During erythrocyte invasion Plasmodium falciparum merozoites use multiple receptor-ligand interactions in a series of coordinated events, but current knowledge of these interactions is limited. Using real-time imaging of invasion, we established that heparin-like molecules block early, and essential, events in erythrocyte invasion by merozoites. All P. falciparum isolates tested, and parasites using different invasion pathways, were inhibited to comparable levels. Furthermore, it was not possible to select for heparin-resistant parasites. Heparin-like molecules occur naturally on the surface of human erythrocytes, where they may act as receptors for binding of merozoite surface proteins. Consistent with this, we demonstrated that MSP1-42, a processed form of merozoite surface protein 1 (MSP1) involved in invasion, bound heparin in a specific manner; furthermore, binding was observed with the secondary processing fragment MSP1-33, but not MSP1-19. We defined key structural requirements of heparin-like molecules for invasion inhibition and interactions with MSP1-42. Optimal activity required a degree of sulfation >/=2, disulfation of the N-acetylglucosamine or hexuronic acid residue, and a minimum chain length of 6 monosaccharides. These findings have significant implications for understanding P. falciparum invasion of erythrocytes, and the development of novel therapeutics and vaccines.

The long-term expression and the ability of a therapeutic gene to confer survival advantage to transduced cells are mandatory requirements for successful anti-HIV gene therapy. In this context, we developed lentiviral vectors (LVs) expressing the F12-Vif derivative Chim3. We recently showed that Chim3 inhibits HIV-1 replication in primary cells by both blocking the accumulation of retrotranscripts, independently of either human APOBEC3G (hA3G) or Vif, and preserving the antiviral function of hA3G. These results were predictive of long-lasting survival of Chim3(+) cells after HIV-1 infection. Furthermore, Vif, like Vpr, deregulates cell cycle progression by inducing a delay in G2 phase. Thus, the aim of this study was to investigate the role of Chim3 on both cell survival and cell cycle regulation after HIV-1 infection. Here, we provide evidence that infected Chim3(+) T-cells prevail over either mock- or empty-LV engineered cells, show reduced G2 accumulation and, as a consequence, ultimately extend their lifespan. Based on these findings, Chim3 rightly belongs to the most efficacious class of antiviral genes. In conclusion, Chim3 usage in anti-HIV gene therapy based on haemopoietic stem cell modification has to be considered as a promising therapeutic intervention to eventually cope HIV-1 infection.

Gene transfer vectors based on retroviruses are commonly used in gene therapy applications because of their unique ability to integrate efficiently into host genomes. This ability also forms the basis of a transformation event that can be induced in transduced cells by transactivation of proto-oncogenes near the vector integration sites. Here, we report on the development of lymphoma in mice generated from embryonic stem cells transduced with an enhanced green fluorescent protein. The cells expressed B220, CD5, Mac1, and IgM on their surfaces and expressed transcription factors characteristic of B cell lymphoma. Importantly, each mouse had a single copy of the provirus in its genome; the copy was integrated into the second intron of the dopamine receptor 3 (D3) gene, and high-level expression of D3 was detected only in the lymphoma cells. Ectopic expression of D3 in murine marrow cells resulted in preferential proliferation of cells at the pre-B-cell stage in response to a D3-specific agonist, but this proliferation was not observed in vivo. Cells co-transduced with D3 and Bcl-x(L) genes had a phenotype similar to that of lymphoma in vivo, suggesting that the leukemogenesis induced by retroviral integration required "second hit" mutations of additional genes.

Hodgkin's lymphoma (HL) relapsing after autologous transplantation (autoSCT) have a dismal outcome. Allogeneic transplantation (alloSCT) employing reduced intensity conditioning (RIC) is a salvage option, but its effectiveness is still unclear. To evaluate the role of RIC alloSCT, we designed a retrospective study based on attending physicians commitment to perform a salvage alloSCT: thus only HL patients HLA-typed immediately after the failed autoSCT were included. Out of 185 patients, 122 found an identical sibling (55%), a matched unrelated (32%) or a haploidentical sibling (13%) donor; 63 patients did not find any donor. Clinical features of both groups did not differ. Two-year progression-free (PFS) and overall survival (OS) were better in the donor group (39.3% versus 14.2%, and 66% versus 42%, respectively, p<0.001) with a median follow-up of 48 months. In multivariable analysis having a donor was significant for better PFS and OS (p<0.001). Patients allografted in complete remission showed a better PFS and OS. This is the largest study comparing RIC alloSCT versus conventional treatment after a failed autoSCT, indicating a survival benefit for patients having a donor.

NA.

T cells contribute to the pathophysiology of ischemic stroke by yet unknown mechanisms. Mice with transgenic T cell receptors (TCR) and mutations in co-stimulatory molecules were used to define the minimal immunological requirements for T cell-mediated ischemic brain damage. Stroke was induced in recombination activating gene 1-deficient (RAG1(-/-)) mice devoid of T and B cells, RAG1(-/-) mice reconstituted with B cells or T cells, TCR-transgenic mice bearing one single CD8+ (2C/RAG2, OTI/RAG1 mice) or CD4+ (OTII/RAG1, 2D2/RAG1 mice) TCR, mice lacking accessory molecules of TCR stimulation (CD28(-/-), PD1(-/-), B7-H1(-/-) mice) or mice deficient in non-classical T cells (NKT and gammadelta T cells) by transient middle cerebral artery occlusion (tMCAO). Stroke outcome was assessed at day 1. RAG1(-/-) mice and RAG1(-/-) mice reconstituted with B cells developed significantly smaller brain infarctions compared to controls but thrombus formation after FeCl(3)-induced vessel injury was unimpaired. In contrast, TCR-transgenic mice and mice lacking co-stimulatory TCR signals were fully susceptible to tMCAO similar to mice lacking NKT and gammadelta T cells. These findings were corroborated by adoptive transfer experiments. Our data demonstrate that T cells critically contribute to cerebral ischemia but their detrimental effect neither depends on antigen recognition nor TCR co-stimulation or thrombus formation.

Allogeneic hematopoietic stem cell transplantation (HSCT) has advanced to a common procedure for treating patients of increasing age with malignancies and immunodeficiency disorders by redirecting the immune system. Unfortunately, cure is often hampered by relapse of the underlying disease, graft versus host disease or severe opportunistic infections, which account for the majority of deaths after HSCT. Enhancing immune reconstitution is therefore an area of intensive research. An increasing variety of approaches has been explored preclinically and clinically: the application of cytokines, keratinocyte growth factor, growth hormone, cytotoxic lymphocytes and mesenchymal stem cells or blockade of sex hormones. New developments of allogeneic HSCT, e.g. umbilical cord blood or haploidentical graft preparations leading to prolonged immunodeficiency have further increased the need to improve immune reconstitution. While a slow T cell reconstitution is regarded as primarily responsible for deleterious infections with viruses and fungi, GVHD and relapse, the importance of innate immune cells for disease and infection control is currently re-evaluated. The ground gets prepared for an individualized therapy partially based on genetic features of the underlying disease. We provide an update on selected issues of development in this fast evolving field without claiming completeness.

Eighty percent of infant ALL cases harbor MLL gene rearrangements (MLL-r). Cooperating leukemogenic events are largely unknown. We explored the role of promoter CpG island hypermethylation in the biology and therapeutic targeting of MLL-r infant ALL. The HpaII-tiny fragment Enrichment by Ligation-mediated PCR (HELP) assay was used to examine genome-wide methylation of a cohort of MLL-r infant leukemia samples (n=5), other common childhood ALL (n=5) and normals (n=5). Unsupervised analysis showed tight clustering of samples into their known biological groups, indicating large differences in methylation patterns. Global hypermethylation was seen in the MLL-r cohort compared to both the normals and the other ALLs, with ratios of significantly (p<0.001) hyper- to hypomethylated CpG's of 1.7 and 2.9 respectively. A subset of 7 differentially hypermethylated genes was assayed by qRT-PCR, confirming relative silencing in 5 of 7. In cell line treatment assays with the DNA methyltransferease inhibitor (DNMTi) decitabine, MLL-r (but not MLL-wt) cell lines showed dose and time dependent cytotoxicity and re-expression of 4 of the 5 silenced genes. MSP confirmed promoter hypermethylation at baseline, and a relative decrease in methylation after treatment. DNMTi may represent a novel molecularly targeted therapy for MLL-r infant ALL.

Although the potential role of Pim2 as a cooperative oncogene has been well described in lymphoma, its role in leukemia has remained largely unexplored. Here we show that high expression of Pim2 is observed in patients with acute promyelocytic leukemia (APL). To further characterize the cooperative role of Pim2 with PML-RARalpha, we used a well-established PML-RARalpha (PRalpha) mouse model. Pim2 coexpression in PRalpha-positive hematopoietic progenitor cells (HPC) induces leukemia in recipient mice after a short latency. Pim2/PRalpha cells were able to repopulate mice in serial transplantations and to induce disease in all of these recipients. Neither Pim2 nor PRalpha alone were sufficient to induce leukemia upon transplantation in this model. The disease induced by Pim2 over-expression in PRalpha cells contained a slightly higher fraction of immature myeloid cells, when compared to the previously described APL disease induced by PRalpha. However, it also clearly resembled an APL-like phenotype and showed signs of differentiation upon ATRA treatment in vitro. These results support the hypothesis that Pim2, which is also a known target of Flt3-ITD (another gene that cooperates with PML-RARalpha), cooperates with PRalpha to induce APL-like disease.

Determining how normal and leukemic stem cells behave in-vivo, in a dynamic and noninvasive way, remains a major challenge. Most optical tracking technologies rely on the use of fluorescent or bioluminescent reporter genes, which need to be stably expressed in the cells of interest. Because gene transfer in primary leukemia samples represents a major risk to impair their capability to engraft in a xenogenic context, we evaluated the possibility to use gene transfer-free labeling technologies. The lipophilic dye carbocyanine DiR was selected among 4 near infrared (NIR) staining technologies. Unfortunately we report here a massive transfer of the dye occurring toward the neighbor cells both in-vivo and in-vitro. We further demonstrate that all lipophilic dyes tested in this study (DiI, DiD, DiR and PKH26) can give rise to micro-environmental contamination, including when used in suboptimal concentration, after extensive washing procedures and in the absence of phagocytosis or marked cell death. This was observed from all cell types tested. Eventually, we show that this microenvironmental contamination is mediated both by direct cell-cell contacts and diffusible micro-particles. We conclude that tracking of labeled-cells using non-genetically encoded markers should always be accompanied by drastic cross validation using multimodality approaches.

All-trans retinoic acid (ATRA) induces granulocytic differentiation and apoptosis in acute promyelocytic leukemia (APL) cells, although the detailed mechanisms are not fully understood. We investigated ATRA-induced cellular responses mediated by the transcription factor FOXO3A in APL cells. FOXO3A was constitutively phosphorylated and localized in the cytoplasm in both APL-derived NB4 cells and primary APL cells. Upon treating the cells with ATRA, FOXO3A phosphorylation was reduced and FOXO3A translocated into the nucleus. Additionally, the expression of TNF-related apoptosis-inducing ligand (TRAIL), a target molecule for FOXO3A, was increased at the transcriptional and protein levels. As expected, transfection of a short hairpin RNA (shRNA) oligonucleotide specific for FOXO3A significantly inhibited ATRA-induced granulocytic differentiation and apoptosis in NB4 cells. In NB4-derived ATRA-resistant NB4/RA cells, neither FOXO3A nuclear localization nor subsequent TRAIL induction was observed after ATRA treatment. Furthermore, forced expression of active FOXO3A in the nucleus induced TRAIL production and apoptosis in NB4/RA cells. We conclude that activation of FOXO3A is an essential event for ATRA-induced cellular responses in NB4 cells. FOXO3A is a promising target for therapeutic approaches to overcome ATRA resistance in APL.

Here, we define an endothelial cell (EC) lumen signaling complex involving Cdc42, Par6b, Par3, junction adhesion molecules (Jam)-B and -C, membrane-type 1- matrix metalloproteinase (MT1-MMP), and integrin, alpha2beta1, which coassociate to control human EC tubulogenesis in 3D collagen matrices. Blockade of both Jam-B and Jam-C using antibodies, siRNA, or dominant negative mutants completely interferes with lumen and tube formation resulting from a lack of Cdc42 activation, inhibition of Cdc42-GTP-dependent signal transduction, and blockade of MT1-MMP-dependent proteolysis. This process requires interdependent Cdc42 and MT1-MMP signaling which involves Par3 binding to the Jam-B and Jam-C cytoplasmic tails, an interaction that is necessary to physically couple the components of the lumen signaling complex. MT1-MMP proteolytic activity is necessary for Cdc42 activation during EC tube formation in 3D collagen matrices but not on 2D collagen surfaces while Cdc42 activation is necessary for MT1-MMP to create vascular guidance tunnels and tube networks in 3D matrices through proteolytic events. This work reveals a novel interdependent role for Cdc42-dependent signaling and MT1-MMP-dependent proteolysis, a process that selectively occurs in 3D collagen matrices and which requires EC lumen signaling complexes, to control human EC tubulogenesis during vascular morphogenesis.

Through its binding with protein S (PS), a key element of the coagulation/fibrinolysis cascade, the C4b-binding protein (C4BP) has been hypothesized to be involved in the susceptibility to venous thrombosis (VT). To identify genetic factors that may influence the plasma levels of the three C4BP existing isoforms, alpha(7)beta(1), alpha(6)beta(1), alpha(7)beta(0), we conducted a genome-wide association study by analyzing 283,437 single nucleotide polymorphisms (SNPs) in the GAIT study composed of 352 individuals. Three SNPs at the C4BPB/C4BPA locus were found genome-wide significantly associated with alpha(7)beta(0) levels. One of these SNPs was further found to explain ~11% of the variability of mRNA C4BPA expression in the Gutenberg Heart Study composed of 1,490 individuals, with no effect on C4BPB mRNA expression. The allele associated with increased alpha(7)beta(0) plasma levels and increased C4BPA expression was further found associated with increased risk of VT (OR = 1.24 [1.03 - 1.53]) in two independent case-control studies (MARTHA and FARIVE) gathering 1706 cases and 1379 controls. This SNP was not associated to free PS nor total PS. In conclusion, we observed strong evidence that the C4BPB/C4BPA locus is a new susceptibility locus for VT through a PS independent mechanism that remains to be elucidated.

Repopulation of immunodeficient mice remains the primary method to assay human hematopoietic stem cells (HSCs). Here we report that female NOD/SCID/IL-2Rg(c)null mice are far superior in detecting human HSCs (Lin(-)CD34(+)CD38(-)CD90(+)CD45RA(-)) in comparison to male recipients. When multiple HSCs were transplanted, female recipients displayed a trend (1.4-fold) towards higher levels of human chimerism (female vs. male: injected femur - 44.4+/-9.3 vs 32.2+/-6.2, n=12f, 24m, p=0.1). Strikingly, this effect was dramatically amplified at limiting cell doses where female recipients had ~11-fold higher chimerism from single HSCs (female vs. male: injected femur - 8.1+/-2.7 vs 0.7+/-0.7, n=28f, 20m, p=0.0001). Secondary transplants from primary recipients indicate that females more efficiently support the self-renewal of human HSCs. Therefore, gender associated factors play a pivotal role in the survival, proliferation and self-renewal of human HSC in the xenograft model and recipient gender must be carefully monitored in the future design of experiments requiring human HSC assays.

Post-transplant non-Hodgkin lymphoma is a life-threatening complication after transplantation. While pharmacologically suppressed adaptive immunity plays a major role in its development, the role of innate immunity in post-transplant lymphoma is unknown. We assessed the 158 V/F poly-morphism in the Fc-gamma receptor 3A gene (FCGR3A); killer cell immunoglobulin-like receptor (KIR)-genotype; KIR ligand status; and a single nucleotide polymorphism affecting the production of IFN-gamma [+874 A/T] in 236 patients with post-transplant lymphoma reported to the Collaborative Transplant Study. In addition, polymorphisms in the IL-10 and TGF-beta genes previously associated with lymphoma development were also typed. Using a split cohort approach gene/allele frequen-cies were related to the 5-year patient survival after the diagnosis of lymphoma and compared to 100 control solid-organ transplant recipients. FCGR3A and KIR genotype significantly influenced survival after diagnosis of post-transplant lymphoma: the hazard of dying was reduced in homozygous carriers of the high affinity V allele (hazard ratio 0.49, 95% confidence interval 0.29-0.82, p=0.006), whereas carrying a genotype including KIR2DL2/KIR2DS2 increased the risk of dying (HR 1.49, 95% CI 1.07-2.05, p=0.02). KIR ligands and cytokine polymorphisms had no effect on survival. None of the genetic loci analyzed emerged as risk factors for lymphoma development.

We investigated the functional relationships between miR-221/222 cluster and p27, a key regulator of cell cycle, in chronic lymphocytic leukemia (CLL). The enforced expression of miR-221/222 in the CLL cell line MEC1 induced a significant down-regulation of p27 protein and conferred a proliferative advantage to the transduced cells that exhibited a faster progression into the S phase of the cell cycle. Accordingly, the expression of miR-221/miR-222 and p27 was found to be inversely related in leukemic cells obtained from the peripheral blood (PB) of 38 CLL patients. Interestingly, when miR-221/222 and p27 protein were evaluated in different anatomic compartments (lymph nodes or bone marrow) of the same patients, increased expression of the two miRNAs became apparent as compared to PB. This finding was paralleled by a low expression of p27. In addition, when CLL cells were induced in vitro to enter cell cycle (e.g. using CpG-ODN), a significant increase of miR-221/222 expression and a marked down-regulation of p27 protein was evident. These data indicate that the miR-221/222 cluster modulates the expression of p27 protein in CLL cells and lead to suggest that miR-221/222 and p27 may represent a regulatory loop that helps maintaining CLL cells in a resting condition.