Per- and polyfluoroalkyl substances (PFAS) are persistent endocrine-disrupting chemicals (EDCs) linked to adverse reproductive outcomes. While the placenta is a known target of PFAS toxicity, most in vitro studies use two-dimensional (2D) cell culture models, often focusing on late-pregnancy tissue or blood PFAS concentrations and examining single compound exposures. In this study, we measure placenta PFAS concentrations in early pregnancy and design a placenta-relevant PFAS mixture to assess its impact on trophoblast function using a three-dimensional (3D) trophoblast spheroid model. PFAS levels in first-trimester placental tissue were quantified using liquid chromatography/triple quadrupole mass spectrometry. Six PFAS: perfluorononanoic acid (PFNA), perfluorooctanesulfonic acid (PFOS), perfluorobutanoic acid (PFBA), perfluorooctanoic acid (PFOA), perfluorohexanesulfonic acid (PFHxS), and perfluorodecanoic acid (PFDA); were selected based on their placenta concentrations and relevance to pregnancy complications to design the placenta real-life PFAS mixture. Next, trophoblast spheroids were propagated from two different cell lines, JEG-3 and HTR-8/SVneo, to assess the effects of PFAS mixture on trophoblast viability, apoptosis, invasion, hormone production, and gene expression. While trophoblast spheroid viability remained largely unaffected, we observed changes in trophoblast function. PFAS exposure significantly increased invasiveness in JEG-3 spheroids at 48 h, but markedly reduced it in HTR-8/SVneo spheroids at 96 h across varying concentrations. Additionally, pregnancy-specific hormone e.g. β-hCG production declined after 48 h of PFAS mixture exposure in JEG-3 spheroids. Gene expression analysis revealed altered apoptosis and proliferation pathways in both trophoblast spheroids. Overall, our study highlights that physiologically relevant 3D trophoblast models can contribute to the broader comprehension of PFAS-associated reproductive health risk assessments.

Hintergrund Die anti-GBM Erkrankung tritt sehr selten im Kindesalter auf. Antikörper gegen die glomeruläre und alveoläre Basalmembran verursachen eine aggressiv verlaufende Autoimmunerkrankung. Bei jungen Patienten tritt die Erkrankung vorrangig als pulmorenales Syndrom auf. Eine überwiegend pulmonale Manifestation ist selten.

Ovarian cancer has the highest mortality rate among female reproductive tract malignancies. A complex network, including the interaction between tumor and immune cells, regulates the tumor microenvironment, survival, and growth. The role of mast cells (MCs) in ovarian tumor pathophysiology is poorly understood. We aimed to understand the effect of MCs on tumor cell migration and growth using in vitro and in vivo approaches. Wound healing assays using human tumor cell lines (SK-OV-3, OVCAR-3) and human MCs (HMC-1) were conducted. Murine ID8 tumor cells were injected into C57BL6/J wildtype (WT) and MC-deficient C57BL/6-KitW-sh/W-sh (KitW-sh) mice. Reconstitution of KitW-sh was performed by the transfer of WT bone marrow-derived MCs (BMMCs). Tumor development was recorded by high-frequency ultrasonography. In vitro, we observed a diminished migration of human ovarian tumor cells upon direct or indirect MC contact. In vivo, application of ID8 cells into KitW-sh mice resulted in significantly increased tumor growth compared to C57BL6/J mice. Injection of BMMCs into KitW-sh mice reconstituted MCs and restored tumor growth. Our data show that MCs have a suppressive effect on ovarian tumor growth and may serve as a new therapeutic target.

BackgroundTo evaluate the benefits of SARS-CoV-2 vaccination in cancer patients it is relevant to understand the adaptive immune response elicited after vaccination. Patients affected by hematologic malignancies are frequently immune-compromised and show a decreased seroconversion rate compared to other cancer patients or controls. Therefore, vaccine-induced cellular immune responses in these patients might have an important protective role and need a detailed evaluation.MethodsCertain T cell subtypes (CD4, CD8, Tfh, γδT), including cell functionality as indicated by cytokine secretion (IFN, TNF) and expression of activation markers (CD69, CD154) were assessed via multi-parameter flow cytometry in hematologic malignancy patients (N=12) and healthy controls (N=12) after a second SARS-CoV-2 vaccine dose. The PBMC of post-vaccination samples were stimulated with a spike-peptide pool (S-Peptides) of SARS-CoV-2, with CD3/CD28, with a pool of peptides from the cytomegalovirus, Epstein-Barr virus and influenza A virus (CEF-Peptides) or left unstimulated. Furthermore, the concentration of spike-specific antibodies has been analyzed in patients.ResultsOur results indicate that hematologic malignancy patients developed a robust cellular immune response to SARS-CoV-2 vaccination comparable to that of healthy controls, and for certain T cell subtypes even higher. The most reactive T cells to SARS-CoV-2 spike peptides belonged to the CD4 and Tfh cell compartment, being median (IQR), 3.39 (1.41-5.92) and 2.12 (0.55-4.14) as a percentage of IFN- and TNF-producing Tfh cells in patients. In this regard, the immunomodulatory treatment of patients before the vaccination period seems important as it was strongly associated with a higher percentage of activated CD4 and Tfh cells. SARS-CoV-2- and CEF-specific T cell responses significantly correlated with each other. Compared to lymphoma patients, myeloma patients had an increased percentage of SARS-CoV-2-specific Tfh cells. T-SNE analysis revealed higher frequencies of γδT cells in patients compared to controls, especially in myeloma patients. In general, after vaccination, SARS-CoV-2-specific T cells were also detectable in patients without seroconversion.ConclusionHematologic malignancy patients are capable of developing a SARS-CoV-2-specific CD4 and Tfh cellular immune response after vaccination, and certain immunomodulatory therapies in the period before vaccination might increase the antigen-specific immune response. A proper response to recall antigens (e.g., CEF-Peptides) reflects immune cellular functionality and might be predictive for generating a newly induced antigen-specific immune response as is expected after SARS-CoV-2 vaccination.

Adaptive immune cells with regulatory function reportedly mediate immune escape in a variety of tumors. Little is known regarding the relevance of the most prominent regulatory cell populations, namely Foxp3+ T regulatory cells (Tregs) and CD19+IL‐10+ B regulatory cells (Bregs), for neuroblastoma (NB) survival. After establishing a novel immunocompetent syngeneic NB mouse model where orthotopic tumors can be generated after intrarenal injection of NB975A cells, we studied the importance of Tregs and Bregs in Foxp3‐DTR mice whose Tregs can be depleted by diphtheria toxin (DT) application as well as in CD19‐specific IL‐10 deficient mice that lack IL‐10+ Bregs (CD19cre +/− × IL‐10 fl/fl mice). We observed Foxp3 Treg cells in tumors from wild type mice. On the contrary, Bregs or B cells were scarce. Specific depletion of Tregs in Foxp3‐DTR mice resulted in an 85% reduction of tumor volume and weight compared to DT‐treated wild type mice and untreated Foxp3‐DTR mice. In contrast, NB tumor growth was not affected in CD19‐specific IL‐10 deficient mice. Similarly, mice lacking mature B cells (μMT mice) and CD19 deficient mice (CD19cre mice) showed no change in growth pattern of NB tumors. In Treg‐depleted mice, reduced tumor growth was associated with an increased concentration of IFN‐gamma, TNF‐alpha, IL‐4, IL‐6, and IL‐10 in isolated splenocytes. In summary, transient ablation of Tregs but not absence of Bregs hindered the growth of NB, strongly suggesting the therapeutic potential of targeting Tregs for this aggressive childhood tumor.

It was postulated that 3D cell culture models more accurately reflect the complex tissue physiology and morphology in comparison to 2D cell monolayers. Currently, there is a shortage of well-characterized and easily maintainable high-throughput experimental models of the human placenta. Here, we characterized three different 3D cultures (e.g., spheroids) derived from trophoblast cell lines and studied their functionality in comparison to primary fetal trophoblasts and placental tissue. The spheroid growth rates of JEG3, BeWo and HTR8/SVneo cell lines were similar among each other and were significantly larger in comparison to primary trophoblast spheroids. All spheroids exhibited migratory properties and shortest distances were registered for JEG3 spheroids. Even though all spheroids displayed invasive capabilities, only the invasive features of HTR8/SVneo spheroids resulted in specific branching. This was in agreement with the invasive properties of the spheroids obtained from primary trophoblasts. Human chorionic gonadotropin production was highest in JEG3 spheroids and only increased when stimulated with cAMP and forskolin in BeWo, but not HTR8/SVneo spheroids. The gene expression analysis confirmed that 3D trophoblast cell cultures and especially HTR8/SVneo spheroids showed considerable similarities with the gene expression profile of primary placental tissue. This study offers a broad characterization of 3D trophoblast spheroids that, in turn, can help in selecting the best model depending on the scientific question that needs to be answered.

We recently identified Galectin-1 (Gal-1), a β-galactoside-binding lectin, as a novel immune regulator in neuroblastoma (NB). Here, we characterized the tolerogenic function of Gal-1 within the CD8+ T cell compartment and further evaluated its relevance as an antigen for effective DNA vaccination against NB in a mouse model. NB cells with Gal-1 knockdown (NXS-2L) exhibited significantly reduced tumor growth compared to NXS-2 NB cells. Administration of anti-CD8 antibodies prevented this antitumor effect, with primary tumor growth comparable to that from Gal-1 (G1)-sufficient NB cells. Peptide epitope screening with online databases and in silico docking experiments predicted the sequences "FDQADLTI" (#1), "GDFKIKCV" (#2), and "AHGDANTI" (#3) to have superior H2-KK binding affinities and "KFPNRLNM" (#4), "DGDFKIKCV" (#5), and "LGKDSNNL" (#6) to have superior H2-DD binding affinities. Minigenes encoding G1-KK (#1-#2-#3), G1-DD (#4-#5-#6) and the triplet with the highest affinity, G1-H (#1-#2-#4), were generated and cloned into a ubiquitin-containing plasmid (pU). Mice receiving pU-G1-KK or pU-G-1H presented a reduction in the s.c. tumor volume and weight of up to 80% compared to control mice; this reduction was associated with increased cytotoxicity of isolated splenocytes from vaccinated animals. Vaccination with pUG1-DD showed a lower capability to suppress primary tumor progression. In conclusion, Gal-1 expression by NB negatively regulates CD8+ T cells. Vaccination with DNA plasmids encoding Gal-1 epitopes overcomes immune escape, enhances CD8+ T cell-dependent immunity and displays effective antitumor activity against NB.

Preterm birth (PTB) is one of the most frequent pregnancy complications. It affects millions of babies each year worldwide and is associated with increased morbidity and mortality. PTB-associated alterations in the maternal immune response may have a direct effect on the developing fetal immune system. Having recently shown that B regulatory (Breg) cells are decreased in number and functionally impaired in maternal blood from women delivering preterm, we now addressed the question whether the adaptive immune system is also altered in cord blood (CB) after the onset of PTB. PTB was associated with increased concentrations of IL-6, TNF-alpha and IL-21 in CB and enhanced IL-6, but decreased IFN-gamma and IL-4 in amniotic fluid (AF) samples compared to term delivery (TD). We found no differences in the frequency of CD19 + B cells, CD4 + T cells or CD4+Foxp3+CD25+T regulatory (Treg) cells in CB cells in PTB vs TD. The frequency of CD86 + B cells was increased, while the percentage of CD24(hi)CD38(hi)CD19 + Breg and CD1d(hi)CD5+ Breg cells and the ability of B cells to convert into Breg cells was diminished in PTB compared to TD. CB B cells from PTB secreted more IL-6, TNF-alpha, IL-9 and IL-2 compared to B cells obtained from term samples. We conclude that, after PTB onset, a shift from immunoregulation towards inflammation takes place in CB cells that are reportedly representative of the fetal compartment. B cells have a substantial contribution herein. This phenomenon might account for the observed enhanced mortality and morbidity in prematurely born infants. Further studies will clarify how to employ this easy-to-obtain information for closely monitoring newborns at risk.

Question We recently introduced the carbohydrate-binding protein Galectin-1 (Gal-1) as a novel tolerogeneic molecule in neuroblastoma (NB). NB-secreted Gal-1 suppressed the maturation of dendritic cells (DC) and inhibited T cell function. Here, we focused on the question whether tumor-derived Gal-1 is the decisive factor to promote tumor growth in our model for NB. Methods C57Bl/6J-derived orthotopic growing NB cells (named NB975A) were used for all culture as well in vivo experiments. Gal-1 knock-down clones (GL) were generated by stable transfection of wt NB975A cells (NBA) with the antisense Gal-1 expression vector (NBA-GL), (kindly provided by Dr. Rabinovich). Blasticidine resistant (5 mikrogram/ml) transfectants were cloned by limited dilution. Protein expression of Gal-1 was determined by Western blot. Orthotopic NB tumors were generated in C57Bl/6J wild type and Gal-1-knock-out (Lgals-/-) female mice by subcapsular tumor cell injection of NBA-GL or wild type NBA (1x106, 0,1 ml PBS) into the left kidney. Primary tumor growth was analyzed by using high frequency ultrasound measurement. Mice were sacrificed 23 days after tumor inoculation and tumor tissue was harvested for further analysis. Results NBA-GL showed 50% reduced protein expression of Gal-1 compared to control wild type NBA cells. In vitro viability and proliferation of the tumor cells was not affected by the transfection. Mice inoculated with NBA-GL cells had significantly smaller primary tumors when compared with control animals that received the NBA cells. Gal-1 sufficient tumor cells injected in Gal-1-deficient host mice grew normally. Conclusion Tumor-derived Gal-1 and not its expression in host is critical for the promotion of tumor growth. Citation Format: Stefan Fest. Tumor-derived Galectin-1 promotes tumor growth in an orthotopic C57Bl/6 NB mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4559.

Purpose of the studyNeuroblastoma (NB) is the most common solid extracranial tumor in childhood. Regulatory T cells (Treg) are known to have immune suppressive capacity and to be involved in tolerance establishment. As they support tumor growth and help some tumors to overcome recognition by the host, they are considered to be an important target. Treg are reportedly increased in blood of NB patients. However, there are no scientific proofs to date that they are involved in neuroblastoma growth. To address this important question we employed Foxp3-DTR mice to selectively deplete Treg upon orthotopic application of syngeneic neuroblastoma cells.MethodsOrthotopic NB tumors were generated in mice Foxp3-DTR mice and in C57Bl/6J controls by subcapsular tumor cell injection of NB975A cells into the left kidney. Primary tumor growth was analysed for 23 days by high frequency ultrasound measurement. To deplete Treg, InjectionDepletion of Diphtheria Toxin (DT, 15 ng/g bodyweight) intraperitoneally every 4 days in Foxp3.DTR mice depleted the Treg pool. Control Foxp3.DTR animals received PBS and control C57B/l6 received DT. All mice were sacrificed 23 days after tumor inoculation. Tumor as wells as immune cells harvested from spleen and inguinal lymph nodes were analyzed by Flow Cytometry, Immunofluorescenceimmunohistochemistry and qRT-PCR.ResultsIn control mice bearing tumors, Treg presence was confirmed at the contact zone between the kidney and the growing, inoculated tumor.Mice devoid of Treg (Foxp3-DTR+DT) showed a significantly reduced tumor growth compared to animals with normal Treg frequencies (Foxp3-DTR+PBS). Tumor growth in wild type controls was not affected by the application of DTR.ConclusionAblation of Treg hindered the growth of NB, strongly suggesting their potential as therapeutic target for this aggressive childhood tumor.Citation Format: Sophie von Lenthe, Christine Weißenborn, Ana Claudia Zenclussen, Stefan Fest, Leonid Metelitsa. Selective depletion of regulatory T cells suppressed neuroblastoma tumor growth in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4570.

PURPOSE:This is the first study to determine the cytomegalovirus (CMV) seronegativity rate for women of childbearing age in Saxony-Anhalt and to determine the prevalence of clinically relevant congenital CMV (cCMV) infection in Central Germany, because there are no valid data available.METHODS:The retrospective study was undertaken between January 2005 and December 2015. For the first time in Germany, the following seven data sources were used to analyze the prevalence of clinically relevant cCMV infection and the rate of CMV seronegative women of childbearing age: CMV Screening in maternity unit, University Women's Hospital, Social Paediatrics Centre (SPC), Malformation Monitoring Centre (MMC), Newborn Hearing Screening (NHS), Neonatal Intensive Care Unit (NICU), and In-house Doctor Department. Key parameters were anti-CMV IgG and IgM, CMV PCR of urine, and clinically relevant symptoms caused by CMV.RESULTS:Between 46 and 52% of women of childbearing age were CMV seronegative. The prevalence of clinically relevant cCMV infection was between 0.008 and 0.04%.CONCLUSIONS:The CMV seronegativity rate of women of childbearing age was confirmed to be in the middle range of estimated data from other sources in Germany. Data from the NICU, SPC, NHS, and MMC show the prevalence of clinically relevant cCMV infection. The risk of all cCMV infections is underestimated. Thus, the true prevalence of clinically relevant and subclinical cCMV infections is >0.04%.

Heme oxygenase (HO)−1 catalyzes the degradation of cytotoxic heme into biliverdin and blocks antitumor immune responses, thus protecting cancer against host defense. Whether this scenario also applies to neuroblastoma (NB), the most common extracranial solid childhood tumor, is not known. Here, we demonstrate for the first time a prognostic relevance of HO‐1 expression in samples from NB patients and show that targeting of HO‐1 prevents both cancer resistance against cellular stress and immune escape in the syngeneic NXS2 A/J mouse model of NB. High HO‐1 RNA expression in NB tissues emerged as unfavorable prognostic marker, in particular for patients older than 18 months as indicated by univariate as well as multivariate survival probability analyses including disease stage and MYCN status. On the basis of this observation we aimed to target HO‐1 by systemic as well as tumor‐specific zinc protoporphyrin‐mediated HO‐1 suppression in a syngeneic immunocompetent NB mouse model. This resulted in 50% reduction of primary tumor growth and a suppression of spontaneous liver metastases. Importantly, HO‐1 inhibition abrogated immune cell paralysis affecting CD4 and CD8 T‐effector cells. This in turn reverted HO‐1‐dependent immune escape mechanisms in NB by increasing NB apoptosis and improved DC maturation. In summary, HO‐1 emerges as a novel immune regulator in NB and emerges as a promising target for the development of therapeutic approaches.

The MYCN oncogene is a strong genetic marker associated with poor prognosis in neuroblastoma (NB). Therefore, MYCN gene amplification and subsequent overexpression provide a possible target for new treatment approaches in NB. We first identified an inverse correlation of MYCN expression with CD45 mRNA in 101 NB tumor samples. KEGG mapping further revealed that MYCN expression was associated with immune-suppressive pathways characterized by a down-regulation of T cell activation and up-regulation of T cell inhibitory gene transcripts. We then aimed to investigate whether DNA vaccination against MYCN is effective to induce an antigen-specific and T cell-mediated immune response. For this purpose, we generated a MYCN-expressing syngeneic mouse model by MYCN gene transfer to NXS2 cells. MYCN-DNA vaccines were engineered based on the pCMV-F3Ub plasmid backbone to drive ubiquitinated full-length MYCN-cDNA and minigene expression. Vaccines were delivered orally with attenuated S. typhimurium strain SL7207 as a carrier. Immunization with both MYCN-DNA vaccines significantly reduced primary tumor growth of MYCN-expressing NB cells in contrast to negative controls. The immune response was mediated by tumor-infiltrating T cells in vivo, which revealed MYCN-specific and MHC class I-restricted lysis of inducible MYCN-expressing NB target cells in vitro. Finally, these antigen-specific T cells also killed MYCN-negative mammary carcinoma cells pulsed with MYCN peptides in contrast to controls. In summary, we demonstrate proof of concept that MYCN can be targeted by DNA vaccination, which may provide an approach to overcoming MYCN immune-suppressive activities in patients with MYCN-amplified disease.

The root of Asparagus cochinchinensis (Lour.) Merr. has been utilized as mucoregulators and expectorants for controlling the airway inflammatory diseases in folk medicine.We investigated whether dioscin and methylprotodioscin isolated from the root of Asparagus cochinchinensis (Lour.) Merr. suppress the gene expression and production of airway MUC5AC mucin induced by phorbol ester and growth factor.Confluent NCI-H292 cells were pretreated with dioscin or methylprotodioscin for 30 min and then stimulated with EGF or PMA for 24 h. The MUC5AC mucin gene expression was measured by RT-PCR. Production of MUC5AC mucin protein was measured by ELISA.(1) Dioscin and methylprotodioscin suppressed the expression of MUC5AC mucin gene induced by EGF or PMA; (2) dioscin suppressed the production of MUC5AC mucin induced by either EGF at 10−5 M (p < 0.05) and 10−6 M (p < 0.05) or PMA at 10−4 M (p < 0.05), 10−5 M (p < 0.05) and 10−6 M (p < 0.05); (3) methylprotodioscin also suppressed the production of MUC5AC mucin induced by either EGF at 10−4 M (p < 0.05) or PMA at 10−4 M (p < 0.05).These results suggest that dioscin and methylprotodioscin isolated from the root of Asparagus cochinchinensis suppress the gene expression and production of MUC5AC mucin, by directly acting on airway epithelial cells, and the results are consistent with the traditional use of Asparagus cochinchinensis as remedy for diverse inflammatory pulmonary diseases.

Evasion from the immune system has emerged as a novel hallmark in the genesis of neuroblastoma as well as in its progression. A profound knowledge of underlying immune regulatory mechanisms in neuroblastoma may be of vital importance to improve current and future immunotherapeutic approaches against this challenging childhood malignancy. This chapter highlights the basic immune escape mechanisms in NB with a particular focus on immune regulatory cells and regulatory molecules that drive immunity to tolerance. (C) 2015 S. Karger AG, Basel

The onset of pregnancy implies the appearance of a new organ, the placenta. One main function of the placenta is to supply oxygen to the fetus via hemoproteins. In this review, we highlight the importance of the enzyme heme oxygenase-1 (HO-1) for pregnancy to be established and maintained. HO-1 expression is pivotal to promote placental function and fetal development, thus determining the success of pregnancy. The deletion of the gene Hmox1 in mice leads to inadequate remodeling of spiral arteries and suboptimal placentation followed by intrauterine growth restriction (IUGR) and fetal lethality. A partial Hmox1 deletion leads to IUGR as well, with heterozygote and wild-type fetuses being born, but Hmox1-/- significantly below the expected Mendelian rate. This strong phenotype is associated with diminished number of pregnancy-protective uterine natural killer (uNK) cells. Pregnant heterozygote females develop gestational hypertension. The protective HO-1 effects on placentation and fetal growth can be mimicked by the exogenous administration of carbon monoxide (CO), a product of heme catalyzed by HO-1. CO application promotes the in situ proliferation of uNK cells, restores placentation and fetal growth, while normalizing blood pressure. Similarly, HO-1 inhibition provokes hypertension in pregnant rats. The HO-1/CO axis plays a pivotal role in sustaining pregnancy and aids in the understanding of the biology of pregnancy and reveals a promising therapeutic application in the treatment of pregnancy complications.