Nitric oxide sustains IL-1β expression in human dendritic cells enhancing their capacity to induce IL-17-producing T-cells

The role played by lung dendritic cells (DCs) which are influenced by external antigens and by their redox state in controlling inflammation is unclear. We studied the role played by nitric oxide (NO) in DC maturation and function. Human DCs were stimulated with a long-acting NO donor, DPTA NONOate, prior to exposure to lipopolysaccharide (LPS). Dose-and time-dependent experiments were performed with DCs with the aim of measuring the release and gene expression of inflammatory cytokines capable of modifying T-cell differentiation, towardsTh1, Th2 and Th17 cells. NO changed the pattern of cytokine release by LPS-matured DCs, dependent on the concentration of NO, as well as on the timing of its addition to the cells during maturation. Addition of NO before LPS-induced maturation strongly inhibited the release of IL-12, while increasing the expression and release of IL-23, IL-1β and IL-6, which are all involved in Th17 polarization. Indeed, DCs treated with NO efficiently induced the release of IL-17 by T-cells through IL-1β. Our work highlights the important role that NO may play in sustaining inflammation during an infection through the preferential differentiation of the Th17 lineage.

Effects of Forskolin on Trefoil factor 1 expression in cultured ventral mesencephalic dopaminergic neurons

Trefoil factor 1 (TFF1) belongs to a family of secreted peptides that are mainly expressed in the gastrointestinal tract. Notably, TFF1 has been suggested to operate as a neuropeptide, however, its specific cellular expression, regulation and function remain largely unknown. We have previously shown that TFF1 is expressed in developing and adult rat ventral mesencephalic tyrosine hydroxylase-immunoreactive (TH-ir) dopaminergic neurons. Here, we investigated the expression of TFF1 in rat ventral mesencephalic dopaminergic neurons (embryonic day 14) grown in culture for 5, 7 or 10 days in the absence (controls) or presence of either glial cell line-derived neurotrophic factor (GDNF), Forskolin or the combination. No TFF1-ir cells were identified at day 5 and only a few at day 7, whereas TH was markedly expressed at both time points. At day 10, several TFF1-ir cells were detected, and their numbers were significantly increased after the addition of GDNF (2.2-fold) or Forskolin (4.1-fold) compared to controls. Furthermore, the combination of GDNF and Forskolin had an additive effect and increased the number of TFF1-ir cells by 5.6-fold compared to controls. TFF1 expression was restricted to neuronal cells, and the percentage of TH/TFF1 co-expressing cells was increased to the same extent in GDNF and Forskolin-treated cultures (4-fold) as compared to controls. Interestingly, the combination of GDNF and Forskolin resulted in a significantly increased co-expression (8-fold) of TH/TFF1, which could indicate that GDNF and Forskolin targeted different subpopulations of TH/TFF1 neurons. Short-term treatment with Forskolin resulted in an increased number of TFF1-ir cells, and this effect was significantly reduced by the MEK1 inhibitor PD98059 or the protein kinase A (PKA) inhibitor H89, suggesting that Forskolin induced TFF1 expression through diverse signaling pathways. In conclusion, distinct populations of cultured dopaminergic neurons express TFF1, and their numbers can be increased by factors known to influence survival and differentiation of dopaminergic cells.

Expression patterns of Bmi-1 and p16 significantly correlate with overall, disease-specific, and recurrence-free survival in oropharyngeal squamous cell carcinoma

BACKGROUND: The objective of this study was to link expression patterns of B-cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1) and p16 to patient outcome (recurrence and survival) in a cohort of 252 patients with oral and oropharyngeal squamous cell cancer (OSCC). METHODS: Expression levels of Bmi-1 and p16 in samples from 252 patients with OSCC were evaluated immunohistochemically using the tissue microarray method. Staining intensity was determined by calculating an intensity reactivity score (IRS). Staining intensity and the localization of expression within tumor cells (nuclear or cytoplasmic) were correlated with overall, disease-specific, and recurrence-free survival. RESULTS: The majority of cancers were localized in the oropharynx (61.1%). In univariate analysis, patients who had OSCC and strong Bmi-1 expression (IRS >10) had worse outcomes compared with patients who had low and moderate Bmi-1 expression (P = .008; hazard ratio [HR], 1.82; 95% confidence interval [CI], 1.167-2.838); this correlation was also observed for atypical cytoplasmic Bmi-1 expression (P = .001; HR, 2.164; 95% CI, 1.389-3.371) and for negative p16 expression (P < .001; HR, 0.292; 95% CI, 0.178-0.477). The combination of both markers, as anticipated, had an even stronger correlation with overall survival (P < .001; HR, 8.485; 95% CI, 4.237-16.994). Multivariate analysis demonstrated significant results for patients with oropharyngeal cancers, but not for patients with oral cavity tumors: Tumor classification (P = .011; HR, 1.838; 95%CI, 1.146-2.947) and the combined marker expression patterns (P < .001; HR, 6.254; 95% CI, 2.869-13.635) were correlated with overall survival, disease-specific survival (tumor classification: P = .002; HR, 2.807; 95% CI, 1.477-5.334; combined markers: P = .002; HR, 5.386; 95% CI, 1.850-15.679), and the combined markers also were correlated with recurrence-free survival (P = .001; HR, 8.943; 95% CI, 2.562-31.220). CONCLUSIONS: Cytoplasmic Bmi-1 expression, an absence of p16 expression, and especially the combination of those 2 predictive markers were correlated negatively with disease-specific and recurrence-free survival in patients with oropharyngeal cancer. Therefore, the current results indicate that these may be applicable as predictive markers in combination with other factors to select patients for more aggressive treatment and follow-up. Cancer 2011;. © 2011 American Cancer Society.

Shear stress modulates the expression of thrombospondin-1 and CD36 in endothelial cells in vitro and during shear stress-induced angiogenesis in vivo

Binding of thrombospondin-1 (TSP-1) to the CD36 receptor inhibits angiogenesis and induces apoptosis in endothelial cells (EC). Conversely, matrix-bound TSP-1 supports vessel formation. In this study we analyzed the shear stress-dependent expression of TSP-1 and CD36 in endothelial cells in vitro and in vivo to reveal its putative role in the blood flow-induced remodelling of vascular networks. Shear stress was applied to EC using a cone-and-plate apparatus and gene expression was analyzed by RT-PCR, Northern and Western blot. Angiogenesis in skeletal muscles of prazosin-fed (50 mg/l drinking water; 4 d) mice was assessed by measuring capillary-to-fiber (C/F) ratios. Protein expression in whole muscle homogenates (WMH) or BS-1 lectin-enriched EC fractions (ECF) was analyzed by Western blot. Shear stress downregulated TSP-1 and CD36 expression in vitro in a force- and time-dependent manner sustained for at least 72 h and reversible by restoration of no-flow conditions. In vivo, shear stress-driven increase of C/F in prazosin-fed mice was associated with reduced expression of TSP-1 and CD36 in ECF, while TSP-1 expression in WMH was increased. Down-regulation of endothelial TSP-1/CD36 by shear stress suggests a mechanism for inhibition of apoptosis in perfused vessels and pruning in the absence of flow. The increase of extra-endothelial (e.g. matrix-bound) TSP-1 could support a splitting type of vessel growth.

Neuronal differentiation of human mesenchymal stem cells: changes in the expression of the Alzheimer's disease-related gene seladin-1

Seladin-1 (SELective Alzheimer's Disease INdicator-1) is an anti-apoptotic gene, which is down-regulated in brain regions affected by Alzheimer's disease (AD). In addition, seladin-1 catalyzes the conversion of desmosterol into cholesterol. Disruption of cholesterol homeostasis in neurons may increase cell susceptibility to toxic agents. Because the hippocampus and the subventricular zone, which are affected in AD, are the unique regions containing stem cells with neurogenic potential in the adult brain, it might be hypothesized that this multipotent cell compartment is the predominant source of seladin-1 in normal brain. In the present study, we isolated and characterized human mesenchymal stem cells (hMSC) as a model of cells with the ability to differentiate into neurons. hMSC were then differentiated toward a neuronal phenotype (hMSC-n). These cells were thoroughly characterized and proved to be neurons, as assessed by molecular and electrophysiological evaluation. Seladin-1 expression was determined and found to be significantly reduced in hMSC-n compared to undifferentiated cells. Accordingly, the total content of cholesterol was decreased after differentiation. These original results demonstrate for the first time that seladin-1 is abundantly expressed by stem cells and appear to suggest that reduced expression in AD might be due to an altered pool of multipotent cells.

Cbfa-1 (Runx-2) and osteocalcin expression by human osteoblasts in heparin osteoporosis in vitro

Heparin may cause adverse effects on bone formation following long-term application. The exact pathomechanism is unclear, but in vitro data suggest an impaired osteoblast function. The transcription axis of Cbfa-1 (Runx-2) and osteocalcin is crucial in maintaining an equilibrium of bone formation and resorption in vivo. We used a human osteoblast cell culture model to further investigate the effect of heparin (low-molecular-weight heparin, dalteparin) on the expression of these two regulators of osteoblast differentiation. At high doses, dalteparin caused a significant inhibition of both osteocalcin and Cbfa-1 expression in vitro. Our data support the hypothesis of a direct inhibition of osteoblast function underlying heparin osteoporosis.

Drug targeting using OX7-immunoliposomes: correlation between Thy1.1 antigen expression and tissue distribution in the rat

OX7 monoclonal antibody F((ab')2) fragments directed against Thy1.1 antigen can be used for drug targeting by coupling to the surface of drug-loaded liposomes. Such OX7-conjugated immunoliposomes (OX7-IL) were used recently for drug delivery to rat glomerular mesangial cells, which are characterized by a high level of Thy1.1 antigen expression. In the present study, the relationship between OX7-IL tissue distribution and target Thy1.1 antigen localization in different organs in rat was investigated. Western blot and immunohistofluorescence analysis revealed a very high Thy1.1 expression in brain cortex and striatum, thymus and renal glomeruli. Moderate Thy1.1 levels were observed in the collecting ducts of kidney, lung tissue and spleen. Thy1.1 was not detected in liver and heart. There was a poor correlation between Thy1.1 expression levels and organ distribution of fluorescence- or (14)C-labeled OX7-IL. The highest overall organ density of OX7-IL was observed in the spleen, followed by lung, liver and kidney. Heart and brain remained negative. With respect to intra-organ distribution, a localized and distinct signal was observed in renal glomerular mesangial cells only. As a consequence, acute pharmacological (i.e. toxic) effects of doxorubicin-loaded OX7-IL were limited to renal glomeruli. The competition with unbound OX7 monoclonal antibody F((ab')2) fragments demonstrated that the observed tissue distribution and acute pharmacological effects of OX7-IL were mediated specifically by the conjugated OX7 antibody. It is concluded that both the high target antigen density and the absence of endothelial barriers are needed to allow for tissue-specific accumulation and pharmacological effects of OX7-IL. The liposomal drug delivery strategy used is therefore specific toward renal glomeruli and can be expected to reduce the risk of unwanted side effects in other tissues.

Transforming growth factor-beta2 upregulates sphingosine kinase-1 activity, which in turn attenuates the fibrotic response to TGF-beta2 by impeding CTGF expression

Transforming growth factor-beta2 (TGF-beta2) stimulates the expression of pro-fibrotic connective tissue growth factor (CTGF) during the course of renal disease. Because sphingosine kinase-1 (SK-1) activity is also upregulated by TGF-beta, we studied its effect on CTGF expression and on the development of renal fibrosis. When TGF-beta2 was added to an immortalized human podocyte cell line we found that it activated the promoter of SK-1, resulting in upregulation of its mRNA and protein expression. Further, depletion of SK-1 by small interfering RNA or its pharmacological inhibition led to accelerated CTGF expression in the podocytes. Over-expression of SK-1 reduced CTGF induction, an effect mediated by intracellular sphingosine-1-phosphate. In vivo, SK-1 expression was also increased in the podocytes of kidney sections of patients with diabetic nephropathy when compared to normal sections of kidney obtained from patients with renal cancer. Similarly, in a mouse model of streptozotocin-induced diabetic nephropathy, SK-1 and CTGF were upregulated in podocytes. In SK-1 deficient mice, exacerbation of disease was detected by increased albuminuria and CTGF expression when compared to wild-type mice. Thus, SK-1 activity has a protective role in the fibrotic process and its deletion or inhibition aggravates fibrotic disease.

Local gene transfer and expression following intramuscular administration of FGF-1 plasmid DNA in patients with critical limb ischemia

NV1FGF is an expression plasmid encoding sp.FGF-1(21-154) currently under investigation for therapeutic angiogenesis in clinical trials. NV1FGF plasmid distribution and transgene expression following intramuscular (IM) injection in patients is unknown. The study involved six patients with chronic critical limb ischemia (CLI) planned to undergo amputation. A total dose of 0.5, 2, or 4 mg NV1FGF was administered as eight IM injections (0.006, 0.25, or 0.5 mg per injection) 3-5 days before amputation. Injected sites (30 cm(3)) were divided into equally sized smaller pieces to assess spatial distribution of NV1FGF sequences (PCR), NV1FGF mRNA (reverse transcriptase-PCR), and fibroblast growth factor-1 (FGF-1)-expressing cells (immunohistochemistry). Data indicated gene expression at all doses. The distribution area was within 5-12 cm for NV1FGF sequences containing the expression cassette, up to 5 cm for NV1FGF mRNA, and up to 3 cm for FGF-1-expressing myofibers. All FGF receptors were detected indicating robust potential for bioactivity after NV1FGF gene transfer. Circulating levels of NV1FGF sequences were shown to decrease within days after injection. Data support demonstration of plasmid-mediated gene transfer and expression in muscles from patients with CLI. FGF-1 expression was shown to be limited to injection sites, which supports the concept of multiple-site injection for therapeutic use.

Expression of trefoil factor 1 in the developing and adult rat ventral mesencephalon

Trefoil factor 1 (TFF1) belongs to a family of secreted peptides with a characteristic tree-looped trefoil structure. TFFs are mainly expressed in the gastrointestinal tract where they play a critical role in the function of the mucosal barrier. TFF1 has been suggested as a neuropeptide, but not much is known about its expression and function in the central nervous system. We investigated the expression of TFF1 in the developing and adult rat midbrain. In the adult ventral mesencephalon, TFF1-immunoreactive (-ir) cells were predominantly found in the substantia nigra pars compacta (SNc), the ventral tegmental area (VTA) and in periaqueductal areas. While around 90% of the TFF1-ir cells in the SNc co-expressed tyrosine hydroxylase (TH), only a subpopulation of the TH-ir neurons expressed TFF1. Some TFF1-ir cells in the SNc co-expressed the calcium-binding proteins calbindin or calretinin and nearly all were NeuN-ir confirming a neuronal phenotype, which was supported by lack of co-localization with the astroglial marker glial fibrillary acidic protein (GFAP). Interestingly, at postnatal (P) day 7 and P14, a significantly higher proportion of TH-ir neurons in the SNc co-expressed TFF1 as compared to P21. In contrast, the proportion of TFF1-ir cells expressing TH remained unchanged during postnatal development. Furthermore, significantly more TH-ir neurons expressed TFF1 in the SNc, compared to the VTA at all four time-points investigated. Injection of the tracer fluorogold into the striatum of adult rats resulted in retrograde labeling of several TFF1 expressing cells in the SNc showing that a significant fraction of the TFF1-ir cells were projection neurons. This was also reflected by unilateral loss of TFF1-ir cells in SNc of 6-hydroxylase-lesioned hemiparkinsonian rats. In conclusion, we show for the first time that distinct subpopulations of midbrain dopaminergic neurons express TFF1, and that this expression pattern is altered in a rat model of Parkinson's disease.

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.

Heterozygous deletion of the PU.1 locus in human AML

The transcription factor PU.1 is essential for myeloid development. Targeted disruption of an upstream regulatory element (URE) decreases PU.1 expression by 80% and leads to acute myeloid leukemia (AML) in mice. Here, we sequenced the URE sequences of PU.1 in 120 AML patients. Four polymorphisms (single nucleotide polymorphisms [SNPs]) in the URE were observed, with homozygosity in all SNPs in 37 patients. Among them, we compared samples at diagnosis and remission, and one patient with cytogenetically normal acute myeloid leukemia M2 was identified with heterozygosity in 3 of the SNPs in the URE at remission. Loss of heterozygosity was further found in this patient at 2 polymorphic sites in the 5' promoter region and in 2 intronic sites flanking exon 4, thus suggesting loss of heterozygosity covering at least 40 kb of the PU.1 locus. Consistently, PU.1 expression in this patient was markedly reduced. Our study suggests that heterozygous deletion of the PU.1 locus can be associated with human AML.

PU.1 is regulated by NF-kappaB through a novel binding site in a 17 kb upstream enhancer element

The majority of patients with acute myeloid leukemia (AML) still die of their disease, and novel therapeutic concepts are needed. Timely expression of the hematopoietic master regulator PU.1 is crucial for normal development of myeloid and lymphoid cells. Targeted disruption of an upstream regulatory element (URE) located several kb upstream in the PU.1 promoter decreases PU.1 expression thereby inducing AML in mice. In addition, suppression of PU.1 has been observed in specific subtypes of human AML. Here, we identified nuclear factor-kappaB (NF-kappaB) to activate PU.1 expression through a novel site within the URE. We found sequence variations of this particular NF-kappaB site in 4 of 120 AML patients. These variant NF-kappaB sequences failed to mediate activation of PU.1. Moreover, the synergistic activation of PU.1 together with CEBPB through these variant sequences was also lost. Finally, AML patients with such variant sequences had suppressed PU.1 mRNA expression. This study suggests that changes of a single base pair in a distal element critically affect the regulation of the tumor suppressor gene PU.1 thereby contributing to the development of AML.

Purification, cDNA structure and biological significance of a single insulin-like growth factor-binding domain protein (SIBD-1) identified in the hemocytes of the spider Cupiennius salei

Cupiennius salei single insulin-like growth factor-binding domain protein (SIBD-1), which exhibits an IGFBP N-terminal domain-like profile, was identified in the hemocytes of the spider C. salei. SIBD-1 was purified by RP-HPLC and the sequence determined by a combination of Edman degradation and 5'-3'- RACE PCR. The peptide (8676.08 Da) is composed of 78 amino acids, contains six intrachain disulphide bridges and carries a modified Thr residue at position 2. SIBD-1 mRNA expression was detected by quantitative real-time PCR mainly in hemocytes, but also in the subesophageal nerve mass and muscle. After infection, the SIBD-1 content in the hemocytes decreases and, simultaneously, the temporal SIBD-1 expression seems to be down-regulated. Two further peptides, SIBD-2 and IGFBP-rP1, also exhibiting IGFBP N-terminal domain variants with unknown functions, were identified on cDNA level in spider hemocytes and venom glands. We conclude that SIBD-1 may play an important role in the immune system of spiders.

Ablation of the tumor suppressor histidine triad nucleotide binding protein 1 is protective against hepatic ischemia/reperfusion injury

The identification of cellular pathways capable of limiting ischemia/reperfusion (I/R) injury remains a frontier in medicine, and its clinical relevance is urgent. Histidine triad nucleotide binding protein 1 (HINT1) is a tumor suppressor that influences apoptosis. Because apoptotic pathways are a feature of I/R injury, we asked whether Hint1 influences hepatic I/R injury. Hint1(-/-) and C57BL/6 mice were subjected to 70% liver ischemia followed by reperfusion for 3 or 24 hours or to a sham operation. The serum aminotransferase levels, histological lesions, apoptosis, reactive oxygen species, and expression of B cell lymphoma 2-associated X protein (Bax), heme oxygenase 1 (HO-1), interleukin-6 (IL-6), IL-10, tumor necrosis factor-a, Src, nuclear factor kappa B (p65/RelA), and c-Jun were quantified. The responses to toll-like receptor ligands and nicotinamide adenine dinucleotide phosphate oxidase activity in Kupffer cells were compared in Hint1(-/-) mice and C57BL/6 mice. After I/R, the levels of serum aminotransferases, parenchymal necrosis, and hepatocellular apoptosis were significantly lower in Hint1(-/-) mice versus control mice. Furthermore, Bax expression decreased more than 2-fold in Hint1(-/-) mice, and the increases in reactive oxygen species and HO-1 expression that were evident in wild-type mice after I/R were absent in Hint1(-/-) mice. The phosphorylation of Src and the nuclear translocation of p65 were increased in Hint1(-/-) mice, whereas the nuclear expression of phosphorylated c-Jun was decreased. The levels of the protective cytokines IL-6 and IL-10 were increased in Hint1(-/-) mice. These effects increased survival after I/R in mice lacking Hint1. Hint1(-/-) Kupffer cells were less activated than control cells after stimulation with lipopolysaccharides. CONCLUSION: The Hint1 protein influences the course of I/R injury, and its ablation in Kupffer cells may limit the extent of the injury.