VEGFR-3 Neutralization Inhibits Ovarian Lymphangiogenesis,Follicle Maturation,and Murine Pregnancy

Lymphatic vessels surround follicles within the ovary, but their roles in folliculogenesis and pregnancy, as well as the necessity of lymphangiogenesis in follicle maturation and health, are undefined. We used systemic delivery of mF4-31C1, a specific antagonist vascular endothelial growth factor receptor 3 (VEGFR-3) antibody to block lymphangiogenesis in mice. VEGFR-3 neutralization for 2 weeks before mating blocked ovarian lymphangiogenesis at all stages of follicle maturation, most notably around corpora lutea, without significantly affecting follicular blood angiogenesis. The numbers of oocytes ovulated, fertilized, and implanted in the uterus were normal in these mice; however, pregnancies were unsuccessful because of retarded fetal growth and miscarriage. Fewer patent secondary follicles were isolated from treated ovaries, and isolated blastocysts exhibited reduced cell densities. Embryos from VEGFR-3–neutralized dams developed normally when transferred to untreated surrogates. Conversely, normal embryos transferred into mF4-31C1–treated dams led to the same fetal deficiencies observed with in situ gestation. Although no significant changes were measured in uterine blood or lymphatic vascular densities, VEGFR-3 neutralization reduced serum and ovarian estradiol concentrations during gestation. VEGFR-3–mediated lymphangiogenesis thus appears to modulate the folliculogenic microenvironment and may be necessary for maintenance of hormone levels during pregnancy; both of these are novel roles for the lymphatic vasculature.Ovarian neovascularization provides a unique environment in which to study physiological adult vasculogenesis apart from the traditional settings of wound healing and cancer pathologies. Lymphatic circulation plays a central role in fluid, lipid, and cellular transport,¹ and lymphatic vessels are present within the ovary and surround follicles during development and maturation,^2–5 but the importance of the lymphatic vasculature and lymphangiogenesis in the ovary is unclear. Consequently, the potential roles of lymphatic vessels in follicle maturation and pregnancy, and the extent of involvement or even necessity of maternal lymphangiogenesis in reproduction, are undefined. This contrasts with ovarian blood angiogenesis, whose critical roles in follicular nourishment and maturation and in the formation and maintenance of the corpus luteum are well appreciated; indeed, oocyte fertilization, embryonic implantation, uterine expansion, and successful gestation all require blood angiogenesis.^6–8 Lymphangiogenesis, which is often concurrent with blood angiogenesis,⁹ may also play an important role in these processes.Adult blood angiogenesis requires signaling via vascular endothelial growth factor receptor 2 (VEGFR-2), most potently by VEGF ligation.^10,11 In murine ovaries, VEGF expression increases during angiogenic growth phases,¹² and blockade of VEGFR-2 signaling in mice effectively prevents angiogenesis, resulting in a marked decrease in ovarian weight, blood vessel density, and number of corpora lutea, and in infertility.^13–15 Because gonadotropin treatment apparently does not correct these deficiencies,¹⁶ it is likely that follicle maturation and successful pregnancy are highly dependent on VEGFR-2–mediated neovascularization in the ovary.^6,17 Vascularization also occurs in the uterine wall and decidua during pregnancy, and significant disruption of angiogenesis by VEGFR-2 blockade in these tissues after fertilization has been shown to greatly reduce pregnancy success.¹⁸VEGFR-3 is expressed primarily on lymphatic endothelial cells in adult tissue,^19,20 and its signaling, via ligation by VEGF-C or VEGF-D, is necessary for lymphangiogenesis by inducing lymphatic endothelial cell proliferation and migration.^19–23 Blockade of VEGFR-3 signaling using a function-blocking antibody such as mF4-31C1 (ImClone Systems; Eli Lilly, Indianapolis, IN) completely blocks the initiation of new lymphatic vessels in adult mice without affecting pre-existing lymphatic morphology or function and without apparently affecting blood angiogenesis.^18,21,22 The ovary contains a dense lymphatic network that has been morphologically assessed in large rodents.^24–26 Recent studies in which murine ovarian lymphatic vessel expansion was impaired during development found the dams to be infertile as adults.³We investigated VEGFR-3–mediated lymphangiogenesis and the roles of new lymphatic vessels and lymphangiogenesis in female reproduction and found that lymphangiogenesis occurs within the murine ovary during reproductive cycles and folliculogenesis and that VEGFR-3 neutralization prevents viable, full-term pregnancies. Using combined in vivo, ex vivo, and in vitro methods, we examined which aspects of female fertility are influenced by inhibited maternal lymphangiogenesis including oocyte and follicular development and maturation, uterine implantation, and embryonic development. After we had eliminated direct effects on fetal and uterine VEGFR-3–mediated neovascularization, our results suggested that the new ovarian lymphatic vessels specifically modulate follicle development and hormone production, demonstrating a critical and novel role for ovarian lymphangiogenesis in reproduction.     

HHV-6 cell receptor CD46 expression on various cell subsets of six blood and graft sources: A prospective series

BackgroundCord Blood (CB) are increasingly used as an alternative stem cells source in adults for allogeneic Stem Cell Transplantation (allo-SCT). The risk of human herpesvirus (HHV-6) reactivation is significantly higher after CB transplant vs unrelated peripheral blood stem cells (PBSC) allo-SCT. Higher HHV-6 cell receptor CD46 expression on progenitor cells in CB may explain this difference.ObjectivesTo prospectively compare the HHV-6 cell receptor CD46 expression on various cell subsets of three freshly harvested blood sources on one hand and of three graft sources on the other hand.Study design52 samples were used for the purpose of this study. They were issued from peripheral blood (PB, n = 10), G-CSF mobilised PB (GCSF-PB, n = 10), cord blood (CB, n = 10), unmanipulated bone marrow (uBM, n = 5), leukapheresis product (LP, n = 10) and thawed CB graft (n = 7). CD46 expression was assessed by FACS analysis on total lymphocytes, monocytes, NK cells, T and B cells subsets, plasmacytoid (pDCs) dendritic cells and stem cells.ResultsAs all cell subsets were found CD46 positive, CD46 mean fluorescence intensity (MFI) was then considered for comparison between the three blood sources and the three graft sources. The most impressive result observed was that HHV-6 cell receptor CD46 expression was significantly reduced in almost all cell components of thawed CB graft compared to other graft sources.ConclusionsThis original study shows strong differences in term of quantitative CD46 expression between several blood and grafts samples. Our results suggest that other factors than the qualitative CD46 expression play a role in the higher HHV-6 reactivation observed after CB transplant in adults.     

The DNA damage response induces antigen presenting cell‐like functions in fibroblasts

The DNA damage response (DDR) alerts the immune system to the danger posed by DNA damage through the induction of damage‐associated molecular pattern molecules, chemokines, and ligands for activating immune receptors such as lymphocyte function‐associated antigen 1 (LFA‐1), NKG2D, and DNAX accessory molecule 1 (DNAM‐1). Here we provide evidence that OVA^257–264‐pulsed fibroblasts gain the ability to activate naïve OT‐I CD8⁺ T cells in response to DNA damage. The ability of fibroblasts to activate OT‐I CD8⁺ T cells depended on the upregulation of ICAM‐1 on fibroblasts and DNAM‐1 expression of CD8⁺ T cells. OVA^257–264‐pulsed fibroblasts were able to induce a protective T‐cell response against B16‐OVA cells in a DDR‐dependent manner. Hence, the DDR may alert the immune system to the presence of potentially dangerous cells by upregulating the expression of ligands that can induce the activation of innate and adaptive immune cells.     

Clinical analysis and prognostic significance of haemophagocytic lymphohistiocytosis‐associated anaplastic large cell lymphoma in children

Haemophagocytic lymphohistiocytosis (HLH) has been rarely described in children treated for an anaplastic large‐cell lymphoma (ALCL). We evaluated the incidence, the clinical and histological characteristics and the prognosis of HLH associated‐ALCL. The medical, biological, cytological and histological data of patients treated for ALK‐positive ALCL in the paediatric department of a single institution between 1975 and 2008 were analysed and assessed for HLH according to diagnosis criteria of the Histiocyte Society. Data concerning a series of 50 consecutive children with ALCL were reviewed. HLH‐associated ALCL was observed in 12% of the patients. Lung involvement was significantly more frequent in HLH‐associated ALCL patients than in the group without HLH (P = 0·004), as well as central nervous system (CNS) and bone marrow involvement (P = 0·001 and P = 0·007 respectively). The histological subtype in children with HLH‐associated ALCL did not differ from that of the group without HLH. There was no significant difference between the two groups in 5‐year EFS and OS (P = 0·91 and P > 0·99 respectively). In conclusion, HLH is not rare in paediatric ALCL. Despite a high incidence of visceral, CNS and bone marrow involvement, HLH does not seem to exert a significant impact on outcome in children treated for ALCL.     

Aggregation-prone c9FTD/ALS poly(GA) RAN-translated proteins cause neurotoxicity by inducing ER stress

The occurrence of repeat-associated non-ATG (RAN) translation, an atypical form of translation of expanded repeats that results in the synthesis of homopolymeric expansion proteins, is becoming more widely appreciated among microsatellite expansion disorders. Such disorders include amyotrophic lateral sclerosis and frontotemporal dementia caused by a hexanucleotide repeat expansion in the C9ORF72 gene (c9FTD/ALS). We and others have recently shown that this bidirectionally transcribed repeat is RAN translated, and the “c9RAN proteins” thusly produced form neuronal inclusions throughout the central nervous system of c9FTD/ALS patients. Nonetheless, the potential contribution of c9RAN proteins to disease pathogenesis remains poorly understood. In the present study, we demonstrate that poly(GA) c9RAN proteins are neurotoxic and may be implicated in the neurodegenerative processes of c9FTD/ALS. Specifically, we show that expression of poly(GA) proteins in cultured cells and primary neurons leads to the formation of soluble and insoluble high molecular weight species, as well as inclusions composed of filaments similar to those observed in c9FTD/ALS brain tissues. The expression of poly(GA) proteins is accompanied by caspase-3 activation, impaired neurite outgrowth, inhibition of proteasome activity, and evidence of endoplasmic reticulum (ER) stress. Of importance, ER stress inhibitors, salubrinal and TUDCA, provide protection against poly(GA)-induced toxicity. Taken together, our data provide compelling evidence towards establishing RAN translation as a pathogenic mechanism of c9FTD/ALS, and suggest that targeting the ER using small molecules may be a promising therapeutic approach for these devastating diseases.