In Vivo Imaging of Fluorescent Probes Linked to Antibodies Against Human and Rat Vascular Endothelial Growth Factor.

PURPOSE: Anti-VEGF therapy has improved functional outcome for many patients with neovascular AMD. A particular challenge in routine clinical application is to find the best treatment regimen as a high degree of interindividual variability of disease activity has been noted. The aim of the study was to investigate fluorescent probes linked to antibodies against VEGF for in vivo imaging in an animal model. METHODS: Bevacizumab, B20-4.1.1 and AF564 were covalently attached to the novel dye 6S-indocyanine green (ICG) maleimide. Binding and proliferation properties were assessed. In a rat model of laser-induced choroidal neovascularization, retinal uptake and topographic localization of antibody-conjugates were analyzed. Distribution and accumulation of the probes were determined by immunohistochemistry and flow cytometry analysis. RESULTS: Antibody-conjugates retained target binding affinity and showed no toxicity. In vivo imaging showed a strong fluorescence immediately following an intravenous or intravitreal injection. While accumulation within the laser lesions was visualized for all three antibody conjugates, the signal strength and the duration of fluorescence varied. In addition, distinct fluorescent spots were also recognized. Patterning and in-depth analyses including histology and flow cytometry data strongly suggest that the fluorescent spots represent labeled microglial cells and/or macrophages. CONCLUSIONS: Pharmacokinetics of fluorescent-labeled bevacizumab, B20-4.1.1 and AF564 can be investigated in vivo. In this model, interpretation of long-term in vivo observations is difficult because of a possible rat-specific immune response and challenges to image localized binding of soluble VEGF. Further investigations in a primate model and the use of appropriate antibodies directed against the VEGF-receptor may represent alternative approaches.

Origin of Foxp3(+) cells during pregnancy.

Pregnancy establishment and maintenance represents a challenge for the maternal immune system because it has to be alert against pathogens while tolerating paternal alloantigens expressed in fetal structures. Regulatory T cells (Tregs) are important for successful implantation and involved in allotolerance towards paternal antigens. The origin and mechanisms leading to Treg generation during pregnancy at different stages remain under discussion. We report an accumulation of Helios(+) Tregs in thymus and in the lymph nodes draining the uterus at early pregnancy. At later pregnancy stages an expanded population of Foxp3(+) Tregs was generated in the periphery as we showed in a Rag-1(-/-) model of cell transfer. Our data suggest that Tregs, predominantly of thymic origin, are needed for pregnancy establishment. At later pregnancy stages an extra thymic Treg population contributes to the Treg pool in the periphery. Our data provides new insights in the origin of Tregs during pregnancy that are essential to understand natural mechanisms of tolerance acquisition.

Blockage of heme oxygenase-1 abrogates the protective effect of regulatory T cells on murine pregnancy and promotes the maturation of dendritic cells.

Regulatory T cells (Treg) play an important role in fetal protection. They expand during normal pregnancy and protect fetal antigens from maternal effector cells. Their effect is associated with the up-regulation of tolerance-associated molecules at the fetal-maternal interface. Among these, Heme Oxygenase-1 (HO-1, coded by Hmox1) is of special importance as its blockage correlates with increased abortion rates and its up-regulation positively affects pregnancy outcome. Here, we aimed to investigate whether the protective effect of Treg is mediated by HO-1 in a mouse model. HO-1 blockage by Zinc Protoporhyrin (ZnPPIX) abrogated the protective effect of Treg transfer. We found that HO-1 is important in maintaining maternal dendritic cells (DCs) in an immature state, which contributes to the expansion of the peripheral Treg population. This brings to light one essential pathway through which Treg mediates the semi-allogeneic fetus tolerance.

The persistence of paternal antigens in the maternal body is involved in regulatory T-cell expansion and fetal-maternal tolerance in murine pregnancy.

PROBLEM: Mammalian pregnancy is a state of immunological tolerance and CD4(+) CD25(+) regulatory T cells (Treg) contribute to its maintenance. Knowing that Treg act in an antigen-specific way during pregnancy, we hypothesized that they are generated after maternal immune cells encounter paternal antigens. METHOD OF STUDY: We mated wild type females with transgenic green fluorescent protein (GFP) males in an allogenic setting and killed them on different days of pregnancy. RESULTS: Presence of paternal and maternal MHC class II(+) cells in vaginal lavage on day 0.5 of pregnancy was confirmed. Thus, antigen presentation may take place early during pregnancy in the periphery either by the direct or indirect pathways. Foxp3(+) cells known to have regulatory activity could be detected on day 2 of pregnancy in lymph nodes and shortly after implantation at the fetal-maternal interface. CONCLUSION: Our data suggest that paternal antigens are processed early during pregnancy, which leads to the generation of Treg. The continuous release of placental antigens into the maternal circulation allows the maintenance of a Treg population which is specific for paternal antigens and mediates tolerance toward the semi-allogeneic fetus until the time point of birth.

PD-1 but not CTLA-4 blockage abrogates the protective effect of regulatory T cells in a pregnancy murine model.

PROBLEM: Regulatory T cells (Treg) play an important role in fetal protection. They expand during normal pregnancy and protect paternal/fetal antigens from rejection by maternal effector cells. Accordingly, the transfer of Treg obtained from BALB/c-mated CBA/J females prevents abortion in DBA/2J-mated animals. The actual mechanism through which Treg mediate their protective effect is still inconclusive. Cytotoxic T lymphocyte antigen-4 (CTLA-4) and Programmed cell death 1 (PD-1) are some of known Treg-associated molecules; however, their role in Treg-mediated fetal protection in murine model has not been investigated. METHOD OF STUDY: Treg obtained from normal pregnant animals (NP; CBA/J x BALB/c) on day 14 were adoptively transferred into abortion-prone mice (AP; CBA/J x DBA/2J) intravenously on day 2 of pregnancy. An amount of 250 microg of either anti-PD-1 or anti-CTLA-4 mAb were injected intraperitoneally on days 0, 3, 6 and 9 of pregnancy. Controls received Treg + IgG or Treg + PBS. NP or AP treated with PBS served as additional controls. RESULTS: Blocking PD-1 abrogated the protective effect of Treg, resulting in a higher median abortion rate in comparison with the Treg/isotype-treated control while CTLA-4 blockage did not interfere with the protective effect of Treg. This was associated with a diminished number of vascular endothelial growth factor-A(+) cells, previously reported as stimulators of lymphocyte extravasation in preterm labor. CONCLUSION: Our data suggest PD-1 as an important mediator in Treg-induced fetal protection in the CBA/J x DBA/2J murine model.

Supporting the hypothesis of pregnancy as a tumor: survivin is upregulated in normal pregnant mice and participates in human trophoblast proliferation.

PROBLEM: Survivin, a tumor-promoting antiapoptotic molecule, is expressed in the human placenta. Here, we analyzed its expression during normal and pathological murine pregnancy and investigated its participation in human first trimester trophoblast cell survival and proliferation. METHOD OF STUDY: We first analyzed the expression of survivin on the mRNA and protein level at the fetal-maternal interface of normal pregnant (CBA/J x BALB/c) and abortion-prone (CBA/J x DBA/2J) mice at different pregnancy stages by RT-PCR and immunohistochemistry. We also evaluated apoptosis in murine trophoblasts in both mating combinations by TUNEL technique. Functional studies were carried out by knockdown survivin by means of siRNA methodology in two human first trimester trophoblast cell lines [Swan.71 (Sw.71) and HTR8 (H8)]. RESULTS: We observed a peak in mRNA levels on day 5 and a peak of protein levels on day 8 of pregnancy in both combinations. The level of survivin in animals from the abortion-prone group was decreased compared with normal pregnant mice on day 8, which was accompanied by elevated apoptosis rates. In later pregnancy stages (days 10 and 14), survivin levels decreased to levels comparable to those observed right after fecundation in both groups. Transfection of human first trimester cell lines (H8 and Sw.71) with siRNA targeting the survivin gene led to a 76-82% reduction of its expression leading to reduced trophoblast cell viability and proliferation. CONCLUSION: Our findings suggest an important role of survivin to promote trophoblast cell survival and proliferation during placentation, thus maintaining pregnancy. The pregnancy-associated expression of a cancer molecule such as survivin supports the 'pseudo-malignancy' hypothesis of pregnancy. Our data may contribute to the better understanding of trophoblast cell development during implantation and placentation.

Mechanisms of action of regulatory T cells specific for paternal antigens during pregnancy.

OBJECTIVE: To investigate whether pregnancy-induced regulatory T cells are generated specifically for paternal antigens or expanded by hormonal changes and to study regulatory T cell-related mechanisms during pregnancy. METHODS: We used murine models of normal, abortion-prone, and pseudopregnancy to characterize regulatory T cells and hormones by methods such as flow cytometry, molecular biology techniques, and chemiluminescence. Antigen specificity was studied in experiments in which animals were vaccinated with paternal antigens or adoptively transferred with regulatory T cells. To analyze regulatory T cell-mediated mechanisms, we used neutralizing antibodies against IL-10 or TGF-beta. RESULTS: Regulatory T cells are activated by male antigens, and minor antigens are protected by linked immunosuppression. Our data exclude the possibility that regulatory T cell expansion during pregnancy is exclusively driven by hormonal changes. An increase in systemic regulatory T cell levels in pseudopregnant females after mating with vasectomized males but not after pseudopregnancy induced mechanically confirms generation of regulatory T cells specific for paternal antigens. As for the mechanisms, neutralizing IL-10 abrogates the protective effect of regulatory T cells, whereas blockage of TGF-beta does not provide the same effect. CONCLUSION: Our data confirm that regulatory T cells act in an antigen-specific manner during pregnancy and strongly suggest that IL-10 is involved in regulatory T cell-mediated protection of the fetus. These data contribute to the knowledge of the basic mechanisms regulating immune tolerance during pregnancy, a major biologic question with important medical implications. LEVEL OF EVIDENCE: II.

Immunology of pregnancy: cellular mechanisms allowing fetal survival within the maternal uterus.

Pregnancy success remains a fascinating phenomenon to immunologists as it defies the immunological rules of rejection. Although it was previously thought that the maternal immune system does not see the fetus, it is now well documented that fetal cells reach the maternal body and encounter host immune cells. Natural tolerance mechanisms following this interaction remain to be fully elucidated. This article reviews the current literature on mechanisms of adaptive immunity, with emphasis on regulatory T cells and heme oxygenase 1 (HO-1). We propose a scenario in which regulatory T cells create a tolerant microenvironment at the fetal-maternal interface characterised by the presence of tolerance-associated molecules such as HO-1, which has been shown to be of vital importance for fetal survival.

Protection from abortion by heme oxygenase-1 up-regulation is associated with increased levels of Bag-1 and neuropilin-1 at the fetal-maternal interface.

Tolerance mechanisms allowing pregnancy success resemble those involved in allograft acceptance. Heme oxygenase (HO) is a tissue-protective molecule, which allows graft acceptance and is known to have antiapoptotic effects on several cell types. We previously reported down-regulated levels of HO-1 and HO-2 in placenta from allopregnant mice undergoing abortion. In this study, we analyzed whether the up-regulation of HO-1 by cobalt-protoporphyrin (Co-PP) during implantation window can rescue mice from abortion. Induction of HO-1 by Co-PP treatment prevented fetal rejection, whereas the down-regulation of HOs by zinc-protoporphyrin application boosted abortion. The beneficial effect of HO-1 induction was not related to a local shift to Th2-profile or to a change in the NO system. Interestingly, the expression of the antiapoptotic/cytoprotective molecule Bag-1 as well as the levels of neuropilin-1, a novel marker for T regulatory cells, were up-regulated after Co-PP treatment. Our data strongly support a very important role for HO-1 in fetal allotolerance and suggest that HO-1 might be protective by up-regulating tissue protective molecules, i.e., Bag-1, and by activating T regulatory cells rather than by changing the local cytokine profile.

Development of a chitinase and v-cathepsin negative bacmid for improved integrity of secreted recombinant proteins.

The application of the baculovirus-insect cell expression system for the production of integral membrane and secreted proteins is often more troublesome than for cytoplasmic proteins. One protein expressed at low levels in insect cells is the Theileria parva sporozoite surface protein p67. Theileria parva is a protozoan parasite, which causes the tick-transmitted disease East Coast fever in cattle. Baculovirus vectors were engineered to produce a secreted form of p67 by replacing the signal peptide of p67 with the honeybee mellitin signal sequence and deleting a putative membrane anchor from the C-terminus. Furthermore, the chitinase and v-cathepsin genes were deleted from the baculovirus expression vector in a bacmid setup, allowing broad scale application of this novel vector. Deletion of the chitinase and v-cathepsin gene had a positive effect on the integrity of both the intracellular and secreted recombinant protein.