Molecular evolution in protobiological systems Final report, Dec. 1, 1961 - Nov. 30, 1964

Arc discharge of irradiated mixtures of simple organic and inorganic compounds for development of theory of molecular evolution in protobiological system

Influence of relative NK-DC abundance on placentation and its relation to epigenetic programming in the offspring

Normal placentation relies on an efficient maternal adaptation to pregnancy. Within the decidua, natural killer (NK) cells and dendritic cells (DC) have a critical role in modulating angiogenesis and decidualization associated with pregnancy. However, the contribution of these immune cells to the placentation process and subsequently fetal development remains largely elusive. Using two different mouse models, we here show that optimal placentation and fetal development is sensitive to disturbances in NK cell relative abundance at the fetal–maternal interface. Depletion of NK cells during early gestation compromises the placentation process by causing alteration in placental function and structure. Embryos derived from NK-depleted dams suffer from intrauterine growth restriction (IUGR), a phenomenon that continued to be evident in the offspring on post-natal day 4. Further, we demonstrate that IUGR was accompanied by an overall reduction of global DNA methylation levels and epigenetic changes in the methylation of specific hepatic gene promoters. Thus, temporary changes within the NK cell pool during early gestation influence placental development and function, subsequently affecting hepatic gene methylation and fetal metabolism.Fil: Freitag, Nancy. Medicine University of Berlin; AlemaniaFil: Zwier, M. V.. University of Groningen; Países BajosFil: Barrientos, Gabriela Laura. Medicine University of Berlin; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Tirado González, Irene. Medicine University of Berlin; AlemaniaFil: Conrad, Melanie L.. Medicine University of Berlin; AlemaniaFil: Rose, Matthias. Medicine University of Berlin; AlemaniaFil: Scherjon, S. A.. University of Groningen; Países BajosFil: Plösch, T.. University of Groningen; Países BajosFil: Blois, Sandra M.. Medicine University of Berlin; Alemani

Investigation of Factors Determining the Enhanced Permeability and Retention Effect in Subcutaneous Xenografts

Liposomal chemotherapy offers several advantages over conventional therapies, including high intratumoral drug delivery, reduced side effects, prolonged circulation time and the possibility to dose higher. The efficient delivery of liposomal chemotherapeutics relies however on the enhanced permeability and retention (EPR) effect, which refers to the ability of macromolecules to extravasate leaky tumor vessels and accumulate in the tumor tissue. Using a panel of human xenograft tumors, we evaluated the influence of the EPR effect on liposomal distribution in vivo by injection of pegylated liposomes radiolabeled with 111In. Liposomal accumulation in tumors and organs was followed over time by SPECT/CT imaging. We observed that fast growing xenografts, which may be less representative of tumor development in patients, showed higher liposomal accumulation as compared to slow growing xenografts. Additionally, several other parameters determining the EPR effect were evaluated, such as blood and lymphatic vessel density, intratumoral hypoxia, and the presence of macrophages. The investigation of various parameters showed a few correlations. Although hypoxia, proliferation and macrophage presence were associated with tumor growth, no hard conclusions or predictions could be made regarding the EPR effect or liposomal uptake. However liposomal uptake was

Placental glycoredox dysregulation associated with disease progression in an animal model of superimposed preeclampsia

Pregnancies carried by women with chronic hypertension are at increased risk of superimposed preeclampsia, but the placental pathways involved in disease progression remain poorly understood. In this study, we used the stroke-prone spontaneously hypertensive rat (SHRSP) model to investigate the placental mechanisms promoting superimposed preeclampsia, with focus on cellular stress and its influence on galectin–glycan circuits. Our analysis revealed that SHRSP placentas are characterized by a sustained activation of the cellular stress response, displaying significantly increased levels of markers of lipid peroxidation (i.e., thiobarbituric acid reactive substances (TBARS)) and protein nitration and defective antioxidant enzyme expression as early as gestation day 14 (which marks disease onset). Further, lectin profiling showed that such redox imbalance was associated with marked alterations of the placental glycocode, including a prominent decrease of core 1 O-glycan expression in trophoblasts and increased decidual levels of sialylation in SHRSP placentas. We also observed significant changes in the expression of galectins 1, 3 and 9 with pregnancy progression, highlighting the important role of the galectin signature as dynamic interpreters of placental microenvironmental challenges. Collectively, our findings uncover a new role for the glycoredox balance in the pathogenesis of superimposed preeclampsia representing a promising target for interventions in hypertensive disorders of pregnancy

Measurement of reaction kinetics of [177Lu]Lu-DOTA-TATE using a microfluidic system

Microfluidic synthesis techniques can offer improvement over batch syntheses which are currently used for radiopharmaceutical production. These improvements are, for example, better mixing of reactants, more efficient energy transfer, less radiolysis, faster reaction optimization, and overall improved reaction control. However, scale-up challenges hinder the routine clinical use, so the main advantage is currently the ability to optimize reactions rapidly and with low reactant consumption. Translating those results to clinical systems could be done based on calculations, if kinetic constants and diffusion coefficients were known. This study describes a microfluidic system with which it was possible to determine the kinetic association rate constants for the formation of [177Lu]Lu-DOTA-TATE under conditions currently used for clinical production. The kinetic rate constants showed a temperature dependence that followed the Arrhenius equation, allowing the determination of Arrhenius parameters for a Lu-DOTA conjugate (A = 1.24 ± 0.05 × 1019 M-1 s-1, EA = 109.5 ± 0.1 × 103 J mol-1) for the first time. The required reaction time for the formation of [177Lu]Lu-DOTA-TATE (99% yield) at 80 °C was 44 s in a microfluidic channel (100 μm). Simulations done with COMSOL Multiphysics® indicated that processing clinical amounts (3 mL reaction solution) in less than 12 min is possible in a micro- or milli-fluidic system, if the diameter of the reaction channel is increased to over 500 μm. These results show that a continuous, microfluidic system can become a viable alternative to the conventional, batch-wise radiolabelling technique

ELMOD3-SH2D6 gene fusion as a possible co-star actor in autism spectrum disorder scenario

Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders characterized by high heritability. It is known that genetic factors contribute to ASD pathogenesis. In particular, copy number variants (CNVs) are involved in ASD susceptibility and can affect gene expression regulation. 2p11.2 microdeletions encompassing ELMOD3, CAPG and SH2D6 genes have been described in four unrelated ASD families. The present study revealed that this microdeletion is responsible for the production of a chimeric transcript generated from the fusion between ELMOD3 and SH2D6. The identified transcript showed significantly higher expression levels in subjects carrying the deletion compared to control subjects, suggesting that it is not subjected to nonsense-mediated decay and might encode for a chimeric protein. In conclusion, this study suggests the possible involvement of this gene fusion, together with the other previously identified variants, in ASD

Influence of tumour size on the efficacy of targeted alpha therapy with 213Bi-[DOTA0,Tyr3]-octreotate

BACKGROUND: Targeted alpha therapy has been postulated to have great potential for the treatment of small clusters of tumour cells as well as small metastases. (213)Bismuth, an α-emitter with a half-life of 46 min, has shown to be effective in preclinical as well as in clinical applications. In this study, we evaluated whether (213)Bi-[DOTA(0), Tyr(3)]-octreotate ((213)Bi-DOTATATE), a (213)Bi-labelled somatostatin analogue with high affinity for somatostatin receptor subtype 2 (SSTR(2)), is suitable for the treatment of larger neuroendocrine tumours overexpressing SSTR(2) in comparison to its effectiveness for smaller tumours. We performed a preclinical targeted radionuclide therapy study with (213)Bi-DOTATATE in animals bearing tumours of different sizes (50 and 200 mm(3)) using two tumour models: H69 (human small cell lung carcinoma) and CA20948 (rat pancreatic tumour). METHODS: Pharmacokinetics was determined for calculation of dosimetry in organs and tumours. H69- or CA20948-xenografted mice with tumour volumes of approximately 120 mm(3) were euthanized at 10, 30, 60 and 120 min post injection of a single dose of (213)Bi-DOTATATE (1.5–4.8 MBq). To investigate the therapeutic efficacy of (213)Bi-DOTATATE, xenografted H69 and CA20948 tumour-bearing mice with tumour sizes of 50 and 200 mm(3) were administered daily with a therapeutic dose of (213)Bi-DOTATATE (0.3 nmol, 2–4 MBq) for three consecutive days. The animals were followed for 90 days after treatment. At day 90, mice were injected with 25 MBq (99m)Tc-DMSA and imaged by SPECT/CT to investigate possible renal dysfunction due to (213)Bi-DOTATATE treatment. RESULTS: Higher tumour uptakes were found in CA20948 tumour-bearing animals compared to those in H69 tumour-bearing mice with the highest tumour uptake of 19.6 ± 6.6 %IA/g in CA20948 tumour-bearing animals, while for H69 tumour-bearing mice, the highest tumour uptake was found to be 9.8 ± 2.4 %IA/g. Nevertheless, as the anti-tumour effect was more pronounced in H69 tumour-bearing mice, the survival rate was higher. Furthermore, in the small tumour groups, no regrowth of tumour was found in two H69 tumour-bearing mice and in one of the CA20948 tumour-bearing mice. No renal dysfunction was observed in (213)Bi-DOTATATE-treated mice after the doses were applied. CONCLUSIONS: (213)Bi-DOTATATE demonstrated a great therapeutic effect in both small and larger tumour lesions. Higher probability for stable disease was found in animals with small tumours. (213)Bi-DOTATATE was effective in different neuroendocrine (H69 and CA20948) tumour models with overexpression of SSTR(2) in mice. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13550-016-0162-2) contains supplementary material, which is available to authorized users

Interaction of pregnancy-specific glycoprotein 1 with integrin Α5β1 is a modulator of extravillous trophoblast functions

Human pregnancy-specific glycoproteins (PSGs) serve immunomodulatory and pro-angiogenic functions during pregnancy and are mainly expressed by syncytiotrophoblast cells. While PSG mRNA expression in extravillous trophoblasts (EVTs) was reported, the proteins were not previously detected. By immunohistochemistry and immunoblotting, we show that PSGs are expressed by invasive EVTs and co-localize with integrin α5. In addition, we determined that native and recombinant PSG1, the most highly expressed member of the family, binds to α5β1 and induces the formation of focal adhesion structures resulting in adhesion of primary EVTs and EVT-like cell lines under 21% oxygen and 1% oxygen conditions. Furthermore, we found that PSG1 can simultaneously bind to heparan sulfate in the extracellular matrix and to α5β1 on the cell membrane. Wound healing assays and single-cell movement tracking showed that immobilized PSG1 enhances EVT migration. Although PSG1 did not affect EVT invasion in the in vitro assays employed, we found that the serum PSG1 concentration is lower in African-American women diagnosed with early-onset and late-onset preeclampsia, a pregnancy pathology characterized by shallow trophoblast invasion, than in their respective healthy controls only when the fetus was a male; therefore, the reduced expression of this molecule should be considered in the context of preeclampsia as a potential therapy

Early expression of pregnancy-specific glycoprotein 22 (PSG22) by trophoblast cells modulates angiogenesis in mice.

Mouse and human pregnancy-specific glycoproteins (PSG) are known to exert immunomodulatory functions during pregnancy by inducing maternal leukocytes to secrete anti-inflammatory cytokines that promote a tolerogenic decidual microenvironment. Many such anti-inflammatory mediators also function as proangiogenic factors, which, along with the reported association of murine PSG with the uterine vasculature, suggest that PSG may contribute to the vascular adaptations necessary for successful implantation and placental development. We observed that PSG22 is strongly expressed around the embryonic crypt on Gestation Day 5.5, indicating that trophoblast giant cells are the main source of PSG22 during the early stages of pregnancy. PSG22 treatment up-regulated the secretion of transforming growth factor beta 1 and vascular endothelial growth factor A (VEGFA) in murine macrophages, uterine dendritic cells, and natural killer cells. A possible role of PSGs in uteroplacental angiogenesis is further supported by the finding that incubation of endothelial cells with PSG22 resulted in the formation of tubes in the presence and absence of VEGFA. We determined that PSG22, like human PSG1 and murine PSG17 and PSG23, binds to the heparan sulfate chains in syndecans. Therefore, our findings indicate that despite the independent evolution and expansion of human and rodent PSG, members in both families have conserved functions that include their ability to induce anti-inflammatory cytokines and proangiogenic factors as well as to induce the formation of capillary structures by endothelial cells. In summary, our results indicate that PSG22, the most abundant PSG expressed during mouse early pregnancy, is likely a major contributor to the establishment of a successful pregnancy.Fil: Blois, Sandra M.. University Medicine of Berlin; AlemaniaFil: Tirado González, Irene. University Medicine of Berlin; AlemaniaFil: Wu, Julie. Uniformed Services University of the Health Sciences; Estados UnidosFil: Barrientos, Gabriela Laura. University Medicine of Berlin; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Johnson, Briana. Uniformed Services University of the Health Sciences; Estados UnidosFil: Warren, James. Uniformed Services University of the Health Sciences; Estados UnidosFil: Freitag, Nancy. University Medicine of Berlin; AlemaniaFil: Klapp, Burghard F.. University Medicine of Berlin; AlemaniaFil: Irmak, Ster. University of Duisburg Essen; AlemaniaFil: Ergun, Suleyman. Julius Maximilians Universitat Wurzburg. Biozentrum; AlemaniaFil: Dveskler, Gabriela S.. Uniformed Services University of the Health Sciences; Estados Unido

Phenotyping placental oxygenation in Lgals1 deficient mice using (19)F MRI

Placental hypoperfusion and hypoxia are key drivers in complications during fetal development such as fetal growth restriction and preeclampsia. In order to study the mechanisms of disease in mouse models, the development of quantitative biomarkers of placental hypoxia is a prerequisite. The goal of this exploratory study was to establish a technique to noninvasively characterize placental partial pressure of oxygen (PO(2)) in vivo in the Lgals1 (lectin, galactoside-binding, soluble, 1) deficient mouse model of preeclampsia using fluorine magnetic resonance imaging. We hypothesized a decrease in placental oxygenation in knockout mice. Wildtype and knockout animals received fluorescently labeled perfluoro-5-crown-15-ether nanoemulsion i.v. on day E14-15 during pregnancy. Placental PO(2) was assessed via calibrated (19)F MRI saturation recovery T(1) mapping. A gas challenge with varying levels of oxygen in breathing air (30%, 60% and 100% O(2)) was used to validate that changes in oxygenation can be detected in freely breathing, anesthetized animals. At the end of the experiment, fluorophore-coupled lectin was injected i.v. to label the vasculature for histology. Differences in PO(2) between breathing conditions and genotype were statistically analyzed with linear mixed-effects modeling. As expected, a significant increase in PO(2) with increasing oxygen in breathing air was found. PO(2) in Lgals1 knockout animals was decreased but this effect was only present at 30% oxygen in breathing air, not at 60% and 100%. Histological examinations showed crossing of the perfluorocarbon nanoemulsion to the fetal blood pool but the dominating contribution of (19)F MR signal is estimated at > 70% from maternal plasma based on volume fraction measurements of previous studies. These results show for the first time that (19)F MRI can characterize oxygenation in mouse models of placental malfunction