Human adipose-derived mesenchymal stem cells reduce inflammatory and T cell responses and induce regulatory T cells in vitro in rheumatoid arthritis.

OBJECTIVES: Adult mesenchymal stem cells were recently found to suppress effector T cell and inflammatory responses and have emerged as attractive therapeutic candidates for immune disorders. In rheumatoid arthritis (RA), a loss in the immunological self-tolerance causes the activation of autoreactive T cells against joint components and subsequent chronic inflammation. The aim of this study is to characterise the immunosuppressive activity of human adipose-derived mesenchymal stem cells (hASCs) on collagen-reactive T cells from patients with RA. METHODS: The effects of hASCs on collagen-reactive RA human T cell proliferation and cytokine production were investigated, as well as effects on the production of inflammatory mediators by monocytes and fibroblast-like synoviocytes from patients with RA. RESULTS: hASCs suppressed the antigen-specific response of T cells from patients with RA. hASCs inhibited the proliferative response and the production of inflammatory cytokines by collagen-activated CD4 and CD8 T cells. In contrast, the numbers of IL10-producing T cells and monocytes were significantly augmented upon hASC treatment. The suppressive activity of hASCs was cell-to-cell contact dependent and independent. hASCs also stimulated the generation of FoxP3 protein-expressing CD4(+)CD25(+) regulatory T cells, with the capacity to suppress collagen-specific T cell responses. Finally, hASCs downregulated the inflammatory response and the production of matrix-degrading enzymes by synovial cells isolated from patients with RA. CONCLUSIONS: The present work identifies hASCs as key regulators of immune tolerance, with the capacity to suppress T cell and inflammatory responses and to induce the generation/activation of antigen-specific regulatory T cells.

Human adult stem cells derived from adipose tissue protect against experimental colitis and sepsis.

BACKGROUND AND AIMS: Inflammatory bowel diseases (IBDs) are associated with uncontrolled innate and adaptive immunity against normal constituents, including commensal bacteria and microbial products. Mesenchymal stem cells (MSCs) suppress effector T cell responses and have beneficial effects in various immune disorders. This work investigates the therapeutic effects of human adipose-derived MSCs (hASCs) in various models of IBD and sepsis. METHODS: Acute and chronic colitis was induced in mice with dextran sulfate sodium. Sepsis was induced by caecal ligation and puncture or by endotoxin injection. Colitic and septic mice were treated intraperitoneally with hASCs or murine ASCs, and diverse disease clinical signs and mortality were determined. The levels of various inflammatory cytokines and chemokines, T helper 1(Th1)-type response and generation of regulatory T cells (Treg) were determined in affected organs. RESULTS: Systemic infusion of ASCs significantly ameliorated the clinical and histopathological severity of colitis, abrogating weight loss, diarrhoea and inflammation, and increasing survival. The therapeutic effect was associated with downregulation of the Th1-driven inflammatory responses. ASCs decreased a wide panel of inflammatory cytokines and chemokines and increased interleukin 10 (IL10), acting on macrophages. hASCs also impaired Th1 cell activation in both colonic mucosa and draining lymph nodes. The induction of IL10-secreting Treg was partially involved in the therapeutic effect of hASCs. Moreover, ASCs protected from severe sepsis by reducing the infiltration of inflammatory cells in various target organs and by downregulating the production of various inflammatory mediators. CONCLUSIONS: hASCs emerge as key regulators of immune/inflammatory responses in vivo and as attractive candidates for cell-based treatments for IBD and sepsis.

Ras-dependent and -independent pathways target the mitogen-activated protein kinase network in macrophages.

Mitogen-activated protein kinases (MAPKs) are activated upon a variety of extracellular stimuli in different cells. In macrophages, colony-stimulating factor 1 (CSF-1) stimulates proliferation, while bacterial lipopolysaccharide (LPS) inhibits cell growth and causes differentiation and activation. Both CSF-1 and LPS rapidly activate the MAPK network and induce the phosphorylation of two distinct ternary complex factors (TCFs), TCF/Elk and TCF/SAP. CSF-1, but not LPS, stimulated the formation of p21ras. GTP complexes. Expression of a dominant negative ras mutant reduced, but did not abolish, CSF-1-mediated stimulation of MEK and MAPK. In contrast, activation of the MEK kinase Raf-1 was Ras independent. Treatment with the phosphatidylcholine-specific phospholipase C inhibitor D609 suppressed LPS-mediated, but not CSF-1-mediated, activation of Raf-1, MEK, and MAPK. Similarly, down-regulation or inhibition of protein kinase C blocked MEK and MAPK induction by LPS but not that by CSF-1. Phorbol 12-myristate 13-acetate pretreatment led to the sustained activation of the Raf-1 kinase but not that of MEK and MAPK. Thus, activated Raf-1 alone does not support MEK/MAPK activation in macrophages. Phosphorylation of TCF/Elk but not that of TCF/SAP was blocked by all treatments that interfered with MAPK activation, implying that TCF/SAP was targeted by a MAPK-independent pathway. Therefore, CSF-1 and LPS target the MAPK network by two alternative pathways, both of which induce Raf-1 activation. The mitogenic pathway depends on Ras activity, while the differentiation signal relies on protein kinase C and phosphatidylcholine-specific phospholipase C activation.

Involvement of the protein tyrosine phosphatase SHP-1 in Ras-mediated activation of the mitogen-activated protein kinase pathway.

Ubiquitously expressed SH2-containing tyrosine phosphatases interact physically with tyrosine kinase receptors or their substrates and relay positive mitogenic signals via the activation of the Ras-mitogen-activated protein kinase (MAPK) pathway. Conversely, the structurally related phosphatase SHP-1 is predominantly expressed in hemopoietic cells and becomes tyrosine phosphorylated upon colony-stimulating factor 1 treatment of macrophages without associating with the colony-stimulating factor 1 receptor tyrosine kinase. Mice lacking functional SHP-1 (me/me and me(v)/me(v)) develop systemic autoimmune disease with accumulation of macrophages, suggesting that SHP-1 may be a negative regulator of hemopoietic cell growth. By using macrophages expressing dominant negative Ras and the me(v)/me(v) mouse mutant, we show that SHP-1 is activated in the course of mitogenic signal transduction in a Ras-dependent manner and that its activity is necessary for the Ras-dependent activation of the MAPK pathway but not of the Raf-1 kinase. Consistent with a role for SHP-1 as an intermediate between Ras and the MEK-MAPK pathway, Ras-independent activation of the latter kinases by bacterial lipopolysaccharide occurred normally in me(v)/me(v) cells. Our results sharply accentuate the diversity of signal transduction in mammalian cells, in which the same signaling intermediates can be rearranged to form different pathways.

Transperineal biopsies of MRI-detected aggressive index lesions in low- and intermediate-risk prostate cancer patients: Implications for treatment decision.

PURPOSE: Multiparametric MRI (mpMRI) has a potential role for the identification of aggressive cancer that can be targeted for biopsy. We report the incidence and severity of discordant information between the pathology found on the transrectal ultrasound (TRUS)-guided biopsy and the mpMRI findings in patients with favorable or intermediate-risk prostate cancer referred for brachytherapy. METHODS AND MATERIALS: From March 2014 to September 2015, 10/44 consecutive patients with low- or intermediate-risk prostate cancer referred for brachytherapy presented an aggressive lesion on mpMRI and underwent an MRI-TRUS fusion-guided transperineal biopsy of the index lesion. RESULTS: A median of two intraprostatic lesions were detected by mpMRI for each patient. Three patients had bilateral disease, and seven had unilateral disease on mpMRI. The median number of cores obtained by MRI-TRUS-guided fusion of the index lesion was 3 (range 2-4). As a result of the re-evaluation consequent to additional information becoming available after the transperineal biopsy, upgrading of Gleason score occurred in 8 of the 10 patients, which changed the risk group in 9 patients. These changes resulted in modification of the proposed treatment in 8 patients. CONCLUSIONS: MpMRI-US fusion-targeted biopsy sampling allows detection and characterization of otherwise undetected aggressive disease, often placing men in higher risk groups and altering the treatment approach.

Suppression of growth factor-mediated MAP kinase activation by v-raf in macrophages: a putative role for the MKP-1 phosphatase.

Many tyrosine kinase growth factor receptors activate the MAP Kinase (MAPK) pathway by stimulating the activity of the RAF kinase. In some, but not all cell types, the expression of activated RAF is sufficient to induce constitutive MAPK activation. In BAC-1.2F5 macrophages the expression of virally activated RAF does not correlate with constitutive MAPK activation; on the contrary, growth factor-mediated stimulation of MAPK activity is suppressed in these cells. Suppression correlates with v-RAF expression, as MAPK activation is normal in a revertant cell line that stopped expressing v-RAF. Inhibition of MAPK activation is associated with lack of ERK-2 tyrosine phosphorylation, and is not due to the suppression of CSF-1-mediated MEK activation. Pretreatment with vanadate restores growth factor-stimulated activation and tyrosine phosphorylation of MAPK in v-RAF-expressing macrophages, indicating the involvement of a tyrosine phosphatase. Interestingly, v-RAF-expressing macrophages contain low constitutive levels of MKP-1 mRNA, an immediate early gene that encodes a MAPK-specific phosphatase and is induced in the parental cell line by CSF-1 treatment. The restoration of MAPK activation by vanadate pretreatment and the presence of MKP-1 mRNA in v-RAF-expressing macrophages raise the intriguing possibility that in macrophages RAF may be feeding back on the MAPK pathway by participating in the control of MKP-1 expression.

v-raf confers CSF-1 independent growth to a macrophage cell line and leads to immediate early gene expression without MAP-kinase activation.

The BAC-1.2F5 macrophage cell line depends on CSF-1 for proliferation and survival. Phosphorylation and activation of the RAF-1 kinase are among the early events in CSF-1 signal transduction. To characterize the role of RAF-1 in CSF-1-induced proliferation, we overexpressed oncogenically activated RAF-1, cellular RAF-1 and RAF-1 kinase-defective mutant proteins in BAC-1.2F5 cells. We were unable to establish stable cell lines expressing either kinase-negative or full length RAF-1 proteins, implying that expression of these molecules is not tolerated in BAC-1.2F5 cells. Oncogenically activated RAF-1 induces CSF-1-independent growth in the absence of autocrine growth factor production. Autonomous growth is not associated with dedifferentiation, since v-raf-expressing macrophages perform the same immunological functions as control cells. Intriguingly, autonomous growth correlates with the suppression of CSF-1-mediated MAP-Kinase activation and with the low constitutive expression of a number of CSF-1-inducible genes, including fos, jun, ets2, and myc, but also the genes for the inflammatory cytokines TNF alpha and IL-1 beta. Many of these genes have AP-1 binding sites in their promoters, and the v-raf-expressing cells contain constitutive AP-1 binding activity. These data indicate that RAF-1, but not MAP-Kinase, is a key component in CSF-1 mitogenic signal transduction, and are consistent with a working hypothesis in which RAF-1 mediates transcriptional activation of genes via AP-1.

Cloning and sequence analysis of the murine Gli3 cDNA.

The zinc finger gene Gli3 plays a role in limb and brain development. To facilitate the molecular analysis of different mouse mutations of this gene, the murine cDNA was isolated and sequenced. This 5113 bp cDNA encodes a putative protein of 1596 amino acids. Comparison of the murine and human GLI3 cDNA revealed an overall homology of 85% between the deduced amino acid sequences. More importantly, several regions of the protein, including the zinc fingers, are more highly conserved ( > 95%), suggesting that these represent functional domains in the Gli3 protein.

Evidence for genetic control of Sonic hedgehog by Gli3 in mouse limb development.

Sonic hedgehog (Shh) expression in the developing limb is associated with the zone of polarising activity (ZPA), and both are restricted to the posterior part of the limb bud. We show that the expression patterns of Shh and Gli3, a member of the Gli-family believed to function in transcriptional control, appear to be mutually exclusive in limb buds of mouse embryos. In the polydactyly mouse mutant extra toes (Xt), possessing a null mutation of Gli3, Shh is additionally expressed in the anterior region of the limb bud. The transcript of Ptc, the putative receptor for Shh protein, can be detected anteriorly as well. Other genes known to be involved in limb outgrowth and patterning, like Fibroblast growth factor (Fgf), Bone morphogenetic protein (Bmp), and Hoxd are misexpressed in relation to the ectopic Shh expression domain in Xt limb buds. This data suggest that Gli3 is a regulator of Shh expression in mouse limb development.

Dual-conjugate wavefront generation for adaptive optics.

We present results of the isoplanatic performance of an astronomical adaptive optics system in the laboratory, by using a dual layer turbulence simulator. We describe how the performance of adaptive correction degrades with off--axis angle. These experiments demonstrate that it is now possible to produce quantifiable multi-layer turbulence in the laboratory as a precursor to constructing multi-conjugate adaptive optics.

[Clinical experiences with dopamine after heart surgery]. / Klinische Erfahrung mit Dopamin nach kardio-chirurgischen Eingriffen

15 patients have been treated with dopamine (2.0--6.0 gamma/kg/min) in the initial phase of cardiogenic shock after cardiac surgery. Indication was a systolic blood pressure of less than 85 mmHg associated with oligurie and peripheral vasoconstriction. 12 patients survived the cardiocirculatory crisis and the early postoperative period. Dopamine alone increased the arterial blood pressure in 6 patients from 52.4 to 80.1 mmHg and the urine flow from 25.2 ml/hr to 181.2 ml/hr. To obtain an optimal perfusion pressure additional application of Noradrenalin was used in 8 patients. In these patients the urine flow rose from 9.1 ml/hr to 131 ml/hr. In one patient no reaction, neither to dopamine, nor in combination with Noradrenalin was seen. The effect of pulse rate, central venous pressure and arterial oxygen tension has been discussed. Dopamine seems to be a useful substance in surgical patients with temporary cardiac insufficiency.

[Reconstructive measures to prevent lower extremity amputation for atherosclerotic occlusive disease in old age and the results obtained between 1973 to 1977 (author's transl)]. / Das "amputationsreife Bein" im Greisenalter: Rekonstruktive Möglichkeiten und Ergebnisse der Jahre 1973--1977.

Within a 5-year-period 102 patients over the age of 70 were admitted with atherosclerotic occlusive disease. All patients had rest pain or necrosis. In 12,8% only a primary amputation could be performed. The procedures and results of reconstructive surgery on 89 patients were demonstrated: mortality rate of 11,2%, a secondary amputation rate of 13,5% and improvement of 75,3%. The problems after amputation in the senium are discussed.

Expression profile of Gli family members and Shh in normal and mutant mouse limb development.

Gli genes represent a small family, encoding zinc-finger proteins of the Krüppel-type. The family consists of Gli(1), Gli2, and Gli3, all of which are expressed in the developing mouse limb bud. To assess the role of the Gli family and Sonic hedgehog (Shh) in mouse limb development, we compared the expression domains of all three Gli genes and of Shh. Although each Gli gene has its own distinct expression pattern in limb buds, at 10.5-11.5 dpc all three genes were found not to be expressed in the posterior region, the presumptive Shh expression domain. This transient mutually exclusive expression suggested a potential interaction between Gli genes and Shh. To address this matter, we analysed the expression of Gli genes and Shh in two polydactyly mouse mutants, Extra toes (Xt) and Hemimelic-extra toes (Hx) which express Shh ectopically in the anterior region of the limb field. Since Xt mice lack Gli3 expression, the ectopic Shh expression is genetically linked to the absence of Gli3. In Hx mice we found a down-regulation of Gli3 in the anterior region of the limb bud. In both mutants Gli2 expression pattern was not altered, whereas Gli1 expression was anteriorly up-regulated adjacent to the ectopic Shh domain. These results strongly suggest a positive regulation of Gli1 by Shh and a negative interaction between Shh and Gli3.

Muscle development during vertebrate limb outgrowth.

Limb development has become one of the model systems for studying vertebrate development. One crucial aspect in limb development is the origin, differentiation and patterning of muscle. Much progress has been made in recent years towards understanding this process. One of the general observations is that the genes involved in limb muscle development appear to be very similar to those involved in muscle development in other regions of the embryo. In this review, we summarize some of the genes and mechanisms that regulate limb muscle development and discuss various avenues along which a deeper understanding can be gained of how muscle cells originate and differentiate in different tissues during vertebrate development.

[Reconstructive surgery of the peripheral arteries in elderly people]. / Rekonstruktionseingriffe an der peripheren arteriellen Strombahn im Greisenalter.

The results of reconstructive surgery of atherosclerotic occlusive disease in the senium are reported on 71 patients. Mortality rate of 14.68% and improvement of 63.38% showed clearly better results than described in literature with an amputation mortality rate between 26% and 60%. In the authors' opinion also in the senium the reconstructive surgery of the peripheral arterial system should be preferred if general and local operability are given.

Laser beacon wave-front sensing without focal anisoplanatism.

Wave-front sensing from artificial beacons is normally performed by formation of a focused spot in the atmosphere and sensing of the wave-front distortions produced during the beam's return passage. We propose an alternative method that senses the distortions produced during the outgoing path by forming an intensity pattern in the atmosphere that is then viewed from the ground. A key advantage of this method is that a parallel beam is used, and therefore the wave-front measurements will not suffer from the effects of focal anisoplanatism. We also envisage other geometries, all based on the concept of projecting a pupil pattern onto the atmosphere.

Ras/MAP kinase-dependent and -independent signaling pathways target distinct ternary complex factors.

Transcriptional activation of the immediate early genes c-fos and egr-1 by extracellular signals appears to be mediated by ternary complex factors (TCFs). In BAC-1 macrophages, growth factor stimulation leads to the retardation of protein-DNA complexes containing distinct TCFs. One TCF is recognized by Elk-1 antisera, whereas the other is immunologically related to SAP-1. The appearance and decay of hyperphosphorylated TCF/Elk-1-containing complexes after stimulation coincide with the activation of mitogen-activated protein kinase (MAPK) and the induction and repression of c-fos and egr-1, whereas modified TCF/SAP-1-containing complexes decay more slowly. Suppression of MAPK activation in macrophages and fibroblasts correlates with the failure to induce TCF/Elk-1 hyperphosphorylation without blocking TCF/SAP-1 modification. Accordingly the modified Elk-1 complex is generated in vitro by activated MAPK, whereas that of SAP-1 is not. Expression of a dominant-negative Ras mutant (RasAsn17) in BAC-1 cells does not affect CSF-1-induced TCF/SAP-1 modification while suppressing TCF/Elk-1 phosphorylation. Neither PKC down-regulation by TPA nor inhibition of Gi proteins by pertussis toxin pretreatment influences CSF-1-induced signaling to TCFs. These data indicate the existence of two separate signaling pathways for the modification of distinct TCFs: one dependent on Ras and MAPK and converging on TCF/Elk-1, and the other targeting TCF/SAP-1 independently of Ras and MAPK.

[Effects of bacterial peritonitis on the low-pressure system in man]. / Auswirkungen der bakteriellen Peritonitis auf das Niederdrucksystem des Menschen.

The effective compliance (C) of the "low-pressure system" has been measured in ten patients with bacterial peritonitis by means of plotting pressure and volume on the first postoperative day (deltaV = 500 ml/10 min). The value of C is decreased in one group of patients, while it increases to as much as five times that of "normal patients" in the other. The cause might be a summation effect of hypovolemia, blood volume shift from intra- to extrathoracic space, and endotoxin reaction on the tone of vascular smooth muscle. The augmentation of the intravascular volume results in an increase of portal venous pressure, which is less than central venous pressure. Splanchnic pooling through increased vascular resistance does not appear.

IFN-gamma/lipopolysaccharide activation of macrophages is associated with protein kinase C-dependent down-modulation of the colony-stimulating factor-1 receptor.

IFN gamma/LPS treatment increases macrophage tumoricidal and microbicidal activity and inhibits CSF-1-induced macrophage proliferation. The mechanism underlying the latter effect was investigated in the CSF-1-dependent mouse macrophage cell line, BAC-1.2F5. IFN-gamma and LPS together dramatically reduced the total number of CSF-1 receptors (CSF-1R) via selective degradation of the cell surface form. Processing and transport of intracellular CSF-1R to the cell surface were unaffected. IFN-gamma alone had no effect but significantly enhanced LPS-induced CSF-1R down-regulation. The reduction in CSF-1R number was protein kinase C-dependent and involved changes in serine phosphorylation of the receptor at different sites. CSF-1R down-modulation by this mechanism may be important in switching off the energy-consuming processes of CSF-1R-mediated proliferation and chemotaxis in activated macrophages.