Early T cell signalling is reversibly altered in PD-1+ T lymphocytes infiltrating human tumors

To improve cancer immunotherapy, a better understanding of the weak efficiency of tumor-infiltrating T lymphocytes (TIL) is necessary. We have analyzed the functional state of human TIL immediately after resection of three types of tumors (NSCLC, melanoma and RCC). Several signalling pathways (calcium, phosphorylation of ERK and Akt) and cytokine secretion are affected to different extents in TIL, and show a partial spontaneous recovery within a few hours in culture. The global result is an anergy that is quite distinct from clonal anergy induced in vitro, and closer to adaptive tolerance in mice. PD-1 (programmed death -1) is systematically expressed by TIL and may contribute to their anergy by its mere expression, and not only when it interacts with its ligands PD-L1 or PD-L2, which are not expressed by every tumor. Indeed, the TCR-induced calcium and ERK responses were reduced in peripheral blood T cells transfected with PD-1. Inhibition by sodium stibogluconate of the SHP-1 and SHP-2 phosphatases that associate with several inhibitory receptors including PD-1, relieves part of the anergy apparent in TIL or in PD-1-transfected T cells. This work highlights some of the molecular modifications contributing to functional defects of human TIL.     

Phytochemical,Antiplasmodial, Cytotoxic and Antimicrobial Evaluation of a Southeast Brazilian Brown Propolis Produced by Apis mellifera Bees

Seven phenolic compounds (ferulic acid, caffeic acid, 4-methoxycinnamic acid, 3,4-dimethoxycinnamic acid, 3-hydroxy-4-methoxybenzaldehyde, 3-methoxy-4-hydroxypropiophenone and 1-O,2-O-digalloyl-6-O-trans-p-coumaroyl-β-D-glucopyranoside), a flavanonol (7-O-methylaromadendrin), two lignans (pinoresinol and matairesinol) and six diterpenic acids/alcohol (19-acetoxy-13-hydroxyabda-8(17),14-diene, totarol, 7-oxodehydroabietic acid, dehydroabietic acid, communic acid and isopimaric acid) were isolated from the hydroalcoholic extract of a Brazilian Brown Propolis and characterized by NMR spectral data analysis. The volatile fraction of brown propolis was characterized by CG-MS, composed mainly of monoterpenes and sesquiterpenes, being the major α-pinene (18.4 %) and β-pinene (10.3 %). This propolis chemical profile indicates that Pinus spp., Eucalyptus spp. and Araucaria angustifolia might be its primary plants source. The brown propolis displayed significant activity against Plasmodium falciparum D6 and W2 strains with IC₅₀ of 5.3 and 9.7 μg/mL, respectively. The volatile fraction was also active with IC₅₀ of 22.5 and 41.8 μg/mL, respectively. Among the compounds, 1-O,2-O-digalloyl-6-O-trans-p-coumaroyl-β-D-glucopyranoside showed IC₅₀ of 3.1 and 1.0 μg/mL against D6 and W2 strains, respectively, while communic acid showed an IC₅₀ of 4.0 μg/mL against W2 strain. Cytotoxicity was determined on four tumor cell lines (SK-MEL, KB, BT-549, and SK-OV-3) and two normal renal cell lines (LLC-PK1 and VERO). Matairesinol, 7-O-methylaromadendrin, and isopimaric acid showed an IC₅₀ range of 1.8–0.78 μg/mL, 7.3–100 μg/mL, and 17–18 μg/mL, respectively, against the tumor cell lines but they were not cytotoxic against normal cell lines. The crude extract of brown propolis displayed antimicrobial activity against C. neoformans, methicillin-resistant Staphylococcus aureus, and P. aeruginosa at 29.9 μg/mL, 178.9 μg/mL, and 160.7 μg/mL, respectively. The volatile fraction inhibited the growth of C. neoformans at 53.0 μg/mL. The compounds 3-hydroxy-4-methoxybenzaldehyde, 3-methoxy-4-hydroxypropiophenone and 7-oxodehydroabietic acid were active against C. neoformans, and caffeic and communic acids were active against methicillin-resistant Staphylococcus aureus.     

Combined administration of sodium chloro-2-propionate and insulin on the secretion of glucagon by the pancreas in the diabetic rat

This work was designed to study the effects of sodium 2-chloropropionate (2CP) alone or combined with insulin, in vitro, on glucagon secretion from pancreas isolated from rats, made diabetic by streptozotocin (66 mg/kg i.p.). The pancreata were perfused with a physiological solution containing 2.8 mM glucose (0.5 g/l) and glucagon secretion was stimulated by an arginine infusion (5 mM) for 30 min. When 2CP (1 mM) and/or insulin (4 IU/l) were applied, they were infused from the start of the organ perfusion. In the presence of glucose alone, a marked decrease in glucagon output was observed in diabetic rat pancreas. The arginine perfusion induced a biphasic glucagon secretion both in normal and diabetic rat pancreas; this response was however clearly reduced in diabetic rat pancreas. In diabetic rat pancreas, the infusion of either 2CP or insulin had no effect on glucagon output in presence of glucose alone, nor did it modify the response to arginine. In contrast, the combined infusion of insulin and 2CP induced different effects depending on the conditions: whereas in presence of glucose alone it restored a glucagon output close to that recorded in normal rat pancreas, it did not modify the response to arginine.     

The susceptibility to insulin-dependent diabetes mellitus bis associated with C4 allotypes independently of the association with HLA-DQ alleles in HLA-DR3, 4 heterozygotes

In the genetically homogeneous Danish population, 27 HLA-DR3,4 heterozygous patients with insulin-dependent diabetes mellitus (IDDM) and 19 DR3,4 heterozygous controls without family history of IDDM were investigated for HLA-region markers and Gm and Km immunoglobulin allotypes. The aim was to define susceptibility factors for IDDM development other than HLA-DR using a number of techniques: lymphocytotoxicity (HLA-DR and DQ antigens), cellular methods (Dw and DP typing), restriction fragment length polymorphism (DQ alleles), electrophoresis and immunofixation (BF and C4 allotypes), and passive hemagglutination inhibition (Gm and Km immunoglobulin allotypes). The complement allotype C4A3 and the HLA-DQw8 (DQw3.2) antigen were found in all of the patients, whereas this was the case for only 8 of the 19 controls (P=6 x 10^–6): five lacked C4A3, five others lacked DQw8, and one of the controls lacked both of these factors. Fourteen of the patients had the complement allotype C4B3 versus three of the controls (P=0.01). Previously reported family studies suggest that these alleles are part of the following haplotype: B15, BFS, C4A3, C4B3, DR4, Dw4, DQw8, and these factors were found together in ten of the patients versus one of the controls (P=0.01). The markers usually associated with DR3 did not show significant differences between IDDM patients and controls, and the non-HLA markers studied showed no significant deviation from what was expected. In addition to the susceptibility factor DQw8, the study suggests the existence of susceptibility genes for IDDM near the complement C4 genes on DR4-carrying haplotypes. Since recent works have shown that the structural gene for the monokine tumor necrosis factor alpha (TNF-) is located between the HLA-B and C4 loci and that TNF- might be of importance in IDDM pathogenesis, the hypothesis is put forward that the C4-associated IDDM susceptibility reflects linkage dis-equilibrium between the C4 gene and a gene controlling TNF- production. The high relative risk for IDDM in HLA-DR3,4 heterozygotes might be explained by the combined action of IDDM-specific susceptibility genes on DR4 haplotypes and DR3-linked susceptibility genes associated with predisposition to autoimmunity.     

rs5888 Variant of SCARB1 Gene Is a Possible Susceptibility Factor for Age-Related Macular Degeneration

Major genetic factors for age-related macular degeneration (AMD) have recently been identified as susceptibility risk factors, including variants in the CFH gene and the ARMS2 LOC387715/HTRA1locus. Our purpose was to perform a case-control study in two populations among individuals who did not carry risk variants for CFHY402H and LOC387715 A69S (ARMS2), called “study” individuals, in order to identify new genetic risk factors. Based on a candidate gene approach, we analyzed SNP rs5888 of the SCARB1 gene, coding for SRBI, which is involved in the lipid and lutein pathways. This study was conducted in a French series of 1241 AMD patients and 297 controls, and in a North American series of 1257 patients with advanced AMD and 1732 controls. Among these individuals, we identified 61 French patients, 77 French controls, 85 North American patients and 338 North American controls who did not carry the CFH nor ARMS2 polymorphisms. An association between AMD and the SCARB1 gene was seen among the study subjects. The genotypic distribution of the rs5888 polymorphism was significantly different between cases and controls in the French population (p<0.006). Heterozygosity at the rs5888 SNP increased risk of AMD compared to the CC genotypes in the French study population (odds ratio (OR) = 3.5, CI95%: 1.4–8.9, p<0.01) and after pooling the 2 populations (OR = 2.9, 95% CI: 1.6–5.3, p<0.002). Subgroup analysis in exudative forms of AMD revealed a pooled OR of 3.6 for individuals heterozygous for rs5888 (95% CI: 1.7–7.6, p<0.0015). These results suggest the possible contribution of SCARB1, a new genetic factor in AMD, and implicate a role for cholesterol and antioxidant micronutrient (lutein and vitamin E) metabolism in AMD.     

Regioselectivity of thiouracil alkylation: Application to optimization of Darapladib synthesis

Darapladib is one of the most potent Lp-PLA₂ (Lipoprotein-associated phospholipase A₂) inhibitor with an IC₅₀ of 0.25?nM. We demonstrate that a crucial step of Darapladib synthesis was not correctly described in the literature, leading to the production of wrong regioisomers. Moreover we show that the inhibitory activity is directly linked to the position on N1 since compounds bearing alkylation on different sites have potentially less interaction within the active site of Lp-PLA₂.     

The regulatory role of the IS 1-encoded InsA protein in transposition

We show here that the protein InsA, which is encoded by IS 1 and binds specifically to the terminal inverted repeats of this insertion sequence, negatively regulates IS 1 transposition activity. We demonstrate that it inhibits both IS 1-mediated cointegrate formation and transposition of a synthetic IS 1-based transposon (‘omegon’Ω-on). These results also indicate that the Ω-on which does not itself encode IS 1 transposition functions can be complemented in trans, presumably by the copies of IS 1 resident in the Escherichia coli chromosome. Using insA-lacZ gene fusions, we show that at least part of this effect can be explained by the ability of InsA to repress expression of IS 1-encoded genes both in cis or in trans. The experiments involving Ω-on transposition raise the possibility that InsA inhibits transposition directly by competition with the transposase for their cognate site within the ends of IS 1.