Recombinant human granulocyte-macrophage colony-stimulating factor induces secretion of autoinhibitory monokines by U-937 cells

Colony-stimulating factors are required for survival proliferation, differentiation and functional activation of granulocytes, macrophages and their precursor cells. In the present report, however, we demonstrate antiproliferative activity of recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF) on monoblast cell line U-937 and provide evidence for the involvement of tumor necrosis factor alpha TNF-alpha and interleukin 1 beta (IL 1 beta) in its growth inhibitory action. GM-CSF (but not granulocyte CSF, G-CSF or macrophage CSF, M-CSF) suppressed DNA synthesis and self renewal of U-937 cells. Similarly, medium conditioned by U-937 cells in response to GM-CSF (GM-CSF U-937-CM) was able to reduce clonogenicity and [3H]thymidine uptake by U-937 cells. Since neutralization of GM-CSF present in GM-CSF U-937-CM by monoclonal antibody to GM-CSF did not abrogate the autoinhibitory activity present in GM-CSF U-937-CM, we considered the possibility that other soluble molecules are released by U-937 cells upon GM-CSF stimulation. Neutralization by antibodies to IL 1 beta and TNF-alpha suggested that both monokines could be the antiproliferative principle operating in GM-CSF U-937-CM. Moreover, employing IL 1 beta-specific enzyme-linked immunosorbent assay, TNF-alpha specific radioimmunoassay, Northern analysis using a cloned TNF-alpha-specific cDNA and an oligonucleotide probe for IL 1 beta, we demonstrate GM-CSF-inducible IL 1 beta and TNF-alpha gene expression by U-937 cells at the mRNA and protein level. Although M-CSF expression was induced under similar conditions, M-CSF failed to inhibit growth of U-937 cells.     

Elevation of plasma viscosity induces sustained NO-mediated dilation in the hamster cremaster microcirculation in vivo

 We studied whether a flow-independent increase of luminal wall shear stress (WSS) could dilate hamster arterioles in vivo and which endothelial mediators are potentially involved. To this end the plasma viscosity was elevated by exchanging blood for dextran-erythrocyte solution thereby augmenting WSS. Diameters of small and large arterioles as well as red blood cell velocities were measured before and after exchange of blood for solutions of identical haematocrit containing either high- (HMWD) or low-molecular weight dextran (LMWD). The potential role of endothelial autacoids was investigated by local application of the NO-synthase inhibitor N ^G-nitro-L-arginine (L-NNA), the inhibitor of cyclooxygenase, indomethacin (3 μM), or the K⁺-channel blocker, tetrabutylammonium (TBA, 0.1 mM) to assess the potential effects of EDHF. HMWD (n = 11 animals) increased plasma viscosity by 64 ± 3% and dilated arterioles of all branching orders (A1–A4) significantly [by 24 ± 3% (A1–A2) and 32 ± 3% (A3–A4)]. This dilation compensated fully for the calculated initial increase of WSS. LMWD (n = 6) did not affect plasma viscosity or arteriolar diameters. Tissue treatment with L-NNA (30–300 μM, n = 12) substantially diminished the HMWD-induced dilation in small arterioles (A3–A4; to 13 ± 3%; P<0.05) and virtually abolished it in large ones (A1–A2). Consequently, the calculated WSS increased significantly in these arterioles (by 31 ± 5%). TBA combined with L-NNA (n = 4) did not reduce further the remaining dilation. Indomethacin (n = 6) had no effect on HMWD-induced dilation. We conclude that an increase of WSS induces a mainly NO-mediated arteriolar dilation. This dilation occurs in all arteriolar branching orders and is of sufficient magnitude to compensate for the initial WSS-increase. Thus, any elevations of WSS fulfil the requirement for a signal to change diameter along the arteriolar tree in a coordinated manner. The fully compensating dilation which we observed indicates that WSS is a controlled variable. It does, however, raise questions as to its role as a continuous endothelial stimulus. Received: 2 August 1996 / Received after revision: 24 February 1997 / Accepted: 14 April 1997     

Similarities and differences of human and experimental mouse lymphangiomas.

Lymphangioma is a disfiguring malformation of early childhood. A mouse lymphangioma model has been established by injecting Freund's incomplete adjuvant (FIA) intraperitoneally, but has not been compared with the human disease. We show that, in accordance with studies from the 1960s, the mouse model represents an oil-granuloma, made up of CD45-positive leukocytes and invaded by blood and lymph vessels. Several markers of lymphatic endothelial cells are expressed in both mouse and human, like CD31, Prox1, podoplanin, and Lyve-1. However, the human disease affects all parts of the lymphovascular tree. We observed convolutes of lymphatic capillaries, irregularly formed collectors with signs of disintegration, and large lymph cysts. We observed VEGFR-2 and -3 expression in both blood vessels and lymphatics of the patients, whereas in mouse VEGFR-2 was confined to activated blood vessels. The experimental mouse FIA model represents a vascularized oil-granuloma rather than a lymphangioma and reflects the complexity of human lymphangioma only partially.     

TLR8 and TLR7 are involved in the host's immune response to human parechovirus 1

Toll-like receptors (TLR) have a key role in regulating immunity against microbial agents. Engagement of TLR by bacterial, viral or fungal components leads to the production and release of inflammatory cytokines. In this study we show that mainly TLR8 and also TLR7 act as the host sensors for human parechovirus 1, a single-stranded RNA (ssRNA) virus. Furthermore, we see that the viral ssRNA genome is detected in endosomal compartments by these TLR, which activate signalling that lead to the synthesis of pro-inflammatory molecules by the host.     

1,3-Oxazoline intermediates in reactive processing applications, 1. Melt grafting of poly(ethene-co-methacrylic acid) with 2-substituted 1,3-oxazolines

A novel family of functional ethene copolymers with various side chains were prepared by melt grafting of poly(ethene-co-methacrylic acid), containing 3,00 and 4,25 mol-% of methacrylic acid, with 2-substituted 1,3-oxazolines such as 2-phenyl-1,3-oxazoline, 2-undecyl-1,3-oxazoline, 2-heptadecyl-1,3-oxazoline, and 4-(1,3-oxazolin-2-yl)phenyl 4-methoxybenzoate. ¹H NMR and FTIR studies of the polymer microstructures revealed that carboxylic acid groups reacted with 1,3-oxazolines within few minutes to form esteramide-coupled side chains in very high yields. Torque of the reaction mixture, mechanical and thermal properties of the graft copolymers were measured. In the case of 2-heptadecyl-esteramide-substituted polyethenes, the side-chain cocrystallization accounted for higher crystallinity of the resulting graft copolymers.     

1,3-Oxazoline intermediates in reactive processing applications, 2.multiphase polymer blends via in-situ cationic melt phase polymerization of 2-phenyl-1,3-oxazoline

2-Phenyl-1,3-oxazoline was injected into poly(ethene-co-methacrylic acid), containing 4,25 mol-% methacrylic acid units, at 225°C. In the first stage, 2-phenyl-1,3-oxazoline reacted with carboxylic acid groups to form esteramide side chains via grafting reaction. In the second stage, 2-phenyl-1,3-oxazoline containing methyl 4-nitrobenzensulfonate initiator was added to initiate in-situ cationic polymerization, yielding poly(N-benzoylethylenimine) as dispersed phase with 0,2–3,5 μm average diameter. Morphological, mechanical and thermal properties of poly-(ethene-co-methacrylic acid) blends, prepared by grafting combined with in-situ polymerization of 2-phenyl-1,3-oxazoline, were investigated.     

Transgenic rat model of Huntington's disease

Huntington's disease (HD) is a late manifesting neurodegenerative disorder in humans caused by an expansion of a CAG trinucleotide repeat of more than 39 units in a gene of unknown function. Several mouse models have been reported which show rapid progression of a phenotype leading to death within 3-5 months (transgenic models) resembling the rare juvenile course of HD (Westphal variant) or which do not present with any symptoms (knock-in mice). Owing to the small size of the brain, mice are not suitable for repetitive in vivo imaging studies. Also, rapid progression of the disease in the transgenic models limits their usefulness for neurotransplantation. We therefore generated a rat model transgenic of HD, which carries a truncated huntingtin cDNA fragment with 51 CAG repeats under control of the native rat huntingtin promoter. This is the first transgenic rat model of a neurodegenerative disorder of the brain. These rats exhibit adult-onset neurological phenotypes with reduced anxiety, cognitive impairments, and slowly progressive motor dysfunction as well as typical histopathological alterations in the form of neuronal nuclear inclusions in the brain. As in HD patients, in vivo imaging demonstrates striatal shrinkage in magnetic resonance images and a reduced brain glucose metabolism in high-resolution fluor-deoxy-glucose positron emission tomography studies. This model allows longitudinal in vivo imaging studies and is therefore ideally suited for the evaluation of novel therapeutic approaches such as neurotransplantation.     

Involvement of nitric oxide in angiogenic activities of vascular endothelial growth factor isoforms

We compared effects of vascular endothelial growth factor-121 (VEGF121) and vascular endothelial growth factor-165 (VEGF165) on generation of NO in HUVEC and the involvement of NO in VEGF121- and VEGF165-induced angiogenesis. VEGF stimulated synthesis of NO within seconds, reaching peak concentrations of 450 +/- 25 and 180 +/- 15 nmol/l for VEGF121, and VEGF165, respectively. The VEGF121 increased NO production for about 40 s while VEGF165-stimulated NO release lasted only for about 20 s. Accordingly, cGMP elevation was stronger in VEGF121- than in VEGF165-treated cells. The VEGF121 was a very weak mitogen but strong chemoattractant for HUVEC, whereas VEGF165 potently induced both cell proliferation and migration. NO appeared to be involved in the endothelial migration and morphogenesis but not in the proliferation. NO was also a permissive molecule for VEGF121- but not for VEGF165-induced capillary sprouting in spheroid culture. In conclusion, VEGF121 is a stronger stimulator of endothelial nitric oxide synthase (eNOS) activity, and angiogenic potential of VEGF121 is more reliant on NO contribution.