Cytotoxic effector cells with antitumor activity can be amplified ex vivo from biopsies or blood of patients with renal cell carcinoma for cell therapy use

Adoptive immunotherapy with antitumor effector cells is an attractive therapeutic approach in metastatic renal cell carcinoma (RCC). The aim of the work was to enhance in vitro activation of lymphocytes with optimal cytotoxic activity against tumor cells. We evaluated a procedure based on the use of dendritic cells (DCs) loaded with irradiated tumor cells (DC-Tu) to stimulate lymphocytes. Experimental conditions were established with cells from healthy donors and melanoma cell lines. Procedures were then applied to cells from RCC patients. A total of 30 tumor biopsies, 14 proximal lymph nodes, and 17 peripheral blood samples from 30 patients were used. When lymphocytes were stimulated in vitro with DC-Tu, they responded to tumor cells with an increased cytolytic activity for all the assays with donor cells (n=18). For RCC patients, DC-Tu stimulation improved the final cytotoxic activity in only half of the assays (16/31). When significantly enhanced (>10%, n=8), responder cells resulted in a final 43% cytotoxicity against autologous RCC cells. Mechanism of lysis was at least in part class I mediated. Effector cells have no lytic activity against normal renal cells. Percentage of cells with regulatory T-cell phenotype was not found to be enhanced in the DC-Tu stimulated lymphocytes. Individual differences were observed in the characteristics of DCs generated from RCC patients in contrast to that observed in donors and could explain why lymphocyte stimulation was not improved by DC-Tu in half of the RCC assays. T-cell spreading was suitable for a therapeutic use (>10⁹ cells) irrespective of the procedure (with or without DC-Tu stimulation) or the tissular origin of lymphocytes from patients. Data show that precursors of selective antitumor effector cells are present in patients with RCC and can be amplified in vitro either with or without DC-Tu stimulation. One of these populations could be chosen for an adoptive transfer immunotherapy.This work was supported by grants from the Comité Grand Ouest de La Ligue Contre le Cancer and from the Faculté de Médecine de Rennes.     

Analysis of the Bacteriolytic Enzymes of the Autolytic Lactococcus lactis subsp. cremoris Strain AM2 by Renaturing Polyacrylamide Gel Electrophoresis: Identification of a Prophage-Encoded Enzyme

Lactococcus lactis subsp. cremoris AM2 was previously shown to lyse early and extensively during cheese ripening (M.-P. Chapot-Chartier, C. Deniel, M. Rousseau, L. Vassal, and J.-C. Gripon, Int. Dairy J. 4:251–269, 1994). We analyzed the bacteriolytic activities of autolytic strain AM2 by using renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis performed with two different substrates in the gel, Micrococcus lysodeikticus and L. lactis autoclaved cells. Several lytic activities were detected in L. lactis AM2; a major lytic activity, designated A2 (46 kDa), was found only with the L. lactis cell substrate. This activity appears to be different from major peptidoglycan hydrolase AcmA characterized previously (G. Buist, J. Kok, K. J. Leenhouts, M. Dabrowska, G. Venema, and A. J. Haandrickman, J. Bacteriol. 177:1554–1563, 1995), which has a similar molecular mass. The two enzymes differ in substrate specificity as well as in sensitivity to pH and different chemical compounds. L. lactis AM2 is lysogenic and mitomycin C inducible. Enzyme A2 was shown to be inducible by mitomycin C and to be prophage encoded. It was identified as an enzyme similar to the lysin encoded by lactococcal small isometric temperate bacteriophages. A prophage-cured derivative of L. lactis AM2 was obtained, and this isolate exhibited different autolytic properties than AM2. After prolonged incubation in the stationary phase after growth on M17 medium, the extent of lysis of an AM2 culture was 60%, whereas over the same period there was almost no lysis in a prophage-cured derivative strain culture. These results suggest that the prophage lytic system is involved in the strain AM2 lysis observed in liquid medium and that it could also be involved in the lysis observed during cheese ripening.     

Wood density proxies of adaptive traits linked with resistance to drought in Douglas fir (Pseudotsuga menziesii (Mirb.) Franco)

Key message

Proxies of adaptive traits for resistance to drought were discovered among original annual ring density variables in Douglas fir.

Abstract

A comparison of dead and surviving Douglas fir trees following the 2003 drought was made to define proxies of adaptive traits for resistance to drought. Increment cores were sampled from trees from three French regions: Centre, Midi-Pyrénées and Burgundy. Original tree-ring variables were calculated, based on a sliding density criterion dividing the microdensity profile into high- and low-density segments. Tree rings were analysed at each site in a number of consecutive annual rings before the 2003 drought event. Comparison between pairs of surviving and dead trees and between pairs of randomly selected trees (whether dead or alive) supports the evidence of systematic dissimilarities between surviving and dead trees in a number of original density variables. Correlation analysis between original and conventional ring density variables indicates a weak association. We found that the surviving trees were denser than the dead trees in all three sites, but that the denser part of the ring varied from region to region. We identified several original density variables intended to be used as proxies of adaptive traits in future studies of genetic determinism of Douglas fir resistance to drought.     

Mapping of binding site III in the leptin receptor and modeling of a hexameric leptin.leptin receptor complex

The leptin.leptin receptor (LR) system shows strong similarities to the long chain cytokine interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF) cytokine.cytokine receptor systems. The IL-6 family cytokines interact with their receptors through three different binding sites (I-III). We demonstrated previously that leptin has similar binding sites I-III and mapped the interactions between binding site II and cytokine receptor homology domain II (CRH2) (Peelman, F., Van Beneden, K., Zabeau, L., Iserentant, H., Ulrichts, P., Defeau, D., Verhee, A., Catteeuw, D., Elewaut, D., and Tavernier, J. (2004) J. Biol. Chem. 279, 41038-41046). In this study, we built homology models for the CRH1 and Ig-like domains of the LR. The Ig-like domain shows a large conserved surface patch in the beta-sheet formed by beta-strands 3, 6, and 7. Mutations in this patch almost completely abolished the leptin-induced STAT3-dependent reporter activity. We propose that a conserved cluster of residues Leu370, Ala407, Tyr409, His417, and His418 forms the center of binding site III of the LR. We built a hexameric leptin.LR complex model based on the hexameric IL-6 complex. In this model, a conserved hydrophobic protuberance of Val36, Thr37, Phe41, and Phe43 in the A-B loop of leptin fits perfectly in the CRH2 domain, corresponding to the IL-6 alpha-receptor, and forms the center of binding site I. The 2:4 hexameric leptin.LR complex offers a rational explanation for mutagenesis studies and residue conservation.     

HLA-G2, -G3, and -G4 isoforms expressed as nonmature cell surface glycoproteins inhibit NK and antigen-specific CTL cytolysis

HLA-G is a nonclassical MHC class I molecule that plays a major role in maternal-fetal tolerance. Four membrane-bound (HLA-G1 to -G4) and two soluble (HLA-G5, and -G6) proteins are generated by alternative splicing. Only HLA-G1 has been extensively studied in terms of both expression and function. We provide evidence here that HLA-G2, -G3, and -G4 truncated isoforms reach the cell surface of transfected cells, as endoglycosidase H-sensitive glycoproteins, after a 2-h chase period. Moreover, cytotoxicity experiments show that these transfected cells are protected from the lytic activity of both innate (NK cells) and acquired (CTL) effectors. These findings highlight the immunomodulatory role that HLA-G2, -G3, and -G4 proteins will assume during physiologic or pathologic processes in which HLA-G1 expression is altered.     

The alternative translated MDMXp60 isoform regulates MDM2 activity

Isoforms derived from alternative splicing, mRNA translation initiation or promoter usage extend the functional repertoire of the p53, p63 and p73 genes family and of their regulators MDM2 and MDMX. Here we show cap-independent translation of an N-terminal truncated isoform of hMDMX, hMDMX^p60, which is initiated at the 7th AUG codon downstream of the initiation site for full length hMDMX^FL at position +384. hMDMX^p60 lacks the p53 binding motif but retains the RING domain and interacts with hMDM2 and hMDMX^FL. hMDMX^p60 shows higher affinity for hMDM2, as compared to hMDMX^FL. In vitro data reveal a positive cooperative interaction between hMDMX^p60 and hMDM2 and in cellulo data show that low levels of hMDMX^p60 promote degradation of hMDM2 whereas higher levels stabilize hMDM2 and prevent hMDM2-mediated degradation of hMDMX^FL. These results describe a novel alternatively translated hMDMX isoform that exhibits unique regulatory activity toward hMDM2 autoubiquitination. The data illustrate how the N-terminus of hMDMX regulates its C-terminal RING domain and the hMDM2 activity.