Assessing Restoration Effects on River Hydromorphology Using the Process-based Morphological Quality Index in Eight European River Reaches.

The Morphological Quality Index (MQI) and the Morphological Quality Index for monitoring (MQIm) have been applied to eight case studies across Europe with the objective of analyzing the hydromorphological response to various restoration measures and of comparing the results of the MQI and MQIm as a morphological assessment applied at the reach scale, with a conventional site scale physical-habitat assessment method. For each restored reach, the two indices were applied to the pre-restoration and post-restoration conditions. The restored reach was also compared to an adjacent, degraded reach. Results show that in all cases the restoration measures improved the morphological quality of the reach, but that the degree of improvement depends on many factors, including the initial morphological conditions, the length of the restored portion in relation to the reach length, and on the type of intervention. The comparison with a conventional site scale physical-habitat assessment method shows that the MQI and MQIm are best suited for the evaluation of restoration effects on river hydromorphology at the geomorphologically-relevant scale of the river reach.

New tools for the hydromorphological assessment and monitoring of European streams.

Hydromorphological stream assessment has significantly expanded over the last years, but a need has emerged from recent reviews for more comprehensive, process-based methods that consider the character and dynamics of the river with greater accuracy. With this as a focus, a series of hydromorphological tools have been developed and/or further extended in Europe within the context of the REFORM (REstoring rivers FOR effective catchment Management) project. The aim of this paper is to present the set of REFORM hydromorphological assessment methods and, based on some examples of their application, to illustrate and discuss their synergic use, specific features, limitations and strengths. This assessment and monitoring includes three tools: the Morphological Quality Index (MQI), the Morphological Quality Index for monitoring (MQIm), and the Geomorphic Units survey and classification System (GUS). These tools constitute the assessment phase of an overall multi-scale, process-based hydromorphological framework developed in REFORM. The MQI is aimed at an assessment, classification and monitoring of the current morphological state; the MQIm aims at monitoring the tendency of morphological conditions (enhancement or deterioration); the GUS provides a characterization, classification and monitoring of geomorphic units. A series of examples are used to illustrate the potential range of application, including: (i) an assessment of morphological conditions; (ii) an assessment of the morphological effects of restoration projects; (iii) an evaluation of the geomorphic impacts of interventions for risk mitigation; and (iv) an integrated use of MQI and GUS to assess and characterise morphological conditions. Finally, some of the main features, strengths and peculiarities of the three hydromorphological tools are discussed with the support of examples of their application.

Radiotherapy-induced miR-223 prevents relapse of breast cancer by targeting the EGF pathway.

In breast cancer (BC) patients, local recurrences often arise in proximity of the surgical scar, suggesting that response to surgery may have a causative role. Radiotherapy (RT) after lumpectomy significantly reduces the risk of recurrence. We investigated the direct effects of surgery and of RT delivered intraoperatively (IORT), by collecting irradiated and non-irradiated breast tissues from BC patients, after tumor removal. These breast tissue specimens have been profiled for their microRNA (miR) expression, in search of differentially expressed miR among patients treated or not with IORT. Our results demonstrate that IORT elicits effects that go beyond the direct killing of residual tumor cells. IORT altered the wound response, inducing the expression of miR-223 in the peri-tumoral breast tissue. miR-223 downregulated the local expression of epidermal growth factor (EGF), leading to decreased activation of EGF receptor (EGFR) on target cells and, eventually, dampening a positive EGF-EGFR autocrine/paracrine stimulation loop induced by the post-surgical wound-healing response. Accordingly, both RT-induced miR-223 and peri-operative inhibition of EGFR efficiently prevented BC cell growth and reduced recurrence formation in mouse models of BC. Our study uncovers unknown effects of RT delivered on a wounded tissue and prompts to the use of anti-EGFR treatments, in a peri-operative treatment schedule, aimed to timely treat BC patients and restrain recurrence formation.

Hemangiopericitoma de aurícula izquierda: Caso clínico / Primary left atrial hemangiopericytoma: Report of one case

We report a 41-year-old male presenting with progressive dyspnea lasting one month. A CAT scan disclosed a left atrial mass, that was surgically excised. The pathological study of the surgical piece showed a primary hemangiopericytoma. One month later, the patient consulted for cervical pain and a positron emission tomography showed multiple metastases. The patient died two months later.

p27(kip1) functional regulation in human cancer: a potential target for therapeutic designs.

The mitotic cell cycle is a tightly regulated process that ensures the correct division of one cell into two daughter cells. Progress along the different phases of the cell cycle is positively regulated by the sequential activation of a family of serine-threonine kinases called CDKs (Cyclin Dependent Kinases). Their activity is counteracted by small proteins known as CDK inhibitors (CKI) that ensure the correct timing of CDK activation in the different phases of the cell cycle. The present review will deal with the role of one of this CKI, p27(kip1), in human cancer, focusing in particular on the mechanisms underlying its functional inactivation in tumor cells. p27(kip1) protein downregulation is usually achieved by proteasomal degradation and is often correlated to a worse prognosis in several types of human cancers, resulting in the reduction of disease free and overall survival. More recently, it has been proposed that p27(kip1) protein, rather than degraded, can be functionally inactivated. The mechanisms and the implications of these two types of p27(kip1) deregulation will be discussed and some potential therapeutic approaches targeting p27(kip1) functions will be proposed.

Regulation of Id2 gene expression by the type 1 IGF receptor and the insulin receptor substrate-1.

The Id family of helix-loop-helix proteins is known to be involved in the proliferation and differentiation of several types of cells. The type 1 IGF receptor (IGF-IR) induces either proliferation or differentiation in 32D cells, a murine hemopoietic cell line, depending on the availability of the appropriate substrates for the receptor. We have previously reported that the IGF-IR regulates the expression of the Id2 gene in 32D cells. We now show that the IGF-IR controls the increase in Id2 gene expression through at least three pathways. These three pathways originate from the tyrosine residue at 950, a domain in the C-terminus, and the activation of the insulin receptor substrate-1 (IRS-1) by the receptor. IRS-1 is the preponderant signal, and its effect on Id2 gene expression requires a functional phosphotyrosine binding domain. With wild-type IRS-1, Id2 gene expression is increased, even in those cells that express IGF-I receptors defective in Id2 signaling. Rapamycin, an inhibitor of p70(S6K), a downstream effector of IRS-1 signaling, partially inhibits (but does not completely abrogate) the increase in Id2 gene expression. A mutant IRS-1 with a deletion of the Pleckstrin domain is as effective as wild-type IRS-1 in up-regulating Id2 gene expression. In addition, it seems to increase the stability of p70(S6K). Our results indicate that the IGF-IR regulates Id2 gene expression through different pathways. At least in 32D cells, increased Id2 gene expression seems to correlate more with inhibition of differentiation than with proliferation.

The role of the insulin receptor substrate-1 in the differentiation of rat hippocampal neuronal cells.

H19-7/IGF-IR cells are rat hippocampal cells expressing a human IGF-I receptor, which differentiate to a neuronal phenotype when stimulated by IGF-I at 39 degrees C. H19-7/IGF-IR cells have low levels of expression of insulin receptor substrate-l (IRS-1), a major substrate of the IGF-IR. IGF-I induces serine-phosphorylation and down-regulation of the endogenous IRS-1 upon differentiation of H19-7/IGF-IR cells. The profound influence of IRS-1 on differentiation of H19-7/IGF-IR cells was confirmed by transfecting these cells with a plasmid expressing mouse IRS-1. Over-expression of wild type IRS-1 in H19-7/IGF-IR cells abolishes IGF-I-induced differentiation at 39 degrees C. A mutant of IRS-1 lacking the PTB domain loses the ability to inhibit the differentiation program. H19-7/IGF-IR/IRS-1 cells at 39 degrees C show a stronger and prolonged activation of Akt, when compared to H19-7/IGF-IR cells. The role of Akt in the inhibition of the differentiation program was confirmed by using the inhibitor of Class I PI3 kinases LY29400, which restores IGF-I-induced differentiation of H19-7/IGF-IR/IRS-1 cells. H19-7/IGF-IR/IRS-1 cells show a strong reduction in MAP kinases signaling, which is related to the superactivation of Akt. This was confirmed by expressing in H19-7/IGF-IR cells a constitutively active Akt, which inhibited MAP kinases activation in these cells. These experiments confirm the importance of MAPK in the mechanism of IGF-I-mediated differentiation of H19-7/IGF-IR cells

Regulation of Id gene expression by type I insulin-like growth factor: roles of Stat3 and the tyrosine 950 residue of the receptor.

Id proteins are known to play important roles in the proliferation and differentiation of many cell types. The type 1 insulin-like growth factor receptor (IGF-IR), activated by its ligand, induces the differentiation of 32D IGF-IR cells, a murine hematopoietic cell line, expressing a human IGF-IR. Expression in 32D IGF-IR cells of a dominant negative mutant of Stat3 (DNStat3) inhibits IGF-I-mediated differentiation. DNStat3 causes a dramatic increase in Id2 gene expression. This increase, however, is IGF-I dependent and is abrogated by a mutation at tyrosine 950 of the IGF-IR. These results indicate that in 32D cells, the IGF-IR regulates the expression of the Id2 gene and that this regulation is modulated by both positive and negative signals. Our results also suggest that in this model, Id2 proteins influence the differentiation program of cells but are not sufficient for the full stimulation of their proliferation program.

Efficient in vitro and in vivo gene regulation of a retrovirally delivered pro-apoptotic factor under the control of the Drosophila HSP70 promoter.

We have developed a self-inactivating retroviral vector system with an internal, inducible Drosophila HSP70 promoter. This vector system delivers the desired transgene into cells rapidly and efficiently. It generates mixed populations of transduced cells where the transgene is inducible, and does not require the isolation of specific clones. Since the transgene is not expressed (or poorly expressed) at the restrictive condition (34 degrees C), mixed populations can be selected in which tumor suppressors or other inhibitory genes can be strongly induced upon changing the conditions (39 degrees C or the plant amino acid L-canavanine). This retroviral vector should be very useful for the expression of sequences that are poorly tolerated by cells, and is also active in animals.

Regulation of Id2 gene expression by the insulin-like growth factor I receptor requires signaling by phosphatidylinositol 3-kinase.

The Id proteins play an important role in proliferation, differentiation, and tumor development. We report here that Id gene expression can be regulated by the insulin-like growth factor I receptor (IGF-IR), a receptor that also participates in the regulation of cellular proliferation and differentiation. Specifically, we found that the IGF-IR activated by its ligand was a strong inducer of Id2 gene expression in 32D murine hemopoietic cells. This activation was not simply the result of cellular proliferation, as Id2 gene expression was higher in 32D cells stimulated by IGF-I than in cells exponentially growing in interleukin-3. The up-regulation of Id2 gene expression was largely dependent on the presence of insulin receptor substrate-1, a major substrate of the IGF-IR and a potent activator of the phosphatidylinositol 3-kinase (PI3K) pathway. The role of PI3K activity in the up-regulation of Id2 gene expression by the IGF-IR was confirmed by different methods and in different cell types. In 32D cells, the up-regulation of Id2 gene expression by the PI3K pathway correlated with interleukin-3 independence and inhibition of differentiation.

Retinoic acid induces neuronal differentiation of embryonal carcinoma cells by reducing proteasome-dependent proteolysis of the cyclin-dependent inhibitor p27.

Retinoic acid (RA) treatment of embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1) induces growth arrest and terminal differentiation along the neuronal pathway. In the present study, we provide a functional link between RA and p27 function in the control of neuronal differentiation in NT2/D1 cells. We report that RA enhances p27 expression, which results in increased association with cyclin E/cyclin-dependent kinase 2 complexes and suppression of their activity; however, antisense clones, which have greatly reduced RA-dependent p27 inducibility (NT2-p27AS), continue to synthesize DNA and are unable to differentiate properly in response to RA as determined by lack of neurite outgrowth and by the failure to modify surface antigens. As to the mechanism involved in RA-dependent p27 upregulation, our data support the concept that RA reduces p27 protein degradation through the ubiquitin/proteasome-dependent pathway. Taken together, these findings demonstrate that in embryonal carcinoma cells, p27 expression is required for growth arrest and proper neuronal differentiation.

Key role of the cyclin-dependent kinase inhibitor p27kip1 for embryonal carcinoma cell survival and differentiation.

Hexamethylen-bisacetamide (HMBA) represents the prototype of a group of hybrid polar compounds, which induce differentiation in a variety of transformed cells including human embryonal carcinoma cells. Therefore, HMBA has been used in the differentiation therapy of cancer for patients with both hematological and solid malignancies. Upon HMBA treatment, the embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1) accumulates in G1 and undergoes terminal differentiation. Here we demonstrate that growth arrest and differentiation of NT2/D1 cells induced by HMBA involve increased expression of the cyclin-dependent kinase inhibitor p27, enhanced association of p27 with cyclin E/CDK2 complexes and suppression of kinase activity associated to cyclin E/CDK2 (but not to cyclin D3/CDK4). When HMBA differentiation was induced in the presence of p27 antisense oligonucleotides, NT2/D1 cells failed to arrest growth properly and, in parallel with the reduction of the anti-apoptotic Bcl-2 gene expression, cells underwent massive programmed cell death. Conversely, constitutive expression of p27 into NT2/D1 cells induced a marked reduction in the growth potential of these cells and partially reproduced HMBA-induced modification of surface antigen expression (down-regulation of SSEA-3 expression and up-regulation of VINIS-53 expression). Expression of p21 induced growth arrest but not differentiation. Likewise, inhibition of CDK2 by transfection of a dominant negative CDK2 in NT2/D1 cells or treatment with the kinase inhibitor olomucine induced growth arrest but not differentiation. Therefore, we propose that p27 represents a crucial molecule in HMBA signaling that cannot be replaced by p21. Furthermore, the results obtained with CDK2 inhibitors demonstrate that the block of CDK2 activity is sufficient for growth arrest but not for cell differentiation and suggest that, at least in these cells, growth arrest and differentiation are regulated by two overlapping but different pathways.

Overexpressed cyclin D3 contributes to retaining the growth inhibitor p27 in the cytoplasm of thyroid tumor cells.

The majority of thyroid carcinomas maintain the expression of the cell growth suppressor p27, an inhibitor of cyclin-dependent kinase-2 (Cdk2). However, we find that 80% of p27-expressing tumors show an uncommon cytoplasmic localization of p27 protein, associated with high Cdk2 activity. To reproduce such a situation, a mutant p27 devoid of its COOH-terminal nuclear-localization signal was generated (p27-NLS). p27-NLS accumulates in the cytoplasm and fails to induce growth arrest in 2 different cell lines, indicating that cytoplasm-residing p27 is inactive as a growth inhibitor, presumably because it does not interact with nuclear Cdk2. Overexpression of cyclin D3 may account in part for p27 cytoplasmic localization. In thyroid tumors and cell lines, cyclin D3 expression was associated with cytoplasmic localization of p27. Moreover, expression of cyclin D3 in thyroid carcinoma cells induced cytoplasmic retention of cotransfected p27 and rescued p27-imposed growth arrest. Endogenous p27 also localized prevalently to the cytoplasm in normal thyrocytes engineered to stably overexpress cyclin D3 (PC-D3 cells). In these cells, cyclin D3 induced the formation of cytoplasmic p27-cyclin D3-Cdk complexes, which titrated p27 away from intranuclear complexes that contain cyclins A-E and Cdk2. Our results demonstrate a novel mechanism that may contribute to overcoming the p27 inhibitory threshold in transformed thyroid cells.

Modulation of in vivo growth of thyroid tumor-derived cell lines by sense and antisense vascular endothelial growth factor gene.

Vascular endothelial growth factor A (VEGF) is a potent mitogen for endothelial cells in vitro and promotes neo-angiogenesis in vivo. VEGF overexpression occurs in most human malignancies including thyroid carcinomas in which elevated VEGF expression is associated with a high tumorigenic potential. To investigate the role of VEGF in angiogenesis associated with development of thyroid carcinomas, we constitutively expressed VEGF121 into a poorly tumorigenic cell line (NPA) expressing minimal levels of endogenous VEGF. Here we report that VEGF overexpressing NPA cells showed the same growth potential as untransfected NPA in vitro but formed well-vascularized tumors when injected subcutaneously into nude mice with markedly reduced latency compared to parental cells. A complementary approach was to suppress VEGF expression in a highly tumorigenic anaplastic cell line (ARO) by the transfection of an antisense construct. Antisense-transfected ARO cells expressed reduced constitutive levels of VEGF, showed the same growth potential as untransfected ARO cells in vitro and formed small tumors characterized by minimal vascularization, extensive necrosis and longer latency compared to parental or vector-transfected ARO cells in vivo. Finally, we investigated the expression of both VEGF tyrosine kinase receptors (Flt-1 and Flk-1/KDR) in tumor specimens by RT - PCR. Expression of (Flt-1 and Flk-1/KDR) was low in tissue specimens derived from NPA tumors, but was found enhanced in NPA VEGF tumors; conversely, the expression of VEGF receptors was high in tissue specimens derived from ARO tumors but was decreased in tumors derived from VEGF depleted ARO cells. These results clearly demonstrate that VEGF indirectly promotes the growth of thyroid tumors by stimulating angiogenesis.

Regulation of thymosin beta10 expression by TSH and other mitogenic signals in the thyroid gland and in cultured thyrocytes.

OBJECTIVE: To investigate the expression of thymosin beta10 - a small conserved acidic protein involved in the inhibition of actin polymerization - in human and experimental thyroid goiters as well as the regulation exerted by TSH on thymosin beta10 expression in thyroid follicular cells both in vivo and in vitro. DESIGN: To this aim, we have used 5 bioptic specimens from patients affected by thyroid goiter, a well known experimental model of thyroid goitrogenesis (rat fed with the drug propylthiouracil) and a cultured rat thyroid cell line (PC Cl 3 cells) as a model system. RESULTS: We report that the mRNA expression of thymosin beta10 is markedly enhanced in human goiters compared with normal thyroid. In vivo results showed that the steady-state level of thymosin beta10 mRNA is up-regulated in the thyroid gland of propylthiouracil-fed rats in parallel with follicular cell proliferation: iodide administration to goitrous rats, which induced a marked involution of thyroid hyperplasia, reduced the mRNA level of thymosin beta10. Finally, in vitro studies showed that in cultured rat thyrocytes, the expression of thymosin beta10 mRNA is induced in a time- and dose-dependent manner by the activation of pathways which are mitogenic for thyroid cells (i.e. the protein kinase (PK) A and PKC pathways). CONCLUSION: Taken together, the findings reported here demonstrate that thymosin beta10 expression is regulated by extracellular signals that stimulate growth of thyroid cells both in vitro and in vivo, and suggest a role for this protein in thyroid diseases characterized by proliferation of follicular cells.

Protein tyrosine phosphatase-eta expression is upregulated by the PKA-dependent and is downregulated by the PKC-dependent pathways in thyroid cells.

We have recently reported the isolation of a rat cDNA encoding a receptor-type tyrosine phosphatase, which appears to be a marker of thyroid differentiation. To elucidate the molecular mechanisms underlying r-PTPeta expression in normal thyroid cells both in vitro and in vivo, we investigated the regulation of r-PTPeta expression in cultured thyrocytes (the rat cell line PC Cl 3) and in an animal model of TSH-dependent thyroid goitrogenesis. In vitro studies showed that mRNA expression of r-PTPeta in thyroid cells is induced in a time- and dose-dependent manner by the activation of growth- and differentiation-linked PKA pathways (TSH and forskolin), whereas it is down-regulated by the activation of the proliferative dedifferentiating PKC-dependent transduction pathway (TPA). However, the regulation of r-PTPeta expression by TSH and TPA, respectively, is observed only in normal thyroid cells, but is lost in transformed thyroid cells. In vivo studies with thiouracil-fed rats demonstrated that increased serum levels of TSH up-regulated r-PTPeta mRNA expression in parallel with the stimulation of thyroid growth and function. The reduction of blood TSH levels due to iodide refeeding to goitrous rats determined a marked down-regulation of r-PTPeta expression, in parallel with involution of thyroid hyperplasia. Taken together these results demonstrate that the phosphatase r-PTPeta is regulated by the two main thyroid regulatory pathways and suggest that it may play an important role in the growth and differentiation of thyroid cells.

The quantification of patch test responses: a comparison between echographic and colorimetric methods.

Patch testing is widely used both for clinical and experimental purposes. Although the clinical grading employed routinely is of practical value, the lack of objectivity makes it unsuitable for research purposes and dose-response analysis studies. Instrumental measuring techniques have been applied to patch test evaluation, because they enable objective quantification of different biophysical aspects of the inflammatory reaction by means of a continuous assessment scale, providing data suitable for statistical analysis. In order to compare the colorimetric and echographic methods for the evaluation of reactions of different intensity, we performed patch tests with 5% nickel sulfate on the flexor aspect of the forearm in 120 nickel-sensitive patients. Clinical and instrumental measurements were performed at 72 h. Numerical values corresponding to instrumental measurements were compared to the positivity degree, as assessed clinically. Whereas echographic parameters, expressing the intensity of oedema and inflammatory infiltration, enabled a distinction between +, +2 and +3 reactions, colorimetric a* values, describing erythema, failed to distinguish between +2 and +3 reactions. Thus, the use of ultrasound is advisable for the quantification of skin reactions of great intensity, whereas the colorimetric method could be usefully employed for dose-response studies assessing minimal eliciting concentrations of allergens, and for the evaluation of clinically undetectable reactions.

Objective assessment of the minimal irritant reaction induced by a 70% solution of glycolic acid.

BACKGROUND/AIMS: Glycolic acid has become a very popular cutaneous improving agent, but, despite its increasing employment, its biophysiological effects on the skin have not been adequately examined. Recently, we described subclinical skin damage induced by a 70% solution of glycolic acid applied on forearm skin by means of non-invasive measuring techniques. In the present study we instrumentally assessed the cutaneous effects induced by a glycolic acid minimal irritant dose on the volar aspect of the right forearm. METHODS: Twenty subjects, seventeen women and three men, aged 16 to 56, were tested with 40 nl of a 70% solution of glycolic acid applied on one volar forearm area until a clincally evident cutaneous reaction occurred. Visual scoring, TEWL measurement, skin colour, capacitance and echographic recordings were performed before the beginning of the experiment and 30 min and 3, 24, 48 and 72 h after glycolic acid removal. The echo-graphic recordings were carried out using a 20 MHz B scanner (Dermascan C), and then were processed by dedicated software based on segmentation procedures. Two amplitude intervals were selected for the elaboration of the images, the first marking the hyporeflecting areas of the dermis, the second highlighting both the hyperreflecting parts of the epidermis and the lower dermis, separately. RESULTS: Mean application-time before the appearance of minimal inflammatory skin reactions was 25±7 min. Clinical evaluation was confirmed by the analysis of the colorimetric parameter a*. TEWL and capacitance values showed a marked increase, reaching their maximum at 30 min after solution removal, followed by a progressive decrease. Values of both parameters were statistically significant for up to 48 h. The elaboration of echographic images by a low amplitude interval showed dermal edema more pronounced at 30 min and 24 h, while by highlighting hyperreflecting areas an evident reduction of the entry echo for up to 3 h and a prolonged decrease of the reflectivity of the lower dermis were observable. CONCLUSION: Our data demonstrate that despite the persistence of higher TEWL values, glycolic acid induced irritation causes a prolonged increase in capacitance that corresponds to modifications in epidermal reflectivity. Epidermal alterations are associated with dermal edema, as evaluated by ultrasound.