Induction of tumor-specific CTL responses using the C-terminal fragment of Viral protein R as cell penetrating peptide.

The discovery of tumor-associated antigens recognized by T lymphocytes opens the possibility of vaccinating cancer patients with defined antigens. However, one of the major limitation of peptide-based vaccines is the low immunogenicity of antigenic peptides. Interestingly, if these epitopes are directly delivered into the cytoplasm of antigen presenting cells, they can be efficiently presented via the direct MHC class I presentation pathway. To improve antigen entry, one promising approach is the use of cell penetrating peptides (CPPs). However, most studies use a covalent binding of the CPP with the antigen. In the present study, we focused on the C-terminal domain of Vpr which was previously demonstrated to efficiently deliver plasmid DNA into cells. We provide evidence that the peptides Vpr55-91 and Vpr55-82 possess the capacity of delivering proteins and epitopes into cell lines as well as into human primary dendritic cells, without the necessicity for a chemical linkage. Moreover, immunization of HLA-A2 transgenic mice with Vpr55-91 as the sole adjuvant is able to induce antigen-specific cytotoxic T lymphocytes against multiple tumor epitopes.

Interleukin-22 binding protein (IL-22BP) is constitutively expressed by a subset of conventional dendritic cells and is strongly induced by retinoic acid.

Interleukin-22 (IL-22) is mainly produced at barrier surfaces by T cells and innate lymphoid cells and is crucial to maintain epithelial integrity. However, dysregulated IL-22 action leads to deleterious inflammation and is involved in diseases such as psoriasis, intestinal inflammation, and cancer. IL-22 binding protein (IL-22BP) is a soluble inhibitory IL-22 receptor and may represent a crucial regulator of IL-22. We show both in rats and mice that, in the steady state, the main source of IL-22BP is constituted by a subset of conventional dendritic cells (DCs) in lymphoid and non-lymphoid tissues. In mouse intestine, IL-22BP was specifically expressed in lamina propria CD103(+)CD11b(+) DC. In humans, IL-22BP was expressed in immature monocyte-derived DC and strongly induced by retinoic acid but dramatically reduced upon maturation. Our data suggest that a subset of immature DCs may actively participate in the regulation of IL-22 activity in the gut by producing high levels of IL-22BP.

Probing platelet factor 4 alpha-granule targeting.

The storage mechanism of endogenous secretory proteins in megakaryocyte alpha-granules is poorly understood. We have elected to study the granule storage of platelet factor 4 (PF4), a well-known platelet alpha-granule protein. The reporter protein green fluorescent protein (GFP), PF4, or PF4 fused to GFP (PF4-GFP), were transfected in the well-characterized mouse pituitary AtT20 cell line, and in the megakaryocytic leukemic DAMI cell line. These proteins were also transduced using a lentiviral vector, in human CD34+ cells differentiated into megakaryocytes in vitro. Intracellular localization of expressed proteins, and colocalization studies were achieved by laser scanning confocal microscopy and immuno-electronmicroscopy. In preliminary experiments, GFP, a non-secretory protein (no signal peptide), localized in the cytoplasm, while PF4-GFP colocalized with adrenocorticotropin hormone (ACTH)-containing granules in AtT20 cells. In the megakaryocytic DAMI cell line and in human megakaryocytes differentiated in vitro, PF4-GFP localized in alpha-granules along with the alpha granular protein von Willebrand factor (VWF). The signal peptide of PF4 was not sufficient to specify alpha-granule storage of PF4, since when PF4 signal peptide was fused to GFP (SP4-GFP), GFP was not stored into granules in spite of its efficient translocation to the ER-Golgi constitutive secretory pathway. We conclude that the PF4 storage pathway in alpha-granules is not a default pathway, but rather a regular granule storage pathway probably requiring specific sorting mechanisms. In addition PF4-GFP appears as an appropriate probe with which to analyze alpha-granule biogenesis and its alterations in the congenital defect gray platelet syndrome.

Identification of an HLA-A*0201-restricted epitopic peptide from human dystrophin: application in duchenne muscular dystrophy gene therapy.

Dystrophin-based gene therapy treatments aimed at correcting the Duchenne muscular dystrophy phenotype require stable expression of normal dystrophin (DYST) protein in myocytes without immune responses, which would compromise long-term expression. To predict cytotoxic T-cell-mediated responses elicited by transgenes, we used here H-2-negative HLA-A*0201 transgenic mice and identified human DYST epitopes, which elicit HLA-A*0201-restricted cytotoxic T cell activities. Among a series of eight peptides predicted from the human DYST sequence, not shared with the endogenous mouse DYST sequence, four of them were able to bind to HLA-A*0201 molecules and to induce cytotoxic T lymphocyte (CTL) responses. After human DYST DNA transfer in muscle of HLA-A*0201 mice, only the human DYST1281 epitope, located in the spectrin-like repeat 9 domain, induced strong CD8(+) CTL responses. Using the corresponding human DYST1281 peptide/HLA-A*0201 tetramer, we detected human DYST1281-specific CD8(+) T cells in peripheral lymphoid organs and blood of HLA-A*0201 mice injected with human DYST DNA. Our results demonstrate that muscle injection with human DYST DNA systematically triggers CTL responses against this HLA-A*0201-restricted human DYST1281 peptide, which is present in long human DYST isoforms. Identification of such immunodominant human DYST epitopes and use of peptide/HLA tetramers will allow the immunomonitoring of CTL responses in HLA-phenotyped Duchenne muscular dystrophy patients undergoing gene therapy. Finally, the knowledge of HLA-A*0201-restricted human DYST peptides will be of importance to test, in mouse models, new immunomodulatory interventions allowing long-term engraftment of human dystrophin.

Expression of coagulation factor IX in a haematopoietic cell line.

We have developed a gene therapy project for haemophilia B which aims to express factor IX (FIX) in haematopoietic lineage. Haematopoietic stem cells and subsequent megakaryocyte-derived cells represent the target cells of this approach. Our speculation is that platelets can deliver the coagulation factor at the site of injury, and subsequently correct the haemostasis defect. In order to direct FIX expression in cells from the megakaryocytic lineage, we designed a FIX cassette where the FIX cDNA was placed under the control of the tissue-specific glycoprotein IIb (GPIIb) promoter. In stably transfected HEL cells, FIX production was higher when driven by the GPIIb promoter compared to the CMV promoter. Using a cassette containing both the GPIIb promoter and a truncated FIX intron 1, FIX synthesis was dramatically increased in HEL cells. Northern blot analysis demonstrated an increase in FIX mRNA amounts, which paralleled with an increase of FIX antigen in the culture supernatants. Using a one-stage clotting assay and an activation by FXIa and FVIIa/TF, the HEL-derived recombinant FIX was shown to be a biologically active protein. This recombinant protein exhibited a 60-kDa molecular mass and was more heterogeneous than plasma immunopurified FIX (Mononine). The molecular mass difference could be partly explained by a different glycosylation pattern. The GPIIb promoter appears therefore to be a very attractive sequence to specifically direct FIX production in the megakaryocytic compartment of hematopoietic cells. These data also demonstrate that hematopoietic cells may represent potential target cells in an approach to gene therapy of haemophilia B.

The alpha(IIb)beta(3) integrin and GPIb-V-IX complex identify distinct stages in the maturation of CD34(+) cord blood cells to megakaryocytes.

Megakaryocytopoiesis is a complex multistep process involving cell division, endoreplication, and maturation and resulting in the release of platelets into the blood circulation. Megakaryocytes (MK) progressively express lineage-restricted proteins, some of which play essential roles in platelet physiology. Glycoprotein (GP)Ib-V-IX (CD42) and GPIIb (CD41) are examples of MK-specific proteins having receptor properties essential for platelet adhesion and aggregation. This study defined the progressive expression of the GPIb-V-IX complex during in vitro MK maturation and compared it to that of GPIIb, an early MK marker. Human cord blood CD34(+) progenitor cells were cultured in the presence of cytokines inducing megakaryocytic differentiation. GPIb-V-IX expression appeared at day 3 of culture and was strictly dependent on MK cytokine induction, whereas GPIIb was already present in immature CD34(+) cells. Analysis by flow cytometry and of the messenger RNA level both showed that GPV appeared 1 day later than GPIb-IX. Microscopy studies confirmed the late appearance of GPV, which was principally localized in the cytoplasm when GPIb-IX was found on the cell surface, suggesting a delayed program of GPV synthesis and trafficking. Cell sorting studies revealed that the CD41(+)GPV(+) population contained 4N and 8N cells at day 7, and was less effective than CD41(+)GPV(-) cells in generating burst-forming units of erythrocytes or MK colonies. This study shows that the subunits of the GPIb-V-IX complex represent unique surface markers of MK maturation. The genes coding for GPIb-IX and GPV are useful tools to study megakaryocytopoiesis and for tissue-specific or conditional expression in mature MK and platelets. (Blood. 2000;96:4169-4177)

Identification of a GATA-overlapping sequence within the enhancer of the murine GPIIb promoter that induces transcriptional deregulation in human K562 cells.

The human and the murine glycoprotein platelet IIb (GPIIb) promoters are megakaryocyte specific in human and murine cell systems, respectively. Here we show that the murine promoter is, however, highly active when transfected in K562 human cells in which the human promoter is almost inactive. A murine promoter, in which the enhancer element was replaced by the human, retrieves its megakaryocytic specificity in human cell lines. The human and murine GATA-binding sites located in the enhancer region display slight sequence divergence next to the consensus GATA core sequence. Gel shift experiments show that, although the murine and the human GATA sequences both bind GATA-1, the murine sequence alone forms an additional complex (B) not detected with the human sequence. When the murine GATA-containing region is replaced by the human in the context of the murine GPIIb promoter, megakaryocyte specificity is restored in the human cell lines. A G nucleotide 3 to GATA appears crucial because its substitution abrogates B but not GATA-1 binding and restores megakaryocyte specificity to the murine promoter. Conversely, substitution of the human GATA-1 binding sequence by its murine homologue that binds both GATA-1 and complex B induces an abnormal activity for the human promoter in K562 cells. Altogether, our data suggest that limited changes in the GATA-containing enhancer of the GPIIb promoter can induce the recruitment of accessory proteins that could be involved in alteration of a megakaryocyte-restricted gene activation program. (Blood. 2000;96:1348-1357)

Physical training and injury in female cadets at the United States Military Academy.

Women have been enrolled at the United States Military Academy (USMA) since 1976. All cadets are required to participate in a rigorous physical training curriculum with few differences for male and female cadets. The effect this physical training has on the health of women at West Point has been monitored closely. This paper will review the physical training program at USMA and the gender differences that exist. The health effects of this demanding physical training on women will also be discussed.

[Alkaloids of Annonaceae. XXIX. Alkaloids of Annona muricata]. / Alcaloïdes des Annonacées XXIX1: Alcaloïdes de l'Annona muricata L.

From leaves, root - and stem - barks of Annona muricata L., seven isoquinoline alkaloids have been isolated: reticuline (main alkaloid), coclaurine, coreximine, atherosperminine, stepharine. Anomurine and anomuricine, two minor alkaloids, are new tetrahydrobenzylisoquinolines, with 5, 6, 7 substituted ring A. The phytochemical significance of these alkaloids is discussed.