Long-term survival and immunological parameters in metastatic melanoma patients who responded to ipilimumab 10 mg/kg within an expanded access programme

Background

Ipilimumab can result in durable clinical responses among patients with advanced melanoma. However, no predictive marker of clinical activity has yet been identified. We provide preliminary data describing the correlation between immunological parameters and response/survival among patients with advanced melanoma who received ipilimumab 10 mg/kg in an expanded access programme.

Methods

Patients received ipilimumab 10 mg/kg every 3 weeks (Q3W) for four doses (induction) and Q12W from week 24 (W24) as maintenance therapy. Tumor assessments were conducted Q12W. Expression of inducible T cell costimulator (ICOS) on CD4⁺ and CD8⁺ T cells was assessed at baseline, W7, W12 and W24, and the ratio between absolute neutrophils (N) and lymphocytes (L) determined at baseline, W4, W7 and W10.

Results

Median overall survival among 27 patients was 9.6 months (95 % CI 3.2–16.1), with 3- and 4-year survival rates of 20.4 %. Five patients survived >4 years. Patients with an increase in the number of circulating ICOS⁺ T cells at W7 were more likely to experience disease control and have improved survival. An N/L ratio below the median at W7 and W10 was also associated with better survival compared with an N/L ratio above the median.

Conclusions

Ipilimumab can induce long-term survival benefits in heavily pretreated patients with metastatic melanoma. Changes in the number of circulating ICOS⁺ T cells or N/L ratio during ipilimumab treatment may represent early markers of response. However, given the limited sample size, further investigation is required.     

Evaluation of AMCase and CHIT-1 expression in monocyte macrophages lineage

Acidic mammalian chitinase (AMCase) and chitotriosidase (CHIT-1) are two active chitinases expressed in humans. The chitinase activity of AMCase was found to be causative in allergic inflammation and its expression was found to be induced by interleukin-13. CHIT1-1 is expressed by phagocytic cells and extremely high levels are seen in lysosomal storage diseases. Despite that AMCase expression in the inflammation is under investigation, little is known regarding its regulation during macrophages' full maturation and polarization. In this study, we compared AMCase and CHIT-1 modulation during monocyte to macrophage transition and polarization. Gene expression analysis was investigated by real-time PCR from mRNA of human monocytes obtained from buffy coat of healthy volunteers, from mRNA of polarized to classically activated macrophages (or M1), obtained by interferon (IFN)-γ and lipopolysaccharide (LPS) treatment, and from mRNA of alternatively activated macrophages (or M2) obtained by interleukin (IL)-4 exposure. Our results showed that the expression of AMCase and CHIT-1 were differently modulated in HMMs at different stage of maturation. The behavior of these two active chitinase suggests that in the immune response their role is complementary.     

Development of a RNA extraction method from milk for gene expression study in the mammary gland of sheep

The aim of the study was to develop a reliable method for the RNA extraction from milk of Sarda sheep breed and to highlight if the extracted RNA can be used for expression study on mammary genes involved in milk fat synthesis using RT-qPCR. The main result is that a sample of 150 ml of milk provides an optimal amount of RNA (73.5 μg/ml). The highest RNA concentration has been found in the samples analysed within 4 h after collection. The RNA extracted was positively correlated to the number of somatic cells (P < 0.001). The efficiency of the extraction method was confirmed by the results obtained from qPCR which showed a Ct value, for SREBPF1 gene of 26.8 ± 0.15. This research demonstrated that the high-quality of the RNA obtained is suited to use for studies of mammary genes expression in sheep, avoiding any damage caused by mammary gland biopsy.     

Protease-activated Receptor 1 (PAR1) and PAR4 Heterodimers Are Required for PAR1-enhanced Cleavage of PAR4 by α-Thrombin

Thrombin is a potent platelet agonist that activates platelets and other cells of the cardiovascular system by cleaving its G-protein-coupled receptors, protease-activated receptor 1 (PAR1), PAR4, or both. We now show that cleaving PAR1 and PAR4 with α-thrombin induces heterodimer formation. PAR1-PAR4 heterodimers were not detected when unstimulated; however, when the cells were stimulated with 10 nm α-thrombin, we were able to detect a strong interaction between PAR1 and PAR4 by bioluminescence resonance energy transfer. In contrast, activating the receptors without cleavage using PAR1 and PAR4 agonist peptides (TFLLRN and AYPGKF, respectively) did not enhance heterodimer formation. Preventing PAR1 or PAR4 cleavage with point mutations or hirugen also prevented the induction of heterodimers. To further characterize the PAR1-PAR4 interactions, we mapped the heterodimer interface by introducing point mutations in transmembrane helix 4 of PAR1 or PAR4 that prevented heterodimer formation. Finally, we show that mutations in PAR1 or PAR4 at the heterodimer interface prevented PAR1-assisted cleavage of PAR4. These data demonstrate that PAR1 and PAR4 require allosteric changes induced via receptor cleavage by α-thrombin to mediate heterodimer formation, and we have determined the PAR1-PAR4 heterodimer interface. Our findings show that PAR1 and PAR4 have dynamic interactions on the cell surface that should be taken into account when developing and characterizing PAR antagonists.     

Nicotinic acid and DP1 blockade: studies in mouse models of atherosclerosis

The use of nicotinic acid to treat dyslipidemia is limited by induction of a “flushing” response, mediated in part by the interaction of prostaglandin D₂ (PGD₂) with its G-protein coupled receptor, DP1 (Ptgdr). The impact of DP1 blockade (genetic or pharmacologic) was assessed in experimental murine models of atherosclerosis. In Ptgdr^−/−ApoE^−/− mice versus ApoE^−/− mice, both fed a high-fat diet, aortic cholesterol content was modestly higher (1.3- to 1.5-fold, P < 0.05) in Ptgdr^−/−ApoE^−/− mice at 16 and 24 weeks of age, but not at 32 weeks. In multiple ApoE^−/− mouse studies, a DP1-specific antagonist, L-655, generally had a neutral to beneficial effect on aortic lipids in the presence or absence of nicotinic acid treatment. In a separate study, a modest increase in some atherosclerotic measures was observed with L-655 treatment in Ldlr^−/− mice fed a high-fat diet for 8 weeks; however, this effect was not sustained for 16 or 24 weeks. In the same study, treatment with nicotinic acid alone generally decreased plasma and/or aortic lipids, and addition of L-655 did not negate those beneficial effects. These studies demonstrate that inhibition of DP1, with or without nicotinic acid treatment, does not lead to consistent or sustained effects on plaque burden in mouse atherosclerotic models.     

Étude comparée de Saga rhodiensis Salfi 1929 et Saga natoliae Serville 1839 (Orthoptera: Tettigoniidae)

Saga rhodiensis se révèle très proche de S. natoliae et ces deux taxons s’acceptent mutuellement pour la copulation. En outre, ils constituent, avec S. hellenica, un groupe assez homogène au niveau chromosomique. Sur le plan morphologique S. hellenica se distingue aisément des deux autres espèces, et aucun accouplement croisé n’a pu aboutir dans nos élevages. La découverte d’individus femelles de S. natoliae présentant les critères morphologiques de S. rhodiensis bien audelà de l’aire de distribution actuellement connue, nous interroge sur la pertinence de ces critères. Des investigations complémentaires sont nécessaires pour préciser le statut exact de S. rhodiensis.     

Phylogeographical pattern of Euplotes nobilii,a protist ciliate with a bipolar biogeographical distribution

Nuclear (18S and ITS) and mitochondrial (16S) ribosomal RNA gene sequences were determined from genetically distinct wild‐type strains of Antarctic (nine strains), Fuegian (four strains), Greenland (nine strains) and Svalbard (three strains) populations of the marine ciliate, Euplotes nobilii, and analysed for their nucleotide polymorphisms. A close genetic homogeneity was found within and between the Antarctic and Fuegian populations, while more significant levels of genetic differentiation were detected within and between the two Arctic populations, as well as between these populations and the Antarctic/Fuegian ones. The phylogeographical pattern that was derived from these data indicates that gene flow is not limited among Arctic populations; it equally connects the Arctic and Antarctic populations either directly, or through the Fuegian population. This indication reinforces previous evidence from laboratory assays of mating interactions between some of the strains analysed in this work that Southern and Northern polar populations of E. nobilii belong to a unique, panmictic population that substantially share the same gene pool.     

Temperature‐dependent conformational change affecting Tyr11 and sweetness loops of brazzein

The sweet protein brazzein, a member of the Csβα fold family, contains four disulfide bonds that lend a high degree of thermal and pH stability to its structure. Nevertheless, a variable temperature study has revealed that the protein undergoes a local, reversible conformational change between 37 and 3°C with a midpoint about 27°C that changes the orientations and side‐chain hydrogen bond partners of Tyr8 and Tyr11. To test the functional significance of this effect, we used NMR saturation transfer to investigate the interaction between brazzein and the amino terminal domain of the sweet receptor subunit T1R2; the results showed a stronger interaction at 7°C than at 37°C. Thus the low temperature conformation, which alters the orientations of two loops known to be critical for the sweetness of brazzein, may represent the bound state of brazzein in the complex with the human sweet receptor. Proteins 2013; © 2012 Wiley Periodicals, Inc.