Fermented mistletoe extract as a multimodal antitumoral agent in gliomas

In Europe, commercially available extracts from the white-berry mistletoe (Viscum album L.) are widely used as a complementary cancer therapy. Mistletoe lectins have been identified as main active components and exhibit cytotoxic effects as well as immunomodulatory activity. Since it is still not elucidated in detail how mistle toe extracts such as ISCADOR communicate their effects, we analyzed the mechanisms that might be responsible for their antitumoral function on a molecular and functional level. ISCADOR-treated glioblastoma (GBM) cells down-regulate central genes involved in glioblastoma progression and malignancy such as the cytokine TGF-β and matrix-metalloproteinases. Using in vitro glioblastoma/immune cell co-cultivation assays as well as measurement of cell migration and invasion, we could demonstrate that in glioblastoma cells, lectin-rich ISCADOR M and ISCADOR Q significantly enforce NK-cell-mediated GBM cell lysis. Beside its immune stimulatory effect, ISCADOR reduces the migratory and invasive potential of glioblastoma cells. In a syngeneic as well as in a xenograft glioblastoma mouse model, both pretreatment of tumor cells and intratumoral therapy of subcutaneously growing glioblastoma cells with ISCADOR Q showed delayed tumor growth. In conclusion, ISCADOR Q, showing multiple positive effects in the treatment of glioblastoma, may be a candidate for concomitant treatment of this cancer

A Renormalization Proof of the KAM Theorem for Non-Analytic Perturbations

We shall use a Renormalization Group (RG) scheme in order to prove the classical KAM result in the case of a non-analytic perturbation (the latter will be assumed to have continuous derivatives up to a sufficiently large order). We shall proceed by solving a sequence of problems in which the perturbations are analytic approximations of the original one. We shall finally show that the sequence of the approximate solutions will converge to a differentiable solution of the original problem.Comment: 33 pages, no figure

VAV1 and BAFF, via NFκB pathway, are genetic risk factors for myasthenia gravis

Objective To identify novel genetic loci that predispose to early‐onset myasthenia gravis (EOMG) applying a two‐stage association study, exploration, and replication strategy. Methods Thirty‐four loci and one confirmation loci, human leukocyte antigen (HLA)‐DRA, were selected as candidate genes by team members of groups involved in different research aspects of MG. In the exploration step, these candidate genes were genotyped in 384 EOMG and 384 matched controls and significant difference in allele frequency were found in eight genes. In the replication step, eight candidate genes and one confirmation loci were genotyped in 1177 EOMG patients and 814 controls, from nine European centres. Results Allele frequency differences were found in four novel loci: CD86, AKAP12, VAV1, B‐cell activating factor (BAFF), and tumor necrosis factor‐alpha (TNF‐α), and these differences were consistent in all nine cohorts. Haplotype trend test supported the differences in allele frequencies between cases and controls. In addition, allele frequency difference in female versus male patients at HLA‐DRA and TNF‐α loci were observed. Interpretation The genetic associations to EOMG outside the HLA complex are novel and of interest as VAV1 is a key signal transducer essential for T‐ and B‐cell activation, and BAFF is a cytokine that plays important roles in the proliferation and differentiation of B‐cells. Moreover, we noted striking epistasis between the predisposing VAV1 and BAFF haplotypes; they conferred a greater risk in combination than alone. These, and CD86, share the same signaling pathway, namely nuclear factor‐kappaB (NFκB), thus implicating dysregulation of proinflammatory signaling in predisposition to EOMG

Sleep enhances numbers and function of monocytes and improves bacterial infection outcome in mice.

Sleep strongly impacts both humoral and cellular immunity; however, its acute effects on the innate immune defense against pathogens are unclear. Here, we elucidated in mice whether sleep affects the numbers and functions of innate immune cells and their defense against systemic bacterial infection. Sleep significantly increased numbers of classical monocytes in blood and spleen of mice that were allowed to sleep for six hours at the beginning of the normal resting phase compared to mice kept awake for the same time. The sleep-induced effect on classical monocytes was neither caused by alterations in corticosterone nor myelopoiesis, bone marrow egress or death of monocytes and did only partially involve Gαi-protein coupled receptors like chemokine receptor 2 (CCR2), but not the adhesion molecules intercellular adhesion molecule 1 (ICAM-1) or lymphocyte function-associated antigen 1 (LFA-1). Notably, sleep suppressed the expression of the clock gene Arntl in splenic monocytes and the sleep-induced increase in circulating classical monocytes was abrogated in Arntl-deficient animals, indicating that sleep is a prerequisite for clock-gene driven rhythmic trafficking of classical monocytes. Sleep also enhanced the production of reactive oxygen species by monocytes and neutrophils. Moreover, sleep profoundly reduced bacterial load in blood and spleen of mice that were allowed to sleep before systemic bacterial infection and consequently increased survival upon infection. These data provide the first evidence that sleep enhances numbers and function of innate immune cells and therewith strengthens early defense against bacterial pathogens

Comparative Study of MSCA-1 and CD146 Isolated Periosteal Cell Subpopulations

Background/Aims: Periosteal tissue is a valuable source of multipotent stem cells for bone tissue engineering. To characterize these cells in detail, we generated an immortalized human cranial periosteal cell line and observed an increased MSCA-1 and CD146 expression, as well as an earlier and stronger mineralization compared to the parental cells. Further, we detected a higher osteogenic potential of MSCA-1high compared to MSCA-1low cranial periosteal cell (CPC) fractions. In the present study, a possible synergism of MSCA-1 and CD146 for periosteal cell mineralization was investigated. Methods: MSCA-1/CD146 positive and negative CPCs were magnetically isolated (MACS) or sorted by flow cytometry (FACS) and subjected to osteogenic differentiation. The expression of osteogenic marker genes in the four subpopulations was analyzed by quantitative real-time PCR. Furthermore, the co-expression of osteogenic markers/antigens was analyzed by multispectral imaging flow cytometry (ImageStream, AMNIS). The mineralization potential was assessed by the quantification of alizarin stainings. Results: While the total cell yield after separation was higher using MACS compared to the FACS approach, the isolation of MSCA-1+/- and CD146+/- subpopulations was more efficient with the FACS separation. The accuracy of the FACS separation of the four distinguished cell subpopulations was confirmed by multispectral imaging flow cytometry. Further, we detected increasing levels of MSCA-1 and CD146 during in vitro differentiation in all subpopulations. However, MSCA-1 expression was significantly higher in the MSCA-1+/CD146+ and MSCA-1+/ CD146- subpopulations, while CD146 expression remained clearly lower in these fractions. Significantly higher gene expression levels of osteogenic markers, ALP and RUNX2, were detected in MSCA-1+ compared to MSCA-1- CPCs at different time points during in vitro differentiation. Staining and quantification of calcium phosphate precipitates revealed a significantly higher mineralization potential of MACS separated MSCA-1+ and CD146- CPCs, compared to their respective counterparts. FACS sorted CPCs displayed earlier mineralization in both MSCA-1+ fractions (d13), while later (d28) only the CD146+/MSCA-1- fraction had a significantly lower calcium phosphate concentration compared to all other fractions. Conclusion: Our results demonstrate, that MSCA-1+ cells isolated from CPCs represent a subpopulation with a higher osteogenic potential. In contrast, we found a lower osteogenic potential in CD146+ CPCs. In conclusion, only MSCA-1, but not CD146, is a suitable marker for the isolation of osteoprogenitors from CPCs

Immunomonitoring of Human Breast Milk Cells During HCMV-Reactivation

BACKGROUND: Breast milk leukocytes may play a role in protecting the infant from pathogens. The dynamics and the role of lymphocytes in human cytomegalovirus (HCMV)-seropositive mothers shedding HCMV into breast milk during the first months postpartum (p.p.) are mostly unclear. METHODS: Breast milk cells were analyzed by Pappenheim panoptic and alpha-naphthyl acetate esterase staining as well as by imaging and polychromatic flow cytometry to simultaneously establish their morphological and phenotypic properties. The latter were characterized in HCMV-seropositive and seronegative mothers´ breast milk cells at different time points p.p. RESULTS: Panoptic staining of breast milk cells revealed the presence of monocytes/macrophages, granulocytes and lymphocytes. Imaging flow cytometry data combining phenotypic and morphological analysis identified NKT-like cells, NK cells, epithelial cells, T cells and monocytes/macrophages. HCMV-seropositive but not -seronegative mothers had significantly higher T cell frequencies in mature milk. CONCLUSIONS: The presence of lymphocyte subsets in breast milk may be more influenced by the HCMV-seropositivity of the mother than previously recognized

Localization and Functionality of the Inflammasome in Neutrophils

Neutrophils represent the major fraction of circulating immune cells and are rapidly recruited to sites of infection and inflammation. The inflammasome is a multiprotein complex that regulates the generation of IL-1 family proteins. The precise subcellular localization and functionality of the inflammasome in human neutrophils are poorly defined. Here we demonstrate that highly purified human neutrophils express key components of the NOD-like receptor family, pyrin domain containing 3 (NLRP3), and absent in melanoma 2 (AIM2) inflammasomes, particularly apoptosis-associated speck-like protein containing a CARD (ASC), AIM2, and caspase-1. Subcellular fractionation and microscopic analyses further showed that inflammasome components were localized in the cytoplasm and also noncanonically in secretory vesicle and tertiary granule compartments. Whereas IL-1β and IL-18 were expressed at the mRNA level and released as protein, highly purified neutrophils neither expressed nor released IL-1α at baseline or upon stimulation. Upon inflammasome activation, highly purified neutrophils released substantially lower levels of IL-1β protein compared with partially purified neutrophils. Serine proteases and caspases were differentially involved in IL-1β release, depending on the stimulus. Spontaneous activation of the NLRP3 inflammasome in neutrophils in vivo affected IL-1β, but not IL-18 release. In summary, these studies show that human neutrophils express key components of the inflammasome machinery in distinct intracellular compartments and release IL-1β and IL-18, but not IL-1α or IL-33 protein. Targeting the neutrophil inflammasome may represent a future therapeutic strategy to modulate neutrophilic inflammatory diseases, such as cystic fibrosis, rheumatoid arthritis, or sepsis