The secretome of induced pluripotent stem cells reduces lung fibrosis in part by hepatocyte growth factor.

INTRODUCTION Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible fibrotic lung disease, resulting in respiratory insufficiency and reduced survival. Pulmonary fibrosis is a result of repeated alveolar epithelial microinjuries, followed by abnormal regeneration and repair processes in the lung. Recently, stem cells and their secretome have been investigated as a novel therapeutic approach in pulmonary fibrosis. We evaluated the potential of induced pluripotent stem cells (iPSC) conditioned media (iPSC-cm) to regenerate and repair the alveolar epithelium in vitro and improve bleomycin induced lung injury in vivo. METHODS IPSC-cm was collected from cultured iPSC derived from human foreskin fibroblasts and its biological effects on alveolar epithelial wound repair was studied in an alveolar wound healing assay in vitro. Furthermore, iPSC-cm was intratracheally instilled 7 days after bleomycin induced injury in the rat lungs and histologically and biochemically assessed 7 days after instillation. RESULTS iPSC-cm increased alveolar epithelial wound repair in vitro compared with medium control. Intratracheal instillation of iPSC-cm in bleomycin-injured lungs reduced the collagen content and improved lung fibrosis in the rat lung in vivo. Profibrotic TGFbeta1 and alpha-smooth muscle actin (alpha-sma) expression were markedly reduced in the iPSC-cm treated group compared with control. Antifibrotic hepatocyte growth factor (HGF) was detected in iPSC-cm in biologically relevant levels, and specific inhibition of HGF in iPSC-cm attenuated the antifibrotic effect of iPSC-cm, indicating a central role of HGF in iPSC-cm. CONCLUSION iPSC-cm increased alveolar epithelial wound repair in vitro and attenuated bleomycin induced fibrosis in vivo, partially due to the presence of HGF and may represent a promising novel, cell free therapeutic option against lung injury and fibrosis.

The secretome of endothelial progenitor cells promotes brain endothelial cell activity through PI3-kinase and MAP-kinase

BACKGROUND Angiogenesis and vascular remodelling are crucial events in tissue repair mechanisms promoted by cell transplantation. Current evidence underscores the importance of the soluble factors secreted by stem cells in tissue regeneration. In the present study we investigated the effects of paracrine factors derived from cultured endothelial progenitor cells (EPC) on rat brain endothelial cell properties and addressed the signaling pathways involved. METHODS Endothelial cells derived from rat brain (rBCEC4) were incubated with EPC-derived conditioned medium (EPC-CM). The angiogenic response of rBCEC4 to EPC-CM was assessed as effect on cell number, migration and tubular network formation. In addition, we have compared the outcome of the in vitro experiments with the effects on capillary sprouting from rat aortic rings. The specific PI3K/AKT inhibitor LY294002 and the MEK/ERK inhibitor PD98059 were used to study the involvement of these two signaling pathways in the transduction of the angiogenic effects of EPC-CM. RESULTS Viable cell number, migration and tubule network formation were significantly augmented upon incubation with EPC-CM. Similar findings were observed for aortic ring outgrowth with significantly longer sprouts. The EPC-CM-induced activities were significantly reduced by the blockage of the PI3K/AKT and MEK/ERK signaling pathways. Similarly to the outcome of the rBCEC4 experiments, inhibition of the PI3K/AKT and MEK/ERK pathways significantly interfered with capillary sprouting induced by EPC-CM. CONCLUSION The present study demonstrates that EPC-derived paracrine factors substantially promote the angiogenic response of brain microvascular endothelial cells. In addition, our findings identified the PI3K/AKT and MEK/ERK pathways to play a central role in mediating these effects.

Bone marrow-derived mesenchymal stromal cells improve vascular regeneration and reduce leukocyte-endothelium activation in critical ischemic murine skin in a dose-dependent manner

BACKGROUND AIMS Stem cells participate in vascular regeneration following critical ischemia. However, their angiogenic and remodeling properties, as well as their role in ischemia-related endothelial leukocyte activation, need to be further elucidated. Herein, we investigated the effect of bone marrow-derived mesenchymal stromal cells (BM-MSCs) in a critically ischemic murine skin flap model. METHODS Groups received either 1 × 10(5), 5 × 10(5), or 1 × 10(6) BM-MSCs or cell-free conditioned medium (CM). Controls received sodium chloride. Intravital fluorescence microscopy was performed for morphological and quantitative assessment of micro-hemodynamic parameters over 12 days. RESULTS Tortuosity and diameter of conduit-arterioles were pronounced in the MSC groups (P < 0.01), whereas vasodilation was shifted to the end arteriolar level in the CM group (P < 0.01). These effects were accompanied by angiopoietin-2 expression. Functional capillary density and red blood cell velocity were enhanced in all treatment groups (P < 0.01). Although a significant reduction of rolling and sticking leukocytes was observed in the MSC groups with a reduction of diameter in postcapillary venules (P < 0.01), animals receiving CM exhibited a leukocyte-endothelium interaction similar to controls. This correlated with leukocyte common antigen expression in tissue sections (P < 0.01) and p38 mitogen-activated protein kinase expression from tissue samples. Cytokine analysis from BM-MSC culture medium revealed a 50% reduction of pro-inflammatory cytokines (interleukin [IL]-1β, IL-6, IL-12, tumor necrosis factor-α, interferon-γ) and chemokines (keratinocyte chemoattractant, granulocyte colony-stimulating factor) under hypoxic conditions. DISCUSSION We demonstrated positive effects of BM-MSCs on vascular regeneration and modulation of endothelial leukocyte adhesion in critical ischemic skin. The improvements after MSC application were dose-dependent and superior to the use of CM alone.

A g-factor metric for k-t-GRAPPA- and PEAK-GRAPPA-based parallel imaging.

PURPOSE The aim of this work is to derive a theoretical framework for quantitative noise and temporal fidelity analysis of time-resolved k-space-based parallel imaging methods. THEORY An analytical formalism of noise distribution is derived extending the existing g-factor formulation for nontime-resolved generalized autocalibrating partially parallel acquisition (GRAPPA) to time-resolved k-space-based methods. The noise analysis considers temporal noise correlations and is further accompanied by a temporal filtering analysis. METHODS All methods are derived and presented for k-t-GRAPPA and PEAK-GRAPPA. A sliding window reconstruction and nontime-resolved GRAPPA are taken as a reference. Statistical validation is based on series of pseudoreplica images. The analysis is demonstrated on a short-axis cardiac CINE dataset. RESULTS The superior signal-to-noise performance of time-resolved over nontime-resolved parallel imaging methods at the expense of temporal frequency filtering is analytically confirmed. Further, different temporal frequency filter characteristics of k-t-GRAPPA, PEAK-GRAPPA, and sliding window are revealed. CONCLUSION The proposed analysis of noise behavior and temporal fidelity establishes a theoretical basis for a quantitative evaluation of time-resolved reconstruction methods. Therefore, the presented theory allows for comparison between time-resolved parallel imaging methods and also nontime-resolved methods. Magn Reson Med, 2014. © 2014 Wiley Periodicals, Inc.

The matrix metalloproteinase inhibitor RS-130830 attenuates brain injury in experimental pneumococcal meningitis.

BACKGROUND Pneumococcal meningitis (PM) is characterized by high mortality and morbidity including long-term neurofunctional deficits. Neuropathological correlates of these sequelae are apoptosis in the hippocampal dentate gyrus and necrosis in the cortex. Matrix metalloproteinases (MMPs) play a critical role in the pathophysiology of PM. RS-130830 (Ro-1130830, CTS-1027) is a potent partially selective inhibitor of MMPs of a second generation and has been evaluated in clinical trials as an anti-arthritis drug. It inhibits MMPs involved in acute inflammation but has low activity against MMP-1 (interstitial collagenase), MMP-7 (matrilysin) and tumour necrosis factor α converting enzyme (TACE). METHODS A well-established infant rat model of PM was used where live Streptococcus pneumoniae were injected intracisternally and antibiotic treatment with ceftriaxone was initiated 18 h post infection (hpi). Treatment with RS-130830 (75 mg/kg bis in die (bid) i.p., n = 40) was started at 3 hpi while control littermates received the vehicle (succinylated gelatine, n = 42). RESULTS Cortical necrosis was significantly attenuated in animals treated with RS-130830, while the extent of hippocampal apoptosis was not influenced. At 18 hpi, concentrations of interleukin (IL)-1β and IL-10 were significantly lower in the cerebrospinal fluid of treated animals compared to controls. RS-130830 significantly reduced weight loss and leukocyte counts in the cerebrospinal fluid of survivors of PM. CONCLUSION This study identifies MMP inhibition, specifically with RS-130830, as an efficient strategy to attenuate disease severity and cortical brain injury in PM.

PreImplantation factor promotes neuroprotection by targeting microRNA let-7

Dysfunction and loss of neurons are the major characteristics of CNS disorders that include stroke, multiple sclerosis, and Alzheimer's disease. Activation of the Toll-like receptor 7 by extracellular microRNA let-7, a highly expressed microRNA in the CNS, induces neuronal cell death. Let-7 released from injured neurons and immune cells acts on neighboring cells, exacerbating CNS damage. Here we show that a synthetic peptide analogous to the mammalian PreImplantation factor (PIF) secreted by developing embryos and which is present in the maternal circulation during pregnancy inhibits the biogenesis of let-7 in both neuronal and immune cells of the mouse. The synthetic peptide, sPIF, destabilizes KH-type splicing regulatory protein (KSRP), a key microRNA-processing protein, in a Toll-like receptor 4 (TLR4)-dependent manner, leading to decreased production of let-7. Furthermore, s.c. administration of sPIF into neonatal rats following hypoxic-ischemic brain injury robustly rescued cortical volume and number of neurons and decreased the detrimental glial response, as is consistent with diminished levels of KSRP and let-7 in sPIF-treated brains. Our results reveal a previously unexpected mechanism of action of PIF and underscore the potential clinical utility of sPIF in treating hypoxic-ischemic brain damage. The newly identified PIF/TLR4/KSRP/let-7 regulatory axis also may operate during embryo implantation and development.

Stimulation of monocytes by placental microparticles involves toll-like receptors and nuclear factor kappa-light-chain-enhancer of activated B cells.

Human pregnancy is accompanied by a mild systemic inflammatory response, which includes the activation of monocytes circulating in maternal blood. This response is exaggerated in preeclampsia, a placental-dependent disorder specific to human pregnancies. We and others showed that placental syncytiotrophoblast membrane microparticles (STBM) generated in vitro from normal placentas stimulated peripheral blood monocytes, which suggest a contribution of STBM to the systemic maternal inflammation. Here, we analyzed the inflammatory potential of STBM prepared from preeclamptic placentas on primary monocytes and investigated the mode of action in vitro. STBM generated in vitro by placental villous explants of normal or preeclamptic placentas were co-incubated with human peripheral blood monocytes. In some cases, inhibitors of specific cellular functions or signaling pathways were used. The analysis of the monocytic response was performed by flow cytometry, enzyme-linked immunoassays, real-time PCR, and fluorescence microscopy. STBM derived from preeclamptic placentas up-regulated the cell surface expression of CD54, and stimulated the secretion of the pro-inflammatory interleukin (IL)-6 and IL-8 in a similar, dose-dependent manner as did STBM prepared from normal placentas. STBM bound to the cell surface of monocytes, but phagocytosis was not necessary for activation. STBM-induced cytokine secretion was impaired in the presence of inhibitors of toll-like receptor (TLR) signaling or when nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation was blocked. Our results suggest that the inflammatory reaction in monocytes may be initiated by the interaction of STBM with TLRs, which in turn signal through NF-κB to mediate the transcription of genes coding for pro-inflammatory factors.

Consistent nuclear expression of thyroid transcription factor 1 in subependymal giant cell astrocytomas suggests lineage-restricted histogenesis.

Thyroid transcription factor 1 (TTF-1) is encoded by the NKX2-1 homeobox gene. Besides specifying thyroid and pulmonary organogenesis, it is also temporarily expressed during embryonic development of the ventral forebrain. We recently observed widespread immunoreactivity for TTF-1 in a case of subependymal giant cell astrocytoma (SEGA, WHO grade I) – a defining lesion of the tuberous sclerosis complex (TSC). This prompted us to investigate additional SEGAs in this regard. We found tumor cells in all 7 specimens analyzed to be TTF-1 positive. In contrast, we did not find TTF-1 immunoreactivity in a cortical tuber or two renal angiomyolipomas resected from TSC patients. We propose our finding of consistent TTF-1 expression in SEGAs to indicate lineage-committed derivation of these tumors from a regionally specified cell of origin. The medial ganglionic eminence, ventral septal region, and preoptic area of the developing brain may represent candidates for the origin of SEGAs. Such lineagerestricted histogenesis may also explain the stereotypic distribution of SEGAs along the caudate nucleus in the lateral ventricles.

Bone-Conditioned Medium Changes Gene Expression in Bone-Derived Fibroblasts.

PURPOSE Autologous bone is used for augmentation in the course of oral implant placement. Bone grafts release paracrine signals that can modulate mesenchymal cell differentiation in vitro. The detailed genetic response of the bone-derived fibroblasts to these paracrine signals has remained elusive. Paracrine signals accumulate in bone-conditioned medium (BCM) prepared from porcine cortical bone chips. MATERIALS AND METHODS In this study, bone-derived fibroblasts were exposed to BCM followed by a whole genome expression profiling and downstream quantitative reverse transciptase polymerase chain reaction of the most strongly regulated genes. RESULTS The data show that ADM, IL11, IL33, NOX4, PRG4, and PTX3 were differentially expressed in response to BCM in bone-derived fibroblasts. The transforming growth factor beta (TGF-β) receptor 1 antagonist SB431542 blocked the effect of BCM on the expression of the gene panel, except for IL33. CONCLUSION These in vitro results extend existing evidence that cortical bone chips release paracrine signals that provoke a robust genetic response in mesenchymal cells that is not exclusively mediated via the TGF-β receptor. The present data provide further insights into the process of graft consolidation.

Factor structure of the Bern Psychopathology Scale in a sample of patients with schizophrenia spectrum disorders.

BACKGROUND The Bern Psychopathology Scale (BPS) is based on a system-specific approach to classifying the psychopathological symptom pattern of schizophrenia. It consists of subscales for three domains (language, affect and motor behaviour) that are hypothesized to be related to specific brain circuits. The aim of the study was to examine the factor structure of the BPS in patients with schizophrenia spectrum disorders. METHODS One hundred and forty-nine inpatients with schizophrenia spectrum disorders were recruited at the Department of Psychiatry II, Ulm University, Germany (n=100) and at the University Hospital of Psychiatry, Bern, Switzerland (n=49). Psychopathology was assessed with the BPS. The VARCLUS procedure of SAS(®) (a type of oblique component analysis) was used for statistical analysis. RESULTS Six clusters were identified (inhibited language, inhibited motor behaviour, inhibited affect, disinhibited affect, disinhibited language/motor behaviour, inhibited language/motor behaviour) which explained 40.13% of the total variance of the data. A binary division of attributes into an inhibited and disinhibited cluster was appropriate, although an overlap was found between the language and motor behaviour domains. There was a clear distinction between qualitative and quantitative symptoms. CONCLUSIONS The results argue for the validity of the BPS in identifying subsyndromes of schizophrenia spectrum disorders according to a dimensional approach. Future research should address the longitudinal assessment of dimensional psychopathological symptoms and elucidate the underlying neurobiological processes.

The Phosphoinositide 3-Kinase p110α Isoform Regulates Leukemia Inhibitory Factor Receptor Expression via c-Myc and miR-125b to Promote Cell Proliferation in Medulloblastoma.

Medulloblastoma (MB) is the most common malignant brain tumor in childhood and represents the main cause of cancer-related death in this age group. The phosphoinositide 3-kinase (PI3K) pathway has been shown to play an important role in the regulation of medulloblastoma cell survival and proliferation, but the molecular mechanisms and downstream effectors underlying PI3K signaling still remain elusive. The impact of RNA interference (RNAi)-mediated silencing of PI3K isoforms p110α and p110δ on global gene expression was investigated by DNA microarray analysis in medulloblastoma cell lines. A subset of genes with selectively altered expression upon p110α silencing in comparison to silencing of the closely related p110δ isoform was revealed. Among these genes, the leukemia inhibitory factor receptor α (LIFR α) was validated as a novel p110α target in medulloblastoma. A network involving c-Myc and miR-125b was shown to be involved in the control of LIFRα expression downstream of p110α. Targeting the LIFRα by RNAi, or by using neutralizing reagents impaired medulloblastoma cell proliferation in vitro and induced a tumor volume reduction in vivo. An analysis of primary tumors revealed that LIFRα and p110α expression were elevated in the sonic hedgehog (SHH) subgroup of medulloblastoma, indicating its clinical relevance. Together, these data reveal a novel molecular signaling network, in which PI3K isoform p110α controls the expression of LIFRα via c-Myc and miR-125b to promote MB cell proliferation.

Endothelial- and Platelet-Derived Microparticles Are Generated During Liver Resection in Humans.

BACKGROUND Cell-derived plasma microparticles (<1.5 μm) originating from various cell types have the potential to regulate thrombogenesis and inflammatory responses. The aim of this study was to test the hypothesis that microparticles generated during hepatic surgery co-regulate postoperative procoagulant and proinflammatory events. METHODS In 30 patients undergoing liver resection, plasma microparticles were isolated, quantitated, and characterized as endothelial (CD31+, CD41-), platelet (CD41+), or leukocyte (CD11b+) origin by flow cytometry and their procoagulant and proinflammatory activity was measured by immunoassays. RESULTS During liver resection, the total numbers of microparticles increased with significantly more Annexin V-positive, endothelial and platelet-derived microparticles following extended hepatectomy compared to standard and minor liver resections. After liver resection, microparticle tissue factor and procoagulant activity increased along with overall coagulation as assessed by thrombelastography. Levels of leukocyte-derived microparticles specifically increased in patients with systemic inflammation as assessed by C-reactive protein but are independent of the extent of liver resection. CONCLUSIONS Endothelial and platelet-derived microparticles are specifically elevated during liver resection, accompanied by increased procoagulant activity. Leukocyte-derived microparticles are a potential marker for systemic inflammation. Plasma microparticles may represent a specific response to surgical stress and may be an important mediator of postoperative coagulation and inflammation.

Anti-Müllerian hormone and progesterone levels produced by granulosa cells are higher when derived from natural cycle IVF than from conventional gonadotropin-stimulated IVF.

BACKGROUND The study was designed to compare the effect of in vitro FSH stimulation on the hormone production and gene expression profile of granulosa cells (GCs) isolated from single naturally matured follicles obtained from natural cycle in vitro fertilization (NC-IVF) with granulosa cells obtained from conventional gonadotropin-stimulated IVF (c-IVF). METHODS Lutein granulosa cells from the dominant follicle were isolated and cultured in absence or presence of recombinant FSH. The cultures were run for 48 h and six days. Messenger RNA (mRNA) expressions of anti-Müllerian hormone (AMH) and FSH receptor were measured by quantitative polymerase chain reaction (qPCR). AMH protein and progesterone concentration (P4) in cultured supernatant were measured by ELISA and RIA. RESULTS Our results showed that the mRNA expression of AMH was significantly higher in GCs from NC- than from c-IVF on day 6 after treatment with FSH (1 IU/mL). The FSH stimulation increased the concentration of AMH in the culture supernatant of GCs from NC-IVF compared with cells from c-IVF. In the culture medium, the AMH level was correlated significantly and positively to progesterone concentration. CONCLUSIONS Differences in the levels of AMH and progesterone released into the medium by cultured GC as well as in AMH gene expression were observed between GCs obtained under natural and stimulated IVF protocols. The results suggest that artificial gonadotropin stimulation may have an effect on the intra-follicular metabolism. A significant positive correlation between AMH and progesterone may suggest progesterone as a factor influencing AMH secretion.

Immune regulatory and neuroprotective properties of preimplantation factor: From newborn to adult.

Embryonic-maternal interaction from the earliest stages of gestation has a key, sustained role in neurologic development, persisting into adulthood. Early adverse events may be detrimental in adulthood. Protective factors present during gestation could significantly impact post-natal therapy. The role of PreImplantation Factor (PIF) within this context is herein examined. Secreted by viable early embryos, PIF establishes effective embryonic-maternal communication and exerts essential trophic and protective roles by reducing oxidative stress and protein misfolding and by blunting the nocive let-7 microRNA related pathway. PIF's effects on systemic immunity lead to comprehensive immune modulation, not immune suppression. We examine PIF's role in protecting embryos from adverse maternal environment, which can lead to neurological disorders that may only manifest post-nataly: Synthetic PIF successfully translates endogenous PIF features in both pregnant and non-pregnant clinically relevant models. Specifically PIF has neuroprotective effects in neonatal prematurity. In adult relapsing-remitting neuroinflammation, PIF reverses advanced paralysis while promoting neurogenesis. PIF reversed Mycobacterium smegmatis induced brain infection. In graft-vs.-host disease, PIF reduced skin ulceration, liver inflammation and colon ulceration while maintaining beneficial anti-cancer, graft-vs.-leukemia effect. Clinical-grade PIF has high-safety profile even at supraphysiological doses. The FDA awarded Fast-Track designation, and university-sponsored clinical trials for autoimmune disorder are ongoing. Altogether, PIF properties point to its determining regulatory role in immunity, inflammation and transplant acceptance. Specific plans for using PIF for the treatment of complex neurological disorders (ie. traumatic brain injury, progressive paralysis), including neuroprotection from newborn to adult, are presented.

Why babies do not feel pain, or: How structure-derived functional interpretations can go wrong

The response to pain involves a non-conscious, reflexive action and a conscious perception. According to Key (2016), consciousness — and thus pain perception — depends on a neuronal correlate that has a “unique neural architecture” as realized in the human cortex. On the basis of the “bioengineering principle that structure determines function,” Key (2016) concludes that animal species such as fish, which lack the requisite cortex-like neuroanatomical structure, are unable to feel pain. This commentary argues that the relationship between brain structure and brain function is less straightforward than suggested in Key’s target article.