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.

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.

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.

Vitamin D improves endothelial barrier integrity and counteracts inflammatory effects on endothelial progenitor cells.

Endothelial colony-forming cells (ECFCs), a proliferative subpopulation of endothelial progenitor cells, are involved in angiogenesis and endothelial repair. In this study, we investigated endothelial barrier characteristics of ECFCs, whether vitamin D supports cell-cell adhesion and barrier integrity, and how it affects ECFC mobilization and actin dynamics. Although ECFC barrier was disrupted under inflammatory conditions, this effect was rescued by vitamin D treatment, leading to higher stability of an ECFC monolayer. Furthermore, vitamin D enhanced ECFC mobilization toward directional migration. In addition, immunocytochemistry, quantitative real-time PCR, and immunoblotting analysis showed that vitamin D increased endothelial interconnections through vascular endothelial cadherin (VE-cadherin) junctions and by impacting cell dynamics through cofilin and VE-cadherin phosphorylation. Our results suggest that vitamin D treatment efficiently counteracts inflammation in an ECFC monolayer, resulting in higher ECFC barrier integrity. This study provides evidence of a new beneficial effect of vitamin D for ECFC homeostasis.-Schröder-Heurich, B., von Hardenberg, S., Brodowski, L., Kipke, B., Meyer, N., Borns, K., von Kaisenberg, C. S., Brinkmann, H., Claus, P., von Versen-Höynck, F. Vitamin D improves endothelial barrier integrity and counteracts inflammatory effects on endothelial progenitor cells.

S100-alarmin-induced innate immune programming protects newborn infants from sepsis.

The high risk of neonatal death from sepsis is thought to result from impaired responses by innate immune cells; however, the clinical observation of hyperinflammatory courses of neonatal sepsis contradicts this concept. Using transcriptomic, epigenetic and immunological approaches, we demonstrated that high amounts of the perinatal alarmins S100A8 and S100A9 specifically altered MyD88-dependent proinflammatory gene programs. S100 programming prevented hyperinflammatory responses without impairing pathogen defense. TRIF-adaptor-dependent regulatory genes remained unaffected by perinatal S100 programming and responded strongly to lipopolysaccharide, but were barely expressed. Steady-state expression of TRIF-dependent genes increased only gradually during the first year of life in human neonates, shifting immune regulation toward the adult phenotype. Disruption of this critical sequence of transient alarmin programming and subsequent reprogramming of regulatory pathways increased the risk of hyperinflammation and sepsis. Collectively these data suggest that neonates are characterized by a selective, transient microbial unresponsiveness that prevents harmful hyperinflammation in the delicate neonate while allowing for sufficient immunological protection.

In neonates S100A8/S100A9 alarmins prevent the expansion of a specific inflammatory monocyte population promoting septic shock.

The high susceptibility of newborn infants to sepsis is ascribed to an immaturity of the neonatal immune system, but the molecular mechanisms remain unclear. Newborn monocytes massively release the alarmins S100A8/S100A9. In adults, these are major regulators of immunosuppressive myeloid-derived suppressor cells (MDSCs). We investigated whether S100A8/S100A9 cause an expansion of monocytic MDSCs (Mo-MDSCs) in neonates, thereby contributing to an immunocompromised state. Mo-MDSCs have been assigned to CD14+/human leukocyte antigen (HLA)-DR-/low/CD33+ monocytes in humans and to CD11b+/Gr-1int/Ly6G-/Ly6Chi cells in mice. We found monocytes with these phenotypes significantly expanded in their respective newborns. Functionally, however, they did not prove immunosuppressive but rather responded inflammatorily to microbial stimulation. Their expansion did not correlate with high S100A8/S100A9 levels in cord blood. Murine studies revealed an excessive expansion of CD11b+/Gr-1int/Ly6G-/Ly6Chi monocytes in S100A9-/- neonates compared to wild-type neonates. This strong baseline expansion was associated with hyperinflammatory responses during endotoxemia and fatal septic courses. Treating S100A9-/- neonates directly after birth with S100A8/S100A9 alarmins prevented excessive expansion of this inflammatory monocyte population and death from septic shock. Our data suggest that a specific population of inflammatory monocytes promotes fatal courses of sepsis in neonates if its expansion is not regulated by S100A8/S100A9 alarmins.-Heinemann, A. S., Pirr, S., Fehlhaber, B., Mellinger, L., Burgmann, J., Busse, M., Ginzel, M., Friesenhagen, J., von Köckritz-Blickwede, M., Ulas, T., von Kaisenberg, C. S., Roth, J., Vogl, T., Viemann, D. In neonates S100A8/S100A9 alarmins prevent the expansion of a specific inflammatory monocyte population promoting septic shock.

Successful Pregnancy and Neobladder Subsequent to Muscle Invasive Bladder Cancer and Fertility Preserving Surgery: Case Report and Review of the Literature.

We report a successful pregnancy and birth subsequent to fertility-preserving cystectomy and neobladder formation in a muscle-invasive sarcomatoid urothelial carcinoma.

Alarmins MRP8 and MRP14 induce stress tolerance in phagocytes under sterile inflammatory conditions.

Hyporesponsiveness by phagocytes is a well-known phenomenon in sepsis that is frequently induced by low-dose endotoxin stimulation of Toll-like receptor 4 (TLR4) but can also be found under sterile inflammatory conditions. We now demonstrate that the endogenous alarmins MRP8 and MRP14 induce phagocyte hyporesponsiveness via chromatin modifications in a TLR4-dependent manner that results in enhanced survival to septic shock in mice. During sterile inflammation, polytrauma and burn trauma patients initially present with high serum concentrations of myeloid-related proteins (MRPs). Human neonatal phagocytes are primed for hyporesponsiveness by increased peripartal MRP concentrations, which was confirmed in murine neonatal endotoxinemia in wild-type and MRP14(-/-) mice. Our data therefore indicate that alarmin-triggered phagocyte tolerance represents a regulatory mechanism for the susceptibility of neonates during systemic infections and sterile inflammation.

Vitamin D prevents endothelial progenitor cell dysfunction induced by sera from women with preeclampsia or conditioned media from hypoxic placenta.

CONTEXT: Placenta-derived circulating factors contribute to the maternal endothelial dysfunction underlying preeclampsia. Endothelial colony forming cells (ECFC), a sub-population of endothelial progenitor cells (EPCs), are thought to be involved in vasculogenesis and endothelial repair. Low vitamin D concentrations are associated with an increased risk for preeclampsia. OBJECTIVE: We hypothesized that the function of human fetal ECFCs in culture would be suppressed by exposure to preeclampsia-related factors--preeclampsia serum or hypoxic placental conditioned medium--in a fashion reversed by vitamin D. DESIGN, SETTING, PATIENTS: ECFCs were isolated from cord blood of uncomplicated pregnancies and expanded in culture. Uncomplicated pregnancy villous placenta in explant culture were exposed to either 2% (hypoxic), 8% (normoxic) or 21% (hyperoxic) O2 for 48 h, after which the conditioned media (CM) was collected. OUTCOME MEASURES: ECFC tubule formation (Matrigel assay) and migration were examined in the presence of either maternal serum from preeclampsia cases or uncomplicated pregnancy controls, or pooled CM, in the presence or absence of 1,25(OH)2 vitamin D3. RESULTS: 1,25(OH)2 vitamin D3 reversed the adverse effects of preeclampsia serum or CM from hypoxic placenta on ECFCs capillary-tube formation and migration. Silencing of VDR expression by VDR siRNA, VDR blockade, or VEGF pathway blockade reduced ECFC functional abilities. Effects of VDR or VEGF blockade were partially prevented by vitamin D. CONCLUSION: Vitamin D promotes the capillary-like tubule formation and migration of ECFCs in culture, minimizing the negative effects of exposure to preeclampsia-related factors. Further evaluation of the role of vitamin D in ECFC regulation and preeclampsia is warranted.

Diagnostic challenges of hemihematocolpos and dysmenorrhea in adolescents: obstructed hemivagina, didelphys or bicornuate uterus and renal aplasia is a rare female genital malformation.

OBJECTIVES: To develop a clear diagnostic and therapeutic strategy for adolescents presenting with abdominal pain and vaginal tumor caused by congenital female genital anomalies, such as blind hemivagina and uterine anomalies, as the lack of the correct diagnosis of the underlying anatomical genitourinary malformation frequently leads to destructive surgical procedures. METHODS: Retrospective study, study group: patients with double/bicornuate uterus, blind hemivagina and hematocolpos (n = 13), controls: patients with uterine malformation and complete vertical vaginal septum (n = 11), analysis for: menarche, age at onset of symptoms, type of malformation, symptoms leading to admission and diagnostic/surgical techniques applied. RESULTS: Median age at diagnosis study group 19.85 (SD ± 6.23, range 13-23 years) versus controls 26.09 years (SD ± 7.44, 16-36 years); predominance of imperforated hemivagina: 69.2 % right-sided versus 30.8 % left-sided septum; renal agenesis ipsilateral to imperforate hemivagina 100 % study group versus 9.1 % controls; 84.6 % previous surgical interventions in the study group, such as partial removal of the septum and re-obliteration, unilateral salpingo-ovarectomy and vaginal drainage of pyometra. We used a single transvaginal surgical procedure, including removal of the obstructed vaginal septum and marsupialization of the blind hemivagina. CONCLUSIONS: A diagnostic and therapeutic algorithm for young women presenting with progressive dysmenorrhea and abdominal pain and/or vaginal tumor reduces destructive interventions.

Chromosomal mosaicisms in prenatal diagnosis: correlation with first trimester screening and clinical outcome.

OBJECTIVE: To investigate the outcome of pregnancy after detection of chromosomal mosaicism and to determine the correlation between human chorionic gonadotropin (free ß-HCG) and pregnancy-associated plasma protein-A (PAPP-A) levels from first-trimester-screening with pregnancy outcome. METHODS: In a single-center, retrospective survey of the results of prenatal diagnostics performed between January 2000 and March 2011, we identified a total of 40 pregnancies with chromosomal mosaicism. Clinical characteristics and results of first-trimester screening, as well as the outcome of these cases, are described. RESULTS: Out of 40 cases, 21 were defined as confined placental mosaicism, 10 classified as true mosaicism and nine were not classifiable cases. Nuchal translucency (NT) was ≥2.5 mm in 8/30 cases with respective measurements. PAPP-A levels were ≤0.4 MoM in 9/26 cases, with respective measurements, two of them being newborns with growth restriction. Remarkably, in pregnancies of all four children born with severe growth retardation, <3rd percentile PAPP-A levels were below 0.52 MoM. CONCLUSIONS: Our observations show mosaic pregnancy outcomes to be very heterogeneous. Nevertheless, a combination of low PAPP-A and interpretation of chromosomal mosaicism might identify pregnancies at particular risk for fetal growth restriction.

Pathologic ultrasound findings and risk for congenital anomalies in teenage pregnancies.

OBJECTIVE: To detect the number and diagnosis of fetal malformations in teenage pregnancies and to evaluate whether low maternal age or epigenetic factors have an influence on this issue. MATERIALS AND METHODS: We performed a retrospective analysis in a single center for prenatal diagnostics in Northern Germany. We searched our electronic databank for all pregnancies with maternal age under 20 years. Pregnancy outcome and fetal malformations are described. RESULTS: The incidence of teenage pregnancies in our study was 638 patients (4.4%). The total of fetal malformations in teenage pregnancies was 51(8.3%). Chromosomal aberrations were found in 5 cases (0.9%). 9 cases of fetal gastroschisis as one of the most frequent malformations were followed up and neonatal outcome was uneventful. Furthermore we found 16 cases with different heart defects and 30 cases with other malformations. Patients' body mass indices showed an increase over the years and nicotine consumption was testified in more than 50% of the patients. CONCLUSIONS: Teenage pregnancies are at risk for fetal non-chromosomal and chromosomal abnormalities. As these might be detected by first-trimester-screening prenatal care in teenage pregnancies should include at least early ultrasound examination. Epigenetic factors may play a key role in certain fetal malformations.

Combined treatment with TRAIL and PPARγ ligands overcomes chemoresistance of ovarian cancer cell lines.

PURPOSE: Ovarian cancer accounts for the highest mortality among all gynecological cancers, mainly due to the fast developing chemoresistance. The death ligand TRAIL induces apoptosis and is able to sensitize tumor cells to cytostatic drugs without affecting physiological tissue. Combined treatment of TRAIL and the antidiabetic acting PPARγ ligands was shown to induce apoptosis synergistically in different ovarian cancer cell lines. METHODS: To investigate feasible TRAIL-dependent inhibition of proliferation and induction of apoptosis in chemoresistant ovarian cancer cell lines, the drug- and TRAIL-sensitive HEY cell line was utilized to develop subclones with selective resistance against cisplatin, etoposide, docetaxel, paclitaxel, gemcitabine and pemetrexed, as well as against TRAIL as control cell line. Expression of the key factors of the TRAIL signaling pathway, TRAIL receptors 1-4, caspase-8, FLIP and XIAP, was analyzed before and after TRAIL treatment by immunoblotting. RESULTS: Cell proliferation experiments showed TRAIL-dependent inhibition that was further increased by combination treatment with the PPARγ ligands. Simultaneous exposure of TRAIL and the PPARγ ligands also resulted in enhanced induction of apoptosis even in partial TRAIL-resistant HEY cell lines. In the parental HEY cell line, additional treatment with the PPARγ ligands led to an increased protein expression of DR5 and a further decline of XIAP expression. CONCLUSION: Therefore, the combinational treatment with TRAIL and PPARγ ligands might be a promising experimental therapy because the PPARγ ligands, especially d15-PGJ(2), sensitize drug-resistant ovarian cancer cells to TRAIL-induced apoptosis.

Congenital diaphragmatic hernia, etiology and management, a 10-year analysis of a single center.

OBJECTIVE: To analyze congenital diaphragmatic hernia (CDH) during a 10-year period at the University of Kiel, from 1995 through 2004, in order to develop a strategy to improve prenatal diagnosis, to be able to consider endoscopical treatment for selected cases and to assess the current postnatal treatment strategies. METHODS: Data were obtained from the fetal medicine ultrasound department, from the birth registry, from the postmortem registry, from the neonatal intensive care unit, from pediatric surgery and from the genetic database. Data were subselected for chromosomes, genetic syndromes, for isolated CDH and for associated anomalies, the lung to head ratio and lung volumes were assessed. Data were analyzed respectively for gestation at diagnosis, the type of CDH, the perinatal management and the postnatal outcome. RESULTS: There were 29 cases of CDH, in 10/29 (34%) the parents requested termination of pregnancy of which two had already died during pregnancy, 12/19 (63%) survived, which was defined as discharge from the neonatal intensive care unit, seven newborns 7/19 (37%) died in the hospital, 5 of these 5/7 (71%) were delivered in Kiel. A prenatal diagnosis was performed in 16/29 (55%), 1/16 (6%), 7/16 (43%) and 8/16 (50%) in the 1st, 2nd and 3rd trimester, respectively; in 10/29 (34%) diagnosis was performed postpartum, in 3/29 (10%) the diagnosis was performed at autopsy following termination of pregnancy. When the liver was in the abdomen, 9/10 (90%) of the children survived, compared to only 3/8 (43%) when the liver was located in the thorax. A lung to head ratio of 0.81 at 24 weeks resulted in death due to pulmonary hypoplasia. CONCLUSIONS: The overall survival in CDH is around 50%, antenatal endoscopical therapy may only be considered, if the diagnosis is performed in the early second trimester, and selection criteria such as the lung to head ratio, associated defects and the chromosomal status can be applied.

An integrated clinical-genomics approach identifies a candidate multi-analyte blood test for serous ovarian carcinoma.

PURPOSE: Cancer of the ovary confers the worst prognosis among women with gynecologic malignancies, underscoring the need to develop new biomarkers for detection of early disease, particularly those that can be readily monitored in the blood. EXPERIMENTAL DESIGN: We developed an algorithm to identify secreted proteins encoded among approximately 22,500 genes on commercial oligonucleotide arrays and applied it to gene expression profiles of 67 stage I to IV serous papillary carcinomas and 9 crudely enriched normal ovarian tissues, to identify putative diagnostic markers. ELISAs were used to validate increased levels of secreted proteins in patient sera encoded by genes with differentially high expression. RESULTS: We identified 275 genes predicted to encode secreted proteins with increased/decreased expression in ovarian cancers (<0.5- or >2-fold, P < 0.001). The serum levels of four of these proteins (matrix metalloproteinase-7, osteopontin, secretory leukoprotease inhibitor, and kallikrein 10) were significantly elevated in a series of 67 independent patients with serous ovarian carcinomas compared with 67 healthy controls (P < 0.001, Wilcoxon rank sum test). Optimized support vector machine classifiers with as few as two of these markers (osteopontin or kallikrein 10/matrix metalloproteinase-7) in combination with CA-125 yielded sensitivity and specificity values ranging from 96% to 98.7% and 99.7% to 100%, respectively, with the ability to discern early-stage disease from normal, healthy controls. CONCLUSIONS: Our data suggest that this assay combination warrants further investigation as a multi-analyte diagnostic test for serous ovarian adenocarcinoma.

Mesenchymal cells with leukocyte and lymphendothelial characteristics in murine embryos.

The development of lymphatic endothelial cells (LECs) from deep embryonic veins or mesenchymal lymphangioblasts is controversially discussed. Studies employing quail-chick grafting experiments have shown that various mesodermal compartments of the embryo possess lymphangiogenic potential, whereas studies on murine embryos have been in favor of a venous origin of LECs. We have investigated NMRI mice from embryonic day (ED) 9.5 to 13.5 with antibodies against the leukocyte marker CD45, the pan-endothelial marker CD31, and the lymphendothelial markers Prox1 and Lyve-1. Early signs of the development of lymphatics are the Lyve-1- and Prox1-positive segments of the jugular and vitelline veins. Then, lymph sacs, which are found in the jugular region of ED 11.5 mice, express Prox1, Lyve-1, and CD31. Furthermore, scattered cells positive for all of the four markers are present in the mesenchyme of the dermatomes and the mediastinum before lymphatic vessels are present in these regions. Their number increases during development. A gradient of increasing CD31 expression can be seen the closer the cells are located to the lymph sacs. Our studies provide evidence for the existence of scattered mesenchymal cells, which up-regulate lymphendothelial and down-regulate leukocyte characteristics when they integrate into growing murine lymphatics. Such stem cells may also be present in the human and may be the cell of origin in post-transplantation Kaposi sarcoma.

Ovarian surgery and follicular reserve.

In patients with cystic ovarian pathology, such as endometriomas, polycystic ovary syndrome, teratomas or benign tumors, there is a tendency towards abnormal ovarian function. Anovulation and sterility may occur. This review will summarize the normal physiology of the female gonad and the development of oogenesis from initial reproductive age to the menopause. Furthermore, the interaction between ovarian pathology and endocrinology will be described. The main focus is on the technique of ovarian-preserving surgery by laparoscopy. In addition, alternative therapeutic approaches and screening tests will be discussed.