression on viral load and SVR. Four hundred and fifty-one Caucasian HCV-patients treated with PEG-interferon and ribavirin were included in the study. ABCB11 1331T>C was genotyped, and plasma BA levels were determined. The 1331C allele was slightly overrepresented in HCV-patients compared to controls. In HCV-patients, a significant difference between patients achieving SVR vs non-SVR was observed for HCV-2/3 (5 vs 9 μm; P = 0.0001), while median BA levels in HCV-1 were marginally elevated. Normal BA levels <8 μm were significantly associated with SVR (58.3%vs 36.3%; OR 2.48; P = 0.0001). This difference was significant for HCV-2/3 (90.7%vs 67.6%; P = 0.002) but marginal in HCV-1 (38.7%vs 27.8%; P = 0.058). SVR rates were equivalent between ABCB11 genotypes for HCV-1, but increased for HCV-2/3 (TT 100%vs CC 78%; OR 2.01; P = 0.043). IL28B genotype had no influence on these associations. No correlation between BA levels and HCV RNA was detected for any HCV genotype. The higher allelic frequency of ABCB11 1331C in HCV-patients compared to controls may indirectly link increased BA to HCV chronicity. Our data support a role for BA as host factor affecting therapy response in HCV-2/3 patients, whereas a weaker association was found for HCV-1.

Heterologous expression and biochemical characterization of an α1,2-mannosidase encoded by the Candida albicans MNS1 gene

Protein glycosylation pathways, commonly found in fungal pathogens, offer an attractive new area of study for the discovery of antifungal targets. In particular, these post-translational modifications are required for virulence and proper cell wall assembly in Candida albicans, an opportunistic human pathogen. The C. albicans MNS1 gene is predicted to encode a member of the glycosyl hydrolase family 47, with 1,2-mannosidase activity. In order to characterise its activity, we first cloned the C. albicans MNS1 gene into Escherichia coli, then expressed and purified the enzyme. The recombinant Mns1 was capable of converting a Man9GlcNAc2 N-glycan core into Man8GlcNAc2 isomer B, but failed to process a Man5GlcNAc2-Asn N-oligosaccharide. These properties are similar to those displayed by Mns1 purified from C. albicansmembranes and strongly suggest that the enzyme is an ±1,2-mannosidase that is localised to the endoplasmic reticulum and involved in the processing of N-linked mannans. Polyclonal antibodies specifically raised against recombinant Mns1 also immunoreacted with the soluble ±1,2-mannosidases E-I and E-II, indicating that Mns1 could share structural similarities with both soluble enzymes. Due to the high degree of similarity between the members of family 47, it is conceivable that these antibodies may recognise ±1,2-mannosidases in other biological systems as well.

Differential expression of THOC1 and ALY mRNP biogenesis/export factors in human cancers

One key step in gene expression is the biogenesis of mRNA ribonucleoparticle complexes (mRNPs). Formation of the mRNP requires the participation of a number of conserved factors such as the THO complex. THO interacts physically and functionally with the Sub2/UAP56 RNA-dependent ATPase, and the Yra1/REF1/ALY RNA-binding protein linking transcription, mRNA export and genome integrity. Given the link between genome instability and cancer, we have performed a comparative analysis of the expression patterns of THOC1, a THO complex subunit, and ALY in tumor samples.

Demyelination induced by protein kinase C-activating tumor promoters in aggregating brain cell cultures.

The plasticity of mature oligodendrocytes was studied in aggregating brain cell cultures at the period of maximal expression of myelin marker proteins. The protein kinase C (PKC)-activating tumor promoters mezerein and phorbol 12-myristate 13-acetate (PMA), but not the inactive phorbol ester analog 4alpha-PMA, caused a pronounced decrease of myelin basic protein (MBP) content and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) activity. In contrast, myelin/oligodendrocyte protein (MOG) content was affected relatively little. Northern blot analyses showed a rapid reduction of MBP and PLP gene expression induced by mezerein, and both morphological and biochemical findings indicate a drastic loss of compact myelin. During the acute phase of demyelination, only a relatively small increase in cell death was perceptible by in situ end labeling and in situ nick translation. Basic fibroblast growth factor (bFGF) also reduced the levels of the oligodendroglial differentiation markers and enhanced the demyelinating effects of the tumor promoters. The present results suggest that PKC activation resulted in severe demyelination and partial loss of the oligodendrocyte-differentiated phenotype.

Efficacy of cetuximab in metastatic castration-resistant prostate cancer might depend on EGFR and PTEN expression: results from a phase II trial (SAKK 08/07).

PURPOSE: The EGF receptor (EGFR) is overexpressed in the majority of metastatic castration-resistant prostate cancers (mCRPC) and might represent a valid therapeutic target. The combination of docetaxel and cetuximab, the monoclonal antibody against EGFR, has not been tested in patients with prostate cancer. EXPERIMENTAL DESIGN: Patients with mCRPC progressing during or within 90 days after at least 12 weeks of docetaxel were included in this phase II trial. Treatment consisted of docetaxel (75 mg/m(2) every 3 weeks or 35 mg/m(2) on days 1, 8, 15 every 4 weeks) in combination with cetuximab (400 mg/m(2) on day 1 and then 250 mg/m(2) weekly). The primary endpoint was progression-free survival (PFS) at 12 weeks defined as the absence of prostate-specific antigen (PSA), radiographic, or clinical progression. Evaluation of known biomarkers of response and resistance to cetuximab (EGFR, PTEN, amphiregulin, epiregulin) was conducted. RESULTS: Thirty-eight patients were enrolled at 15 Swiss centers. Median age was 68 years and median PSA was 212 ng/mL. PFS at 12 weeks was 34% [95% confidence interval (CI), 19%-52%], PFS at 24 weeks was 20%, and median overall survival (OS) was 13.3 months (95% CI, 7.3-15.4). Seven patients (20%) had a confirmed ≥ 50% and 11 patients (31%) a confirmed ≥ 30% PSA decline. About 47% of enrolled patients experienced grade 3 and 8% grade 4 toxicities. A significantly improved PFS was found in patients with overexpression of EGFR and persistent activity of PTEN. CONCLUSIONS: EGFR inhibition with cetuximab might improve the outcome of patients with mCRPC. A potential correlation between EGFR overexpression, persistent expression of PTEN, and EGFR inhibition should be investigated further.