Constantin von Kaisenberg

The transcription factor Prox1 is a marker for lymphatic endothelial cells in normal and diseased human tissues.

Detection of lymphatic endothelal cells (LECs) has been problematic because of the lack of specific markers. The homeobox transcription factor Prox1 is expressed in LECs of murine and avian embryos. We have studied expression of Prox1 in human tissues with immunofluorescence. In 19‐wk‐old human fetuses, Prox1 and vascular endothelial growth factor receptor‐3 (VEGFR‐3) are coexpressed in LECs of lymphatic trunks and lymphatic capillaries. Prox1 is located in the nucleus, and its expression is mutually exclusive with that of the blood vascular marker PAL‐E. Prox1 is a constitutive marker of LECs and is found in tissues of healthy adults and lymphedema patients. Blood vascular endothelial cells (BECs) of hemangiomas express CD31 and CD34, but not Prox1. A subset of these cells is positive for VEGFR‐3. Lymphatics in the periphery of hemangiomas express Prox1 and CD31, but not CD34. In lymphangiomas, LECs express Prox1, CD31, and VEGFR‐3, but rarely CD34. In the stroma, spindle‐shaped CD34‐positive cells are present. We show that Prox1 is a reliable marker for LECs in normal and pathologic human tissues, coexpressed with VEGFR‐3 and CD31. VEGFR‐3 and CD34 are less reliable markers for LECs and BECs, respectively, because exceptions from their normal expression patterns are found in pathologic tissues.

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. Developmental Dynamics 235:1554–1562, 2006.

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 ∼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.

Biochemical tissue-specific injury markers of the heart and brain in postpartum cord blood

OBJECTIVEnWe sought to establish references ranges and to test the hypothesis that biochemical tissue-specific markers for the heart in umbilical cord blood of newborns with cardiac defects and intrauterine growth restriction (IUGR) are abnormal.nnnSTUDY DESIGNnA prospective study was conducted. Serum samples of the umbilical vein (n = 280) and artery (n = 156) from 599 healthy newborns at 37(+0)-42(+0) weeks of gestation were collected. Total creatine kinase (CK), CK-MB heart type (CK-MB), cardiac troponin T (cTnT), myoglobin, N-terminal pro-B-type natriuretic peptide (NT-proBNP), and S100 were measured. Reference ranges for each marker were constructed. Concentrations of tissue-specific markers from umbilical cord blood of neonates with cardiac defects (n = 10) and IUGR (n = 41) were plotted against the established reference ranges.nnnRESULTSnReference ranges for each studied marker were established for both umbilical artery and vein. In fetuses with cardiac defects, both NT-proBNP (4/6 [66%] in the artery, 7/10 [70%] in the vein) and cTnT (2/10 [20%] in the vein) were increased. In fetuses with IUGR in the vein, NT-proBNP (10/41 [24%]) and cTnT (5/41 [12%]) were increased, whereas S100 (9/41 [21%]) was decreased.nnnCONCLUSIONnIn a subset of neonates with cardiac defects or growth restriction, irrespective of the pH at birth, tissue-specific injury markers for the heart in umbilical cord blood are abnormal.

Human [gamma][delta] T cells are quickly reconstituted after stem-cell transplantation and show adaptive clonal expansion in response to viral infection

To investigate how the human γδ T cell pool is shaped during ontogeny and how it is regenerated after transplantation of hematopoietic stem cells (HSCs), we applied an RNA-based next-generation sequencing approach to monitor the dynamics of the repertoires of γδ T cell antigen receptors (TCRs) before and after transplantation in a prospective cohort study. We found that repertoires of rearranged genes encoding γδ TCRs (TRG and TRD) in the peripheral blood of healthy adults were stable over time. Although a large fraction of human TRG repertoires consisted of public sequences, the TRD repertoires were private. In patients undergoing HSC transplantation, γδ T cells were quickly reconstituted; however, they had profoundly altered TCR repertoires. Notably, the clonal proliferation of individual virus-reactive γδ TCR sequences in patients with reactivation of cytomegalovirus revealed strong evidence for adaptive anti-viral γδ T cell immune responses.

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 byxa0low-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.

Preeclampsia and long-term risk of cardiovascular disease: what do obstetrician-gynecologists know?

BackgroundPreeclampsia (PE), a hypertensive disorder of pregnancy affects 2-8% of women and is associated with increased cardiovascular disease (CVD) risk later in life. There is little information about the knowledge of obstetrician-gynecologists in German outpatient care setting regarding the future health risk of PE and knowledge of the current guidelines on treatment and counseling patients post PE. This study aimed to assess whether obstetrician-gynecologists are aware of PE’s association with maternal long-term adverse outcomes and providing appropriate counseling.MethodsA random sample of 500 obstetrician-gynecologists in the federal state of Lower Saxony was mailed a survey and a reminder with a second copy of the survey. The questionnaire elicited both personal information, and knowledge on future disease risks, e.g. cardiovascular disease (CVD) and current guidelines as well as on counseling practice. Descriptive analysis was used to analyze the responses.ResultsA total of 212 obstetrician-gynecologists (42.4%) responded to the questionnaire. A large proportion of physicians stated that PE was associated with a higher risk for the development for hypertension (86.6%), stroke (78.5%) and kidney disease (78.0%). Of the participants 75.8% reported that women after PE have a shorter life expectancy. Respondents with knowledge of the current guidelines of the German Association of Obstetrics and Gynecology concerning follow up and risk management of PE (45.2%) were more often aware of the development of CVD and stroke and counseled patients on self -blood-pressure measurement, meaning and long-term-risks of PE and attached importance to family history of PE compared to physicians with no knowledge of the guidelines.ConclusionAlthough the majority of obstetrician-gynecologists were aware of higher CVD risk after PE, weaknesses exist in the follow up care and counseling of these patients. These deficiencies would be amendable to directed educational activities to improve the implementation of current guidelines.

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.

Human umbilical cord-derived mesenchymal stem cells utilise Activin-A to suppress Interferon-gamma production by natural killer cells.

Following allogeneic hematopoietic stem cell transplantation (HSCT), interferon (IFN)-γ levels in the recipient’s body can strongly influence the clinical outcome. Human umbilical cord-derived mesenchymal stem cells (UC-MSCs) are lucrative as biological tolerance-inducers in HSCT settings. Hence, we studied the molecular mechanism of how UC-MSCs influence natural killer (NK) cell-mediated IFN-γ production. Allogeneic NK cells were cultured in direct contact with UC-MSCs or cell-free supernatants from mesenchymal stem cell (MSC) cultures (MSC-conditioned media). We found that soluble factors secreted by UC-MSCs strongly suppressed interleukin (IL)-12/IL-18-induced IFN-γ production by NK cells by reducing phosphorylation of STAT4, NF-κB, as well as T-bet activity. UC-MSCs secreted considerable amounts of activin-A, which could suppress IFN-γ production by NK cells. Neutralization of activin-A in MSC-conditioned media significantly abrogated their suppressive abilities. Till date, multiple groups have reported that prostaglandin (PG)-E2 produced by MSCs can suppress NK cell functions. Indeed, we found that inhibition of PGE2 production by MSCs could also significantly restore IFN-γ production. However, the effects of activin-A and PGE2 were not cumulative. To the best of our knowledge, we are first to report the role of activin-A in MSC-mediated suppression of IFN-γ production by NK cells.

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

PurposeOvarian 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.MethodsTo 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.ResultsCell 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.ConclusionTherefore, the combinational treatment with TRAIL and PPARγ ligands might be a promising experimental therapy because the PPARγ ligands, especially d15-PGJ2, sensitize drug-resistant ovarian cancer cells to TRAIL-induced apoptosis.