Scalable generation of functional human iPSC-derived CAR-macrophages that efficiently eradicate CD19-positive leukemia.

BACKGROUND: Macrophages have recently become attractive therapeutics in cancer immunotherapy. The potential of macrophages to infiltrate and influence solid malignancies makes them promising targets for the chimeric antigen receptor (CAR) technology to redirect their stage of polarization, thus enhancing their anticancer capacities. Given the emerging interest for CAR-macrophages, generation of such cells so far mainly depends on peripheral blood monocytes, which are isolated from the respective donor prior to genetic manipulation. This procedure is time-intensive and cost-intensive, while, in some cases, insufficient monocyte amounts can be recovered from the donor, thus hampering the broad applicability of this technology. Hence, we demonstrate the generation and effectiveness of CAR-macrophages from various stem cell sources using also modern upscaling technologies for next generation immune cell farming. METHODS: Primary human hematopoietic stem and progenitor cells and induced pluripotent stem cells were used to derive anti-CD19 CAR-macrophages. Anticancer activity of the cells was demonstrated in co-culture systems, including primary material from patients with leukemia. Generation of CAR-macrophages was facilitated by bioreactor technologies and single-cell RNA (scRNA) sequencing was used to characterize in-depth response and behavior of CAR-macrophages. RESULTS: Irrespective of the stem-cell source, CAR-macrophages exhibited enhanced and antigen-dependent phagocytosis of CD19+ target cancer cells with increased pro-inflammatory responses. Phagocytic capacity of CAR-macrophages was dependent on target cell CD19 expression levels with superior function of CAR-macrophages against CD19+ cancer cell lines and patient-derived acute lymphocytic leukemia cancer cells. scRNA sequencing revealed CAR-macrophages to be distinct from eGFP control cells after co-culture with target cells, which includes the activation of pro-inflammatory pathways and upregulation of chemokines and cytokines associated with adaptive immune cell recruitment, favoring the repolarization of CAR-macrophages to a pro-inflammatory state. Taken together, the data highlight the unique features of CAR-macrophages in combination with the successful upscaling of the production pipeline using a three-dimensional differentiation protocol and intermediate scale bioreactors. CONCLUSION: In summary, our work provides insights into the seminal use and behavior of CAR-macrophages which are derived from various sources of stem cells, while introducing a unique technology for CAR-macrophage manufacturing, all dedicated to the clinical translation of CAR-macrophages within the field of anticancer immunotherapies.

Role of Alpha-Fetoprotein (AFP) in Diagnosing Childhood Cancers and Genetic-Related Chronic Diseases.

Alpha-fetoprotein (AFP) is a protein commonly found during fetal development, but its role extends beyond birth. Throughout the first year of life, AFP levels can remain high, which can potentially mask various conditions from the neurological, metabolic, hematological, endocrine, and early childhood cancer groups. Although AFP reference values and clinical utility have been established in adults, evaluating AFP levels in children during the diagnostic process, treatment, and post-treatment surveillance is still associated with numerous diagnostic pitfalls. These challenges arise from the presence of physiologically elevated AFP levels, inconsistent data obtained from different laboratory tests, and the limited population of children with oncologic diseases that have been studied. To address these issues, it is essential to establish updated reference ranges for AFP in this specific age group. A population-based study involving a statistically representative group of patients could serve as a valuable solution for this purpose.

Inflammation-inducible promoters to overexpress immune inhibitory factors by MSCs.

BACKGROUND: Mesenchymal stromal cells (MSCs) are excessively investigated in the context of inflammation-driven diseases, but the clinical results are often moderate. MSCs are naturally activated by inflammatory signals, which lead to the secretion of immune inhibitory factors in inflamed tissues. Many work groups try to improve the therapeutic outcome of MSCs by genetic modification and the constitutive overexpression of immune modulatory transgenes. However, the ectopic secretion of immune inhibitory transgenes increases the chances of infections, and constitutive transgene expression is not necessary for chronic diseases undergoing different inflammatory stages. METHODS: We designed and tested inflammation-induced promoters to control transgene expression from integrating lentiviral vectors in human umbilical cord MSCs. Therefore, we investigated different combinations of general transcription factor elements to achieve a minimal promoter with low basal activity. The best candidates were combined with interferon-induced GAS or ISRE DNA motifs. The constructs with the highest transgene expression upon addition of pro-inflammatory cytokines were compared to vectorized promoters from inflammation-induced genes (CD317, CXCL9, CXCL10, CXCL11 and IDO1). Finally, we investigated IL10 as a potential immune inhibitory transgene by transcriptome analyses, ELISA and in an acute lung injury mouse model. RESULTS: The synthetic promoters achieved a high and specific transgene expression upon IFN-γ addition. However, the CXCL11 promoter showed synergistic activity upon IFN-γ, TNF-α and IL1-ß treatment and surpassed the transgene expression height of all tested promoters in the study. We observed in transcriptome analyses that IL10 has no effect on MSCs and in ELISA that IL10 is only secreted by our genetically modified and activated CXCL11-IL10-MSCs. Finally, transplanted CXCL11-IL10-MSCs increased CD19+ and CD4+ lymphoid cells, and decreased CD11b+ Ly6g myeloid cells in an ALI mouse model. CONCLUSION: These results provide new insights into MSC inflammatory activation and the subsequent translation into a tool for a tailored expression of transgenes in inflammatory microenvironments. The newly developed promoter elements are potentially interesting for other inflamed tissues, and can be combined with other elements or used in other cell types.

The Role of Alpha-Fetoprotein (AFP) in Contemporary Oncology: The Path from a Diagnostic Biomarker to an Anticancer Drug.

This article presents contemporary opinion on the role of alpha-fetoprotein in oncologic diagnostics and treatment. This role stretches far beyond the already known one-that of the biomarker of hepatocellular carcinoma. The turn of the 20th and 21st centuries saw a significant increase in knowledge about the fundamental role of AFP in the neoplastic processes, and in the induction of features of malignance and drug resistance of hepatocellular carcinoma. The impact of AFP on the creation of an immunosuppressive environment for the developing tumor was identified, giving rise to attempts at immunotherapy. The paper presents current and prospective therapies using AFP and its derivatives and the gene therapy options. We directed our attention to both the benefits and risks associated with the use of AFP in oncologic therapy.

Influence of selected factors on serum AFP levels in pregnant women in terms of prenatal screening accuracy - literature review.

Alpha-fetoprotein (AFP) is one of the biochemical components of the triple (T-3) and quadruple (T-4) test used so far in prenatal screening mainly for trisomy 21 (T21) and neural tube defects (NTDs). Based on many years of experience and data collected during these studies, a variety of factors have been identified that can affect a pregnant woman's serum AFP level, and thus the risk assessment of trisomy 21 (T21) and neural tube defects. These include both unaccounted for purely medical data (e.g., from baseline information about the patient, assisted reproduction methods used, comorbidities and emerging pregnancy pathologies) and errors made during statistical analysis. Since the triple or quadruple test is usually performed between 15 and 20 weeks of pregnancy, most scientific studies are based solely on results from this period of pregnancy - limited data are available for the first and third trimesters of pregnancy. In the era of new improved screening tests, AFP has the potential to become an independent marker for pregnancy well-being evaluation.

Fully Human Herpesvirus-Specific Neutralizing IgG Antibodies Generated by EBV Immortalization of Splenocytes-Derived from Immunized Humanized Mice.

Antiviral neutralizing antibodies (nAbs) are commonly derived from B cells developed in immunized or infected animals and humans. Fully human antibodies are preferred for clinical use as they are potentially less immunogenic. However, the function of B cells varies depending on their homing pattern and an additional hurdle for antibody discovery in humans is the source of human tissues with an immunological microenvironment. Here, we show an efficient method to pharm human antibodies using immortalized B cells recovered from Nod.Rag.Gamma (NRG) mice reconstituting the human immune system (HIS). Humanized HIS mice were immunized either with autologous engineered dendritic cells expressing the human cytomegalovirus gB envelope protein (HCMV-gB) or with Epstein-Barr virus-like particles (EB-VLP). Human B cells recovered from spleen of HIS mice were efficiently immortalized with EBV in vitro. We show that these immortalized B cells secreted human IgGs with neutralization capacities against prototypic HCMV-gB and EBV-gp350. Taken together, we show that HIS mice can be successfully used for the generation and pharming fully human IgGs. This technology can be further explored to generate antibodies against emerging infections for diagnostic or therapeutic purposes.

Downregulation of miR-1270 mediates endothelial progenitor cell function in preeclampsia: Role for ATM in the Src/VE-cadherin axis.

Preeclampsia, a pregnancy-related hypertensive disorder, is associated with endothelial dysfunction and increased cardiovascular risk of the offspring in adulthood. In preeclampsia, endothelial colony-forming cells (ECFC) are reduced in number and function. Recently, we have shown that miR-1270, which is involved in cancer in vitro proliferation, migration, and tumor progression, is downregulated in fetal ECFC from preeclamptic pregnancies. We now hypothesize that miR-1270 dysregulation contributes to vascular endothelial dysfunction occurring after preeclampsia via ATM (ataxia telangiectasia mutated) overexpression, the key kinase of DNA damage repair. Here, we show that miR-1270 silencing in normal ECFC and downregulation in preeclamptic ECFC are accompanied by an increase in the expression levels of ATM. Furthermore, ATM activation correlates with upregulated tyrosine kinase Src leading to phosphorylation and internalization of VE-cadherin (vascular endothelial-cadherin) which subsequently compromises endothelial barrier permeability and morphodynamic cell parameters. Treatment with specific ATM inhibitors reveals a novel role of ATM upstream of tyrosine kinase Src activation. Subsequently, Src phosphorylation and internalization of VE-cadherin compromise endothelial barrier permeability. Our findings suggest that downregulation of miR-1270 contributes to impaired ECFC function via the associated ATM overexpression, which further identifies ATM as a novel and critical factor for ECFC defects in preeclampsia. Our study provides new insights into the understanding of ECFC impairment associated with cardiovascular risk in preeclamptic offspring and identifies potential novel therapeutic targets.

Ex Vivo Generation of CAR Macrophages from Hematopoietic Stem and Progenitor Cells for Use in Cancer Therapy.

Chimeric antigen receptor (CAR) T-cell therapies have shown impressive results in patients with hematological malignancies; however, little success has been achieved in the treatment of solid tumors. Recently, macrophages (MΦs) were identified as an additional candidate for the CAR approach, and initial proof of concept studies using peripheral blood-derived monocytes showed antigen-redirected activation of CAR MΦs. However, some patients may not be suitable for monocyte-apheresis, and prior cancer treatment regimens may negatively affect immune cell number and functionality. To address this problem, we here introduce primary human hematopoietic stem and progenitor cells (HSPCs) as a cell source to generate functional CAR MΦs ex vivo. Our data showed successful CAR expression in cord blood (CB)-derived HSPCs, with considerable cell expansion during differentiation to CAR MΦs. HSPC-derived MΦs showed typical MΦ morphology, phenotype, and basic anti-bacterial functionality. CAR MΦs targeting the carcinoembryonic antigen (CEA) and containing either a DAP12- or a CD3ζ-derived signaling domain showed antigen redirected activation as they secreted pro-inflammatory cytokines specifically upon contact with CEA+ target cells. In addition, CD3ζ-expressing CAR MΦs exhibited significantly enhanced phagocytosis of CEA+ HT1080 cells. Our data establish human HSPCs as a suitable cell source to generate functional CAR MΦs and further support the use of CAR MΦs in the context of solid tumor therapy.

Alpha-fetoprotein (AFP) - new aspects of a well-known marker in perinatology.

Alpha-fetoprotein (AFP) is a serum protein, which is characteristic of the fetal development period and a well-known oncological marker. The predominance of AFP among serum proteins is common in fetal life, whereas after birthing its functions are gradually taken over by albumins. An understanding of the mechanism of AFP transfer between fetus and mother has led to the development of screening tests for identifying neural tube defects and Down's syndrome. Currently, the knowledge on patophysiology and the possible importance of AFP in perinatology and fetal medicine extends far beyond those 2 disease states. Throughout the 50 years of research on AFP, there has been dynamic progress of diagnostic techniques, from the qualitative ones that are used solely for scientific studies to the widely used radioimmunoassays and immunoenzymatic assays (enzyme-linked immunosorbent assay, chemiluminescence immunoassay, time-resolved fluorescence immunoassay). Some genetic mutations cause complete inhibition of AFP production by the fetus. This affects the results of screening tests during pregnancy, and also leads to constantly high levels of AFP in adults, which are not linked to oncogenesis.

Cyclosporine A and Tacrolimus Induce Functional Impairment and Inflammatory Reactions in Endothelial Progenitor Cells.

Immunosuppressants are a mandatory therapy for transplant patients to avoid rejection of the transplanted organ by the immune system. However, there are several known side effects, including alterations of the vasculature, which involve a higher occurrence of cardiovascular events. While the effects of the commonly applied immunosuppressive drugs cyclosporine A (CsA) and tacrolimus (Tac) on mature endothelial cells have been addressed in several studies, we focused our research on the unexplored effects of CsA and Tac on endothelial colony-forming cells (ECFCs), a subgroup of endothelial progenitor cells, which play an important role in vascular repair and angiogenesis. We hypothesized that CsA and Tac induce functional defects and activate an inflammatory cascade via NF-κB signaling in ECFCs. ECFCs were incubated with different doses (0.01 µM-10 µM) of CsA or Tac. ECFC function was determined using in vitro models. The expression of inflammatory cytokines and adhesion molecules was explored by quantitative real-time PCR and flow cytometry. NF-κB subunit modification was assessed by immunoblot and immunofluorescence. CsA and Tac significantly impaired ECFC function, including proliferation, migration, and tube formation. TNF-α, IL-6, VCAM, and ICAM mRNA expression, as well as PECAM and VCAM surface expression, were enhanced. Furthermore, CsA and Tac led to NF-κB p65 subunit phosphorylation and nuclear translocation. Pharmacological inhibition of NF-κB by parthenolide diminished CsA- and Tac-mediated proinflammatory effects. The data of functional impairment and activation of inflammatory signals provide new insight into mechanisms associated with CsA and Tac and cardiovascular risk in transplant patients.

In Vivo Lentiviral Gene Delivery of HLA-DR and Vaccination of Humanized Mice for Improving the Human T and B Cell Immune Reconstitution.

Humanized mouse models generated with human hematopoietic stem cells (HSCs) and reconstituting the human immune system (HIS-mice) are invigorating preclinical testing of vaccines and immunotherapies. We have recently shown that human engineered dendritic cells boosted bonafide human T and B cell maturation and antigen-specific responses in HIS-mice. Here, we evaluated a cell-free system based on in vivo co-delivery of lentiviral vectors (LVs) for expression of a human leukocyte antigen (HLA-DRA*01/ HLA-DRB1*0401 functional complex, "DR4"), and a LV vaccine expressing human cytokines (GM-CSF and IFN-α) and a human cytomegalovirus gB antigen (HCMV-gB). Humanized NOD/Rag1null/IL2Rγnull (NRG) mice injected by i.v. with LV-DR4/fLuc showed long-lasting (up to 20 weeks) vector distribution and expression in the spleen and liver. In vivo administration of the LV vaccine after LV-DR4/fLuc delivery boosted the cellularity of lymph nodes, promoted maturation of terminal effector CD4+ T cells, and promoted significantly higher development of IgG+ and IgA+ B cells. This modular lentigenic system opens several perspectives for basic human immunology research and preclinical utilization of LVs to deliver HLAs into HIS-mice.

Pulmonary transplantation of alpha-1 antitrypsin (AAT)-transgenic macrophages provides a source of functional human AAT in vivo.

Inherited deficiency of the antiprotease alpha-1 antitrypsin (AAT) is associated with liver failure and early-onset emphysema. In mice, in vivo lentiviral transduction of alveolar macrophages (AMs) has been described to yield protective pulmonary AAT levels and ameliorate emphysema development. We here investigated the pulmonary transplantation of macrophages (PMT) transgenic for AAT as a potential therapy for AAT deficiency-associated lung pathology. Employing third-generation SIN-lentiviral vectors expressing the human AAT cDNA from the CAG or Cbx-EF1α promoter, we obtained high-level AAT secretion in a murine AM cell line as well as murine bone marrow-derived macrophages differentiated in vitro (AAT MΦ). Secreted AAT demonstrated a physiologic glycosylation pattern as well as elastase-inhibitory and anti-apoptotic properties. AAT MΦ preserved normal morphology, surface phenotype, and functionality. Furthermore, in vitro generated murine AAT MΦ successfully engrafted in AM-deficient Csf2rb-/- mice and converted into a CD11c+/Siglec-F+ AM phenotype as detected in bronchoalveolar lavage fluid and homogenized lung tissue 2 months after PMT. Moreover, human AAT was detected in the lung epithelial lining fluid of transplanted animals. Efficient AAT expression and secretion were also demonstrated for human AAT MΦ, confirming the applicability of our vectors in human cells.

Induced dendritic cells co-expressing GM-CSF/IFN-α/tWT1 priming T and B cells and automated manufacturing to boost GvL.

Acute myeloid leukemia (AML) patients with minimal residual disease and receiving allogeneic hematopoietic stem cell transplantation (HCT) have poor survival. Adoptive administration of dendritic cells (DCs) presenting the Wilms tumor protein 1 (WT1) leukemia-associated antigen can potentially stimulate de novo T and B cell development to harness the graft-versus-leukemia (GvL) effect after HCT. We established a simple and fast genetic modification of monocytes for simultaneous lentiviral expression of a truncated WT1 antigen (tWT1), granulocyte macrophage-colony-stimulating factor (GM-CSF), and interferon (IFN)-α, promoting their self-differentiation into potent "induced DCs" (iDCtWT1). A tricistronic integrase-defective lentiviral vector produced under good manufacturing practice (GMP)-like conditions was validated. Transduction of CD14+ monocytes isolated from peripheral blood, cord blood, and leukapheresis material effectively induced their self-differentiation. CD34+ cell-transplanted Nod.Rag.Gamma (NRG)- and Nod.Scid.Gamma (NSG) mice expressing human leukocyte antigen (HLA)-A∗0201 (NSG-A2)-immunodeficient mice were immunized with autologous iDCtWT1. Both humanized mouse models showed improved development and maturation of human T and B cells in the absence of adverse effects. Toward clinical use, manufacturing of iDCtWT1 was up scaled and streamlined using the automated CliniMACS Prodigy system. Proof-of-concept clinical-scale runs were feasible, and the 38-h process enabled standardized production and high recovery of a cryopreserved cell product with the expected identity characteristics. These results advocate for clinical trials testing iDCtWT1 to boost GvL and eradicate leukemia.

Enhanced differentiation of functional human T cells in NSGW41 mice with tissue-specific expression of human interleukin-7.

Humanized mouse models have become increasingly valuable tools to study human hematopoiesis and infectious diseases. However, human T-cell differentiation remains inefficient. We generated mice expressing human interleukin-7 (IL-7), a critical growth and survival factor for T cells, under the control of murine IL-7 regulatory elements. After transfer of human cord blood-derived hematopoietic stem and progenitor cells, transgenic mice on the NSGW41 background, termed NSGW41hIL7, showed elevated and prolonged human cellularity in the thymus while maintaining physiological ratios of thymocyte subsets. As a consequence, numbers of functional human T cells in the periphery were increased without evidence for pathological lymphoproliferation or aberrant expansion of effector or memory-like T cells. We conclude that the novel NSGW41hIL7 strain represents an optimized mouse model for humanization to better understand human T-cell differentiation in vivo and to generate a human immune system with a better approximation of human lymphocyte ratios.

Low Ethanol Concentrations Promote Endothelial Progenitor Cell Capacity and Reparative Function.

BACKGROUND: Endothelial progenitor cells (EPCs) are recruited to injured endothelium and contribute to its regeneration. There is evidence that moderate ethanol consumption prevents the development and progression of atherosclerosis in a variety of in vitro and in vivo models and increases the mobilization of progenitor cells. Furthermore, there are studies that identified ethanol at low concentration as a therapeutic tool to mobilize progenitor cells in peripheral blood. At the same time, the cell number of EPCs represents a close link to cardiovascular system constitution and function and contributes to cardiovascular risk. The aim of this study was to evaluate the effect of low dose ethanol on typical features of endothelial colony-forming cells (ECFCs), a proliferative subtype of EPCs. METHODS AND RESULTS: We tested whether ethanol impacts the functional abilities of ECFC (e.g., migration, tube formation, and proliferation) using in vitro assays, the intercommunication of ECFC by exploring cell surface molecules by flow cytometry, and the expression of (anti-)angiogenic molecules by ELISA. Low concentrations of ethanol concentration promoted migration, proliferation, and tubule formation of ECFC. The expression of the cell surface marker VE-cadherin, a protein which plays an important role in cell-cell interaction, was enhanced by ethanol, while (anti-)angiogenic molecule expression was not impacted. CONCLUSION: Ethanol at moderate concentrations increases the angiogenic abilities of endothelial progenitor cells thus possibly contributing to vasoprotection.

CAR-T Cells Targeting Epstein-Barr Virus gp350 Validated in a Humanized Mouse Model of EBV Infection and Lymphoproliferative Disease.

Epstein-Barr virus (EBV) is a latent and oncogenic human herpesvirus. Lytic viral protein expression plays an important role in EBV-associated malignancies. The EBV envelope glycoprotein 350 (gp350) is expressed abundantly during EBV lytic reactivation and sporadically on the surface of latently infected cells. Here we tested T cells expressing gp350-specific chimeric antigen receptors (CARs) containing scFvs derived from two novel gp350-binding, highly neutralizing monoclonal antibodies. The scFvs were fused to CD28/CD3ζ signaling domains in a retroviral vector. The produced gp350CAR-T cells specifically recognized and killed gp350+ 293T cells in vitro. The best-performing 7A1-gp350CAR-T cells were cytotoxic against the EBV+ B95-8 cell line, showing selectivity against gp350+ cells. Fully humanized Nod.Rag.Gamma mice transplanted with cord blood CD34+ cells and infected with the EBV/M81/fLuc lytic strain were monitored dynamically for viral spread. Infected mice recapitulated EBV-induced lymphoproliferation, tumor development, and systemic inflammation. We tested adoptive transfer of autologous CD8+gp350CAR-T cells administered protectively or therapeutically. After gp350CAR-T cell therapy, 75% of mice controlled or reduced EBV spread and showed lower frequencies of EBER+ B cell malignant lymphoproliferation, lack of tumor development, and reduced inflammation. In summary, CD8+gp350CAR-T cells showed proof-of-concept preclinical efficacy against impending EBV+ lymphoproliferation and lymphomagenesis.

Repeated Freezing Procedures Preserve Structural and Functional Properties of Amniotic Membrane for Application in Ophthalmology.

For decades, the unique regenerative properties of the human amniotic membrane (hAM) have been successfully utilized in ophthalmology. As a directly applied biomaterial, the hAM should be available in a ready to use manner in clinical settings. However, an extended period of time is obligatory for performing quality and safety tests. Hence, the low temperature storage of the hAM is a virtually inevitable step in the chain from donor retrieval to patient application. At the same time, the impact of subzero temperatures carries an increased risk of irreversible alterations of the structure and composition of biological objects. In the present study, we performed a comprehensive analysis of the hAM as a medicinal product; this is intended for a novel strategy of application in ophthalmology requiring a GMP production protocol including double freezing-thawing cycles. We compared clinically relevant parameters, such as levels of growth factors and extracellular matrix proteins content, morphology, ultrastructure and mechanical properties, before and after one and two freezing cycles. It was found that epidermal growth factor (EGF), transforming growth factor beta 1 (TGF-ß1), hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), hyaluronic acid, and laminin could be detected in all studied conditions without significant differences. Additionally, histological and ultrastructure analysis, as well as transparency and mechanical tests, demonstrated that properties of the hAM required to support therapeutic efficacy in ophthalmology are not impaired by dual freezing.

Intracranial emergencies during pregnancy requiring urgent neurosurgical treatment.

OBJECTIVE: Despite contemporary diagnostic and therapeutic techniques intracranial emergencies in the obstetric setting pose still a major challenge for the clinicians. There are limited guidelines and differing ethical views. Multidisciplinary teams are needed to support the pregnant woman in a way that she can deliver a viable and healthy child. The aim of the present study was to scrutinize the management of intracranial emergencies during pregnancy which needed urgent neurosurgical treatment. PATIENTS AND METHODS: Data of all pregnant women who presented with newly diagnosed intracranial pathologies and neurological symptoms caused by these pathologies in an emergency setting were collected over a 10-year period (2008-2018). Patient characteristics including maternal age, gestational age, and preoperative work-up of both mother and fetus were recorded. Furthermore, the surgical treatment, mode of delivery, and neonatal and maternal outcomes were analysed. RESULTS: The mean maternal age was 32.7 years and most patients were in their third trimester. There was one twin pregnancy (total of 12 fetuses). Five out of eleven pregnant women suffered from intracerebral haemorrhage (epidural haematoma (1), arteriovenous malformation (1), subarachnoid haemorrhage (2) and intracerebral haemorrhage (1)) and the other six patients had intracranial neoplasms (primary meningeal sarcoma (1), trigeminal schwannoma (1), anaplastic astrocytoma (2), glioblastoma (1) and sphenoid wing meningioma (1)).Neurosurgical procedures were performed via craniotomies in eight patients. A stereotactic biopsy via a frontal burr hole was achieved one patient. The two other patients with subarachnoid haemorrhage due to rupture of PICA aneurysms were treated with coil embolization. Depending on the gestational age and the clinical condition of the pregnant women it was decided to perform an emergency Caesarean section prior to further therapeutic measures in seven patients. Two out of 12 fetuses were unviable. Six women survived, while five women succumbed to the intracranial pathology. CONCLUSION: The individualized treatment approach in this peculiar obstetric scenario needs to consider various issues such as the clinical condition of the pregnant woman, prognosis of the disease, gestational age and the status of the pregnancy. The primary concern in this context must be the mother`s health and safety. Caesarean section is the primary mode of delivery in most cases. While contemporary care can insure survival for the majority of infants, maternal mortality still poses an extraordinary challenge. Interdisciplinary consulting of the patient and/or her family is necessary to develop a treatment strategy for both the expectant woman and her offspring.

Spinal Emergency Surgery During Pregnancy: Contemporary Strategies and Outcome.

BACKGROUND: Low back pain is a common complaint during pregnancy. However, spinal pathologies, which manifest with severe pain, radiculopathy, and acute neurologic deficits because of disk herniation or mass lesions require special attention. Here, we present our interdisciplinary experience in the surgical management of spinal emergencies during pregnancy. METHODS: The data of pregnant women who underwent surgery for spinal pathologies over a 10-year period were collected. Patient-related characteristics such as maternal age, gestational age, preoperative workup, signs and symptoms of mothers, and diagnostic procedures were evaluated. After an interdisciplinary conference, individualized treatment plans regarding available options were developed. Fetal Doppler and cardiotocography were obtained before and after surgery. RESULTS: Nine pregnant women presented with spinal disorders and underwent spinal emergency surgery within the study period. The mean maternal age was 32.2 years. Six women presented with lumbar disk herniations manifesting as severe sciatica or foot drop and 3 patients had thoracic mass lesions resulting in cauda equine syndrome and/or ataxia. The mean gestational age at the time of presentation was 26.5 weeks. Caesarean sections were performed in 3 women prior to the neurosurgical procedure, whereas the pregnancies were maintained in the 6 other patients. Eight infants who were healthy at birth had an unremarkable development. CONCLUSIONS: Surgery for spinal emergencies in pregnancy can be performed safely according to individual treatment plans developed by an interdisciplinary team taking into account the expectant mother's decision. Maintenance of pregnancy is possible and feasible in most patients.

PD-1 Blockade Aggravates Epstein-Barr Virus+ Post-Transplant Lymphoproliferative Disorder in Humanized Mice Resulting in Central Nervous System Involvement and CD4+ T Cell Dysregulations.

Post-transplant lymphoproliferative disorder (PTLD) is one of the most common malignancies after solid organ or allogeneic stem cell transplantation. Most PTLD cases are B cell neoplasias carrying Epstein-Barr virus (EBV). A therapeutic approach is reduction of immunosuppression to allow T cells to develop and combat EBV. If this is not effective, approaches include immunotherapies such as monoclonal antibodies targeting CD20 and adoptive T cells. Immune checkpoint inhibition (ICI) to treat EBV+ PTLD was not established clinically due to the risks of organ rejection and graft-versus-host disease. Previously, blockade of the programmed death receptor (PD)-1 by a monoclonal antibody (mAb) during ex vivo infection of mononuclear cells with the EBV/M81+ strain showed lower xenografted lymphoma development in mice. Subsequently, fully humanized mice infected with the EBV/B95-8 strain and treated in vivo with a PD-1 blocking mAb showed aggravation of PTLD and lymphoma development. Here, we evaluated vis-a-vis in fully humanized mice after EBV/B95-8 or EBV/M81 infections the effects of a clinically used PD-1 blocker. Fifteen to 17 weeks after human CD34+ stem cell transplantation, Nod.Rag.Gamma mice were infected with two types of EBV laboratory strains expressing firefly luciferase. Dynamic optical imaging analyses showed systemic EBV infections and this triggered vigorous human CD8+ T cell expansion. Pembrolizumab administered from 2 to 5 weeks post-infections significantly aggravated EBV systemic spread and, for the M81 model, significantly increased the mortality of mice. ICI promoted Ki67+CD30+CD20+EBER+PD-L1+ PTLD with central nervous system (CNS) involvement, mirroring EBV+ CNS PTLD in humans. PD-1 blockade was associated with lower frequencies of circulating T cells in blood and with a profound collapse of CD4+ T cells in lymphatic tissues. Mice treated with pembrolizumab showed an escalation of exhausted T cells expressing TIM-3, and LAG-3 in tissues, higher levels of several human cytokines in plasma and high densities of FoxP3+ regulatory CD4+ and CD8+ T cells in the tumor microenvironment. We conclude that PD-1 blockade during acute EBV infections driving strong CD8+ T cell priming decompensates T cell development towards immunosuppression. Given the variety of preclinical models available, our models conferred a cautionary note indicating that PD-1 blockade aggravated the progression of EBV+ PTLD.