Publications by authors named "Giorgio Emanuelli"

19 Publications

  • Page 1 of 1

A Xenogenic Bone Derivative as a Potential Adjuvant for Bone Regeneration and Implant Osseointegration: An Study.

Tissue Eng Regen Med 2017 Jun 7;14(3):243-251. Epub 2017 Apr 7.

Department of Medical Sciences, University of Turin, Via Genova 3, 10126 Turin, Italy.

Several clinical conditions may limit the success of bone regeneration and/or implant osseointegration. For this reason, many compounds have been tested for their ability to stimulate this biological process. Synthetic hydroxyapatite (HA), mimicking natural bone hydroxyapatite, and extra-cellular matrix proteins, such as type I collagen, are potential candidates. However, the synthetic origin of HA and the denaturing conditions required for extracting collagen from skin and derma are sources of potential drawbacks. This study examines the effects of a natural bone derivative (NBD) extracted from equine bone and containing both natural, non-synthetic bone hydroxyapatite and native, non-denatured, type I bone collagen as a possible active compound for stimulating bone regeneration and implant osseointegration. The activity of NBD was tested on bone marrow stromal cells (BMSCs), evaluating their growth/viability by the methylthiazol tetrazolium (MTT) assay and their migration potential by a scratch assay. Moreover, expression of the hyaluronic acid receptor (CD44) and the C-X-C chemokine receptor type 4 (CXCR4, CD184) on the surface of BMSCs was assessed by flow cytometry, and the release of Transforming Growth Factor (TGF)-β, Interleukin (IL)-1α and IL-6 was quantified using an enzyme-linked immunosorbent assay (ELISA). The effect of NBD-coated implants on human osteoblasts was tested by measuring alkaline phosphatase (ALP) activity with the p-nitrophenyl phosphate (pNPP) degradation test. NBD stimulated BMSC growth/viability, migration, CD184 surface expression and the release of TGF-β1. NBD-coated implants increased ALP activity of human osteoblasts. These results indicate that NBD may be an adjuvant to accelerate both bone regeneration and osseointegration.
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http://dx.doi.org/10.1007/s13770-017-0029-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6171591PMC
June 2017

Loss of interstitial cells of Cajal network in severe idiopathic gastroparesis.

World J Gastroenterol 2006 Oct;12(38):6172-7

Department of Gastroenterology and Clinical Nutrition, University of Torino, Italy.

Aim: To report a case of severe idiopathic gastroparesis in complete absence of Kit-positive gastric interstitial cells of Cajal (ICC).

Methods: Gastric tissue from a patient with severe idiopathic gastroparesis unresponsive to medical treatment and requiring surgery was analyzed by conventional histology and immunohistochemistry.

Results: Gastric pacemaker cells expressing Kit receptor had completely disappeared while the local level of stem cell factor, the essential ligand for its development and maintenance, was increased. No signs of cell death were observed in the pacemaker region.

Conclusion: These results are consistent with the hypothesis that a lack of Kit expression may lead to impaired functioning of ICC. Total gastrectomy proves to be curative.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4088112PMC
http://dx.doi.org/10.3748/wjg.v12.i38.6172DOI Listing
October 2006

KIT/stem cell factor expression in premalignant and malignant lesions of the colon mucosa in relationship to disease progression and outcomes.

Int J Oncol 2006 Oct;29(4):851-9

Department of Clinical Physiopathology, University of Turin, I-10126 Torino, Italy.

Autocrine/paracrine stimulation of KIT has been observed in colorectal carcinoma (CRC) cell lines. We investigated the expression of KIT and stem cell factor (SCF) in CRC in comparison with premalignant colon lesions and normal colonic mucosa to assess the prognostic and therapeutic relevance of this receptor/ligand system in CRC. Transcript levels of c-kit and the two SCF splicing variants were determined quantitatively by real-time RT-PCR using cDNA obtained from normal, premalignant and malignant snap frozen colon tissue specimens. Immunohistochemistry with specific anti-KIT and anti-SCF antibodies was performed on paraffin-embedded tissue sections in order to localize the relative protein expression in epithelial compartments. Approximately 10% of patients expressed KIT in their adenoma or primary tumor. The majority of KIT-positive carcinomas co-expressed SCF. Real-time RT-PCR showed expression of c-kit and SCF transcripts in all cDNA specimens examined. A significant association between the co-expression of KIT/SCF and a worse clinical outcome was found. In conclusion, KIT expression was observed in a proportion of premalignant and malignant colonic lesions, while it was virtually absent in normal colon mucosa. Moreover, the majority of KIT-positive carcinomas co-expressed SCF, suggesting the possibility of aberrant signaling by an autocrine loop, as confirmed by the negative prognostic value of this association. Therefore, in the subset of CRC patients with concomitant KIT/SCF expression, the activity of Imatinib mesylate, a selective inhibitor of specific tyrosine kinases including KIT, may be exploited in combination with standard therapy.
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October 2006

Antagonistic interactions between gemcitabine and 5-fluorouracil in the human pancreatic carcinoma cell line Capan-2.

Cancer Biol Ther 2006 Oct 4;5(10):1294-303. Epub 2006 Oct 4.

Department of Clinical Physiopathology, University of Turin, and Department of Gastroenterology, San Giovanni Battista Hospital, Turin, Italy.

Although the recently-developed Gemcitabine (GEM) has renewed interest in clinical research in pancreatic carcinoma, it offers modest improvement of tumor-related symptoms and marginal survival advantage, even when combined with other currently-available chemotherapeutic agents such as 5-Fluorouracil (5-FU). We hypothesized that this disappointing result could be due to an interaction between the two drugs affecting cytotoxic activity. We measured in-vitro growth inhibition, cell cycle distribution, gene and protein expression of apoptosis regulators bcl-2, bcl-x and survivin, NFkappaB and telomerase activities of human pancreatic carcinoma cell line Capan-2 following exposure to GEM and 5-FU singly or combined, by MTT assay and median effect analysis, flow cytometry, real-time RT-PCR, Western blotting, electrophoretic mobility shift assay (EMSA) and telomeric repeat amplification protocol (TRAP) assay, respectively. We found cell growth to be inhibited by both drugs, decreasing the percentage of cells in S and G2/M phases and inducing apoptosis, dependent on the levels of bcl-2, bcl-xL and survivin expression in the case of 5-FU, but not for GEM. Moreover, while telomerase activity was reduced equally by both drugs, 5-FU but not GEM effectively downregulated NFkappaB binding activity. Intriguingly, a substantial antagonistic effect was noticed when GEM was combined with 5-FU in the concentration range tested, with the exception of the TRAP assay. These indications of an antagonistic interaction between GEM and 5-FU in some pancreatic cancer context urge further investigation of both genetic and non-genetic differences to identify the variables most relevant for optimal selection and dosing of treatment for the individual patient.
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http://dx.doi.org/10.4161/cbt.5.10.3152DOI Listing
October 2006

Cooperative induction of a tolerogenic dendritic cell phenotype by cytokines secreted by pancreatic carcinoma cells.

J Immunol 2006 Sep;177(5):3448-60

Department of Clinical Physiopathology, University of Turin, Italy.

Ag presentation by dendritic cells (DC) is essential to effective antitumor T cell responses in cancer patients. Depending on their origin, maturation state, and the ambient cytokine milieu, DC can differentiate into distinct subpopulations, which preferentially either induce Th1 cell activation (CD11c+,CD123- myeloid DC (MDC)) or immunosuppressive T cell development (CD11c-,CD123+ plasmacytoid DC (PDC)). The present study was undertaken to characterize the effects of pancreatic carcinoma cell-derived cytokines on immature monocyte-derived DC (iMo-DC) in vitro and in vivo. Medium conditioned by human pancreatic carcinoma cells inhibited iMo-DC proliferation, expression of costimulatory molecules (CD80 and CD40) and of HLA-DR, and functional activity as assessed by MLR and IL-12p70 production. iMo-DC generated from pancreatic carcinoma patients in advanced stages of the disease similarly showed decreased levels of HLA-DR expression and reduced ability to stimulate MLR in response to CD40L and IFN-gamma. Moreover, in tumor-patient peripheral blood, the ratio of MDC to PDC cells was lower than in healthy controls due to reduced numbers of MDC CD11c+ cells. Importantly, rather than a single cytokine, a combination of tumor-derived cytokines was responsible for these effects; these were primarily TGF-beta, IL-10, and IL-6, but not vascular endothelial growth factor. In summary, we have identified an array of pancreatic carcinoma-derived cytokines that cooperatively affect iMo-DC activation in a manner consistent with ineffective antitumor immune responses.
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http://dx.doi.org/10.4049/jimmunol.177.5.3448DOI Listing
September 2006

Botulinum toxin treatment of oesophageal achalasia in the old old and oldest old: a 1-year follow-up study.

Drugs Aging 2005 ;22(9):779-83

Department of Gastroenterology and Clinical Nutrition, University of Torino, Torino, Italy.

Background: Intrasphincteric injection of botulinum toxin (BTX) has become one of the most frequent therapeutic approaches for the treatment of oesophageal achalasia. This treatment seems particularly effective in elderly patients who are not candidates for more invasive procedures.

Aims: There are few or no data on BTX treatment of achalasia in the old old and oldest old. Therefore, we evaluated BTX treatment in a group of patients with achalasia in the extreme age range who were too ill or frail to undergo surgery or pneumatic dilatation.

Patients And Methods: Twelve elderly achalasic patients (age range 81-94 years, average age 86 years) with American Society of Anesthesiologists (ASA) class III-IV status were recruited for the study. After baseline clinical and instrumental evaluations, BTX 100U was injected at time 0 and 1 month later. Clinical follow-up was carried out after 3, 6 and 12 months.

Results: A significant improvement in symptom score was documented at each follow-up step. On the basis of improvements in scores, approximately 70% of patients were considered responders at the end of follow-up.

Conclusions: BTX treatment is an effective treatment in a substantial proportion of achalasic patients >80 years of age, in whom benefits are still detectable after 12 months. BTX is a therapeutic option in patients unsuitable for surgery or pneumatic dilatation.
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http://dx.doi.org/10.2165/00002512-200522090-00006DOI Listing
March 2006

Circulating vascular endothelial growth factor and interferon-gamma-inducible protein-10 levels in pancreatic cancer during chemotherapy.

Anticancer Res 2005 Sep-Oct;25(5):3287-91

Department of Clinical Physiopathology, University of Turin, Italy.

Background: Chemotherapeutic anticancer properties are thought to derive from apoptosis pathway activation and/or cell division arrest, but animal models have also evidenced anti-angiogenic activity in some agents.

Patients And Methods: The impact of gemcitabine, irinotecan and oxaliplatin + 5-FU upon the serum markers vascular endothelial growth factor (VEGF) (pro-angiogenic) and IFN-gamma-inducible protein (IP)-10 (anti-angiogenic) was evaluated by ELISA in locally advanced and/or metastatic cancer versus clinical efficacy and survival.

Results: Patients had higher serum levels of both markers versus controls. No objective response to therapy was observed and no significant difference in either marker occurred during the first month of chemotherapy; analysis by survival showed slight transient VEGF decrease in longer survivors on day 14 and slight increase on day 28 in shorter survivors, who had baseline median IP-10 levels above longer survivors, diverging on day 14 (decrease and increase, respectively). Both groups were below baseline at day 28. Changes in IP-10 were not significant.

Conclusion: These preliminary results provide a rationale for exploring whether continuous or frequent administration of some anti-neoplastic agents may elicit a global anti-angiogenic activity, and whether different administration schedules of the same drug could have a synergistic or an antagonistic effect, which obviously would need to be taken into account in determining combinations with new agents targeting angiogenesis.
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October 2005

Cytokine expression profile in human pancreatic carcinoma cells and in surgical specimens: implications for survival.

Cancer Immunol Immunother 2006 Jun 11;55(6):684-98. Epub 2005 Aug 11.

Department of Clinical Physiopathology, Università di Torino, Via Genova, 3, 10126 Torino, Italy.

Cytokine shedding by tumor cells into the local microenvironment modulates host immune response, tumor growth, and metastasis. The study aimed to verify the hypothesis that the immunological microenvironment of pancreatic carcinoma exists in a prevalently immunosuppressive state, influencing survival. We analyzed expression profiles of pro-inflammatory (IL-1beta, IL-2, IL-6, IL-8, IL-12 p40, IL-18 and IFN-gamma) and anti-inflammatory (IL-10, IL-11, IL-13 and TGF-beta isoforms) cytokines. The study was performed both in vitro, in five pancreatic carcinoma cell lines (real time RT-PCR), and in specimens from 65 patients, comparing tumoral versus non-tumoral pancreatic tissues (real time RT-PCR and immunohistochemistry). Furthermore, cytokines were measured in supernatants and sera (from patients and controls) by ELISA. All cell lines expressed IL-8, IL-18, TGF-beta1, TGF-beta2 and TGF-beta3, but not IFN-gamma and IL-2 transcripts. Expression of IL-1beta, IL-6, IL-10, IL-11, IL-13 and IL-12 mRNA was variable. All the above cytokines were detected as soluble proteins in supernatants, except IL-13. Tumor tissues overexpressed IL-1beta, IL-6, IL-8, IL-10, IL-11, IL-12 p40, IL-18, IFN-gamma, TGF-beta1, TGF-beta2 and TGF-beta3 at the mRNA level and IL-1beta, IL-18, TGF-beta2 and TGF-beta3 also at the protein level. Conversely, non-tumor tissues had stronger RNA and protein expression of IL-13. Survival was significantly longer in patients with high IL-1beta and IL-11 and moderate IL-12 expression. Serum IL-8, IL-10, IL-12, IL-18, TGF-beta1 and TGF-beta2 were higher in patients than in controls, as opposed to IL-1beta and IL-13. Patients with low circulating levels of IL-6, IL-18 and TGF-beta2 survived longer. Pancreatic cancer is characterized by peculiar cytokine expression patterns, associated with different survival probabilities.
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http://dx.doi.org/10.1007/s00262-005-0047-0DOI Listing
June 2006

Noncardiac chest pain of esophageal origin in patients with and without coronary artery disease.

Hepatogastroenterology 2005 May-Jun;52(63):792-5

Servizio di Endoscopia e Motilità Intestinale, Cattedra di Medicina Interna, Dipartimento di Fisiopatologia Clinica, Università di Torino, Italy.

Background/aims: Non-cardiac chest pain is a frequent finding in patients admitted to emergency departments, and it has been shown that many of these patients may have an esophageal cause for their pain. However, little data are available on patients primarily referred to the cardiology unit, and especially those with coronary artery disease. The purpose of this study was to assess the role of esophageal dysfunction in chest pain patients with and without coronary artery disease.

Methodology: Eighty-one patients referred from a cardiology unit for chest pain and no myocardial infarction entered the study. Sixty-one patients had no evidence of coronary artery disease, whereas 20 had coronary artery disease with chest pain at rest. After the cardiological evaluation, the patients underwent esophageal function testing by means of upper endoscopy, manometry, and 24-hour pH-monitoring.

Results: Overall, 10% of patients (2.5% in the coronary artery disease group) had evidence of endoscopic esophagitis, 46% of esophageal motor disorders (12% in the coronary artery disease group), and 10% abnormal pH-monitoring (1% in the coronary artery disease group).

Conclusions: We report that the esophagus might be responsible for non-cardiac chest pain in patients with and without coronary artery disease. In our experience, esophageal motor disorders, and not an increased acid reflux, are the abnormalities most commonly found in these patients.
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December 2005

Human pancreatic carcinoma cells secrete bioactive interleukin-18 after treatment with 5-fluorouracil: implications for anti-tumor immune response.

Cancer Biol Ther 2005 Feb 2;4(2):231-41. Epub 2005 Feb 2.

Department of Gastroenterology and Clinical Nutrition, Azienda Ospedaliera San Giovanni Battista, Molinette, Turin, Italy.

Recently we observed that pancreatic carcinoma cell lines constitutively express Interleukin-18 (IL-18). Bioactive IL-18 induces Interferon (IFN)-gamma production, Fas Ligand (FasL) expression, and inhibits angiogenesis, raising the issue of anti-tumor effects of a tumor-derived cytokine and motivating a more detailed analysis of IL-18 production in pancreatic carcinoma cells. This analysis included the study of effects of chemotherapeutic drugs (5-fluorouracil [5-FU], gemcitabine, cisplatin) commonly used in the treatment of pancreatic cancer patients on IL-18 production and processing. IL-18 expression and post-translational processing were determined using RT-PCR, immunoblot and ELISA in pancreatic carcinoma cell lines and in tumor tissue and serum samples from pancreatic carcinoma patients in the presence and absence of chemotherapeutic drugs. We describe expression of IL-18 in pancreatic carcinoma cells and tissues associated with significantly elevated IL-18 levels in patients sera. Specifically, Capan-2 pancreatic tumor cells produced and secreted precursor IL-18 with no apparent biological activity. However, the chemotherapeutic agent 5-FU, by inducing Caspase-1 and Caspase-3 activation, induced secretion of proteolytically processed mature and degraded IL-18 species, respectively, in Capan-2 cells. Conditioned medium from 5-FU-treated but not control Capan-2 cells induced IFN-gamma production by activated T cells in an IL-18-dependent manner. Furthermore, adjuvant polychemotherapy including 5-FU significantly increased serum levels of mature, bioactive IL-18 in pancreatic carcinoma patients. Treatment of pancreatic cancer cells with 5-FU induced Caspase-dependent processing of pro-IL18 leading to the secretion of biologically active IL-18. These findings delineate a novel mechanism by which chemotherapeutic agents may modulate local anti-tumor cell-mediated immune responses.
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http://dx.doi.org/10.4161/cbt.4.2.1476DOI Listing
February 2005

Ablation of phosphoinositide 3-kinase-gamma reduces the severity of acute pancreatitis.

Am J Pathol 2004 Dec;165(6):2003-11

Dipartimento di Fisiopatologia Clinica, Università di Torino, Via Genova 3, 10126 Torino, Italy.

In pancreatic acini, the G-protein-activated phosphoinositide 3-kinase-gamma (PI3K gamma) regulates several key pathological responses to cholecystokinin hyperstimulation in vitro. Thus, using mice lacking PI3K gamma, we studied the function of this enzyme in vivo in two different models of acute pancreatitis. The disease was induced by supramaximal concentrations of cerulein and by feeding mice a choline-deficient/ethionine-supplemented diet. Although the secretive function of isolated pancreatic acini was identical in mutant and control samples, in both models, genetic ablation of PI3K gamma significantly reduced the extent of acinar cell injury/necrosis. In agreement with a protective role of apoptosis in pancreatitis, PI3K gamma-deficient pancreata showed an increased number of apoptotic acinar cells, as determined by terminal dUTP nick-end labeling and caspase-3 activity. In addition, neutrophil infiltration within the pancreatic tissue was also reduced, suggesting a dual action of PI3K gamma, both in the triggering events within acinar cells and in the subsequent neutrophil recruitment and activation. Finally, the lethality of the choline-deficient/ethionine-supplemented diet-induced pancreatitis was significantly reduced in mice lacking PI3K gamma. Our results thus suggest that inhibition of PI3K gamma may be of therapeutic value in acute pancreatitis.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1618701PMC
http://dx.doi.org/10.1016/s0002-9440(10)63251-8DOI Listing
December 2004

Constitutive expression of interleukin-18 in head and neck squamous carcinoma cells.

Head Neck 2004 Jun;26(6):494-503

Division of Otorhinolaryngology, Department of Clinical Pgysiopathology, University of Torino, via Genova 3, 10126 Torino, Italy.

Background: Interleukin (IL)-18 is a potent immunomodulatory cytokine promoting TH-1 and cytotoxic immune responses through interferon (IFN)-gamma induction. The aim of this study was to investigate the production of IL-18 by squamous cell carcinoma of the head and neck (HNSCC).

Methods: The expression of IL-18 was analyzed by reverse transcriptase-polymerase chain reaction, Western blot, and ELISA in untreated and 5-fluorouracil (5-FU)-treated HNSCC cell lines. Immunohistochemical analysis was performed on tumor specimens from 16 patients with primary invasive HNSCC.

Results: We have demonstrated that HNSCC cell lines express IL-18 at the mRNA, as well as the protein, level. However, the IL-18 protein was expressed intracellularly and predominantly released as an unprocessed inactive 24-kDa form. After exposure to 5-FU, the processed form of IL-18 was detected in the supernatants of both HNSCC cell lines.

Conclusions: These results indicate that HNSCC cells are a potential source of IL-18 cytokine. The finding that the exposure to 5-FU can elicit its processing suggests a novel target for immunomodulatory intervention in patients with HNSCC.
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http://dx.doi.org/10.1002/hed.20011DOI Listing
June 2004

Inhibition of cell survival and invasive potential of colorectal carcinoma cells by the tyrosine kinase inhibitor STI571.

Cancer Biol Ther 2004 Apr 18;3(4):385-92. Epub 2004 Apr 18.

Department of Clinical Physiopathology, University of Torino, Torino, Italy.

Inhibiting tyrosine kinases has recently emerged as a therapeutic modality in several forms of neoplasia. The tyrosine kinase inhibitor STI571 (IMATINIB MESYLATE; GLEEVEC; GLIVEC) is a case in point as it has shown promise in the treatment of malignancies expressing the BCR/ABL fusion protein. In addition to BCR/ABL, STI571 inhibits the tyrosine kinase moieties of several cell surface receptors including the platelet-derived growth factor (PDGF) receptors and c-Kit. Previous work demonstrated that c-Kit activation supports migration, invasion and, survival of certain colorectal carcinoma cells including DLD-1. Here we describe that blocking c-Kit with STI571 inhibits these malignant traits not only in DLD-1 cells but also in two early passage colorectal carcinoma cell strains. Specifically, STI571 inhibited anchorage-independent colony formation and cell scattering in semi-solid medium. Furthermore, it enhanced apoptosis susceptibility and abrogated invasion of DLD-1 cells through Matrigel. In addition, STI571 treatment affected the balance of the Bcl-2 family of apoptosis regulators on favor of a pro-apoptotic phenotype. Specifically, STI571 treatment of DLD-1 cells was associated with lower levels of Bcl-2 expression accompanied by de novo expression of Bcl-xS. Finally, STI571 acted as a chemosensitizing agent in DLD-1 cells when used in combination with 5-fluorouracil.
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http://dx.doi.org/10.4161/cbt.3.4.727DOI Listing
April 2004

Long-term study on the effects of visual biofeedback and muscle training as a therapeutic modality in pelvic floor dyssynergia and slow-transit constipation.

Dis Colon Rectum 2004 Jan 2;47(1):90-5. Epub 2004 Jan 2.

Servizio di Endoscopia e Motilità Intestinale, Cattedra di Medicina Interna, Dipartimento di Fisiopatologia Clinica, Università di Torino, Torino, Italy.

Purpose: Biofeedback training has been shown as an effective therapeutic measure in patients with pelvic floor dyssynergia, at least in the short term. Long-term effects have received less attention. Moreover, its effects in patients with slow-transit constipation have been scarcely investigated. This study was designed to assess in an objective way the medium- and long-term effects of biofeedback and muscle training in patients with pelvic floor dyssynergia and slow-transit constipation.

Methods: Twenty-four patients (14 with pelvic floor dyssynergia and 10 with slow transit) meeting the Rome II criteria for constipation, and unresponsive to conventional treatments, entered the study. Clinical evaluation and anorectal manometry were performed basally and three months after a cycle of electromyographic biofeedback and muscle training; moreover, a clinical interview was obtained one year after biofeedback. Patients with slow-transit constipation also had colonic transit time reassessed at one year.

Results: Clinical variables (abdominal pain, straining, number of evacuations/week, use of laxatives) all significantly improved in both groups at three-month assessment; anorectal manometric variables remained unchanged, apart from a significant decrease of sensation threshold in the pelvic floor dyssynergia group and of the maximum rectal tolerable volume in the slow-transit constipation group. At one-year control, 50 percent of patients with pelvic floor dyssynergia still maintained a beneficial effect from biofeedback, whereas only 20 percent of those complaining of slow-transit constipation did so. Moreover, the latter displayed no improvement in colonic transit time.

Conclusions: In our experience, patients with pelvic floor dyssynergia are likely to have continued benefit from biofeedback training in the time course, whereas its effects on slow-transit constipation seems to be maximal in the short-term course.
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http://dx.doi.org/10.1007/s10350-003-0010-0DOI Listing
January 2004

Mechanisms of the priming effect of low doses of lipopoly-saccharides on leukocyte-dependent platelet aggregation in whole blood.

Thromb Haemost 2003 Nov;90(5):872-81

Department of Clinical Pathophysiology, University of Turin, Italy.

Several studies focused on the ability of bacterial lipopolysac-charides (LPS) in triggering platelet and/or leukocyte activation. The aim of this study was to investigate the molecular mechanisms involved in the aggregation of platelets and in their interaction with leukocytes in whole blood after stimulation with low doses of LPS. LPS did not directly induce platelet aggregation in whole blood, but they primed the aggregation of platelets induced by epinephrine, adenosine diphosphate and arachidonic acid. As shown by cytofluorimetry, platelets neither bind FITC-LPS, nor express the LPS-receptors CD14 and toll-like receptor 4 (TLR4). On the contrary, LPS primed monocytes and to a lesser extent polymorphonuclear neutrophils to adhere to platelets. Both platelet-leukocyte interaction and platelet aggregation in whole blood were inhibited by blockade of CD14 and TLR4. Moreover, the interaction between platelets and leukocytes was inhibited by P-selectin, and by blockade of PAF and reactive oxygen species, suggesting a role of P-selectin and of leukocyte-derived mediators. In conclusion, these results elucidate the mechanisms leading to platelet activation and interaction with leukocytes triggered by LPS. They suggest that the activation of platelets by LPS is mainly dependent on leukocytes and especially monocytes as a result of CD14 and TLR4 engagement. Moreover, we found that leukocyte-platelet interaction was triggered by the synthesis of PAF and the generation of oxygen radicals that induced upregulation of surface expression of P-selectin.
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http://dx.doi.org/10.1160/TH03-02-0085DOI Listing
November 2003

Decreased expression of stem cell factor in esophageal and gastric mucosa after esophagogastric anastomosis for cancer: potential relevance to motility.

Ann Surg Oncol 2003 Aug;10(7):801-9

General Surgery Section, University of Torino, Italy.

Background: Esophageal replacement with gastric tube is a well-established reconstruction of the alimentary tract after esophagectomy in cancer patients. The resulting molecular events in the transposed gastric tube and residual esophagus have yet to be investigated. Stem cell factor (SCF) was recently shown to be critical for signaling in gastrointestinal motility. SCF expression is here correlated with changes in mucosal morphology, acid and biliary reflux, and motility in the residual esophagus and gastric tube.

Methods: Thirteen patients surgically resected for squamous esophageal carcinoma with gastric tube replaced by esophagogastric anastomosis underwent upper endoscopy, esophageal manometry, 24-hour pH monitoring, and bile reflux detection. Esophageal and gastric mucosa samples were examined for SCF expression by immunohistochemical and semiquantitative reverse transcriptase-polymerase chain reaction analysis and for SCF serum levels by enzyme-linked immunosorbent assay.

Results: All patients showed severe residual esophagus hypoperistalsis and no gastric tube motor activity. The 24-hour pH monitoring was positive in most; 24-hour bile detection was mostly negative. SCF levels in the residual esophageal and gastric tube mucosa were dramatically decreased compared with those of normal subjects. The correlation between SCF and slow-wave activity was positive.

Conclusions: Hypomotility of the residual esophagus and gastric tube seems closely associated with disruption of the SCF/c-kit signaling pathway. However, the absence of notable relations between mucosal changes after chronic exposure to acid, biliary gastric content, and SCF expression indicates that this analysis cannot be considered part of endoscopic follow-up.
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http://dx.doi.org/10.1245/aso.2003.11.010DOI Listing
August 2003

Intra-plaque production of platelet-activating factor correlates with neoangiogenesis in human carotid atherosclerotic lesions.

Int J Mol Med 2003 Sep;12(3):327-34

Dipartimento di Fisiopatologia Clinica, Universitá degli Studi di Torino, Torino, Italy.

Platelet-activating factor (PAF) is a phospholipid mediator synthesized by activated inflammatory and endothelial cells. Recently PAF has been shown to contribute to neoangiogenesis in several experimental models. Here we evaluated the presence of PAF and its potential role in neovascularization within human atherosclerotic plaques. The amount of PAF extracted from 18 carotid plaques (266.65+/-40.07 pg/100 mg dry tissue; mean +/- SE) was significantly higher than that extracted from 18 normal arterial specimens (6 from carotid artery and 12 from aorta) (4.72+/-2.31 pg/100 mg dry tissue; mean +/- SE). The levels of PAF significantly correlated with the infiltration of CD68-positive monocytes and the extent of neovascularization, detected as von Willebrand Factor-positive cells. The amount of PAF also correlated with the area occupied by TNF-alpha-expressing cells. The absence of enhanced level of PAF in the circulation of atherosclerotic patients suggests a local production of this mediator within the plaque. The lipid extracts of atherosclerotic plaques containing high levels of PAF-bioactivity, but not those of control arteries, were angiogenic in a murine Matrigel model. WEB 2170, a specific PAF receptor antagonist, significantly prevented angiogenesis induced by the lipid extracts of atherosclerotic plaques. Our results indicate a local production of PAF within the atherosclerotic plaques and suggest that it may contribute to intra-plaque neoangiogenesis.
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September 2003

The membrane attack complex of complement contributes to plasmin-induced synthesis of platelet-activating factor by endothelial cells and neutrophils.

Immunology 2003 Aug;109(4):557-63

Departments of Clinical Pathophysiology and Internal Medicine, University of Turin, Turin, Italy.

Thrombolytic agents, used to restore blood flow to ischaemic tissues, activate several enzymatic systems with pro-inflammatory effects, thus potentially contributing to the pathogenesis of ischaemia-reperfusion injury. Platelet-activating factor (PAF), a phospholipid mediator of inflammation, has been implicated in the pathogenesis of this process. We previously showed that the infusion of streptokinase (SK) induces the intravascular release of PAF in patients with acute myocardial infarction (AMI), and that cultured human endothelial cells (EC) synthesized PAF in response to SK and plasmin (PLN). In the present study, we investigated the role of the membrane attack complex (MAC) of complement in the PLN-induced synthesis of PAF. In vivo, we showed a correlation between the levels of soluble terminal complement components (sC5b-9) and the concentrations of PAF detected in blood of patients with AMI infused with SK. In vitro both EC and polymorphonuclear neutrophils (PMN), incubated in the presence of PLN and normal human serum, showed an intense staining for the MAC neoepitope, while no staining was detected when they were incubated with PLN in the presence of heat-inactivated normal human serum. Moreover, the insertion of MAC on EC and PMN plasmamembrane elicited the synthesis of PAF. In conclusion, our results elucidate the mechanisms involved in PAF production during the activation of the fibrinolytic system, showing a role for complement products in this setting. The release of PAF may increase the inflammatory response, thus limiting the beneficial effects of thrombolytic therapy. Moreover, it may have a pathogenic role in other pathological conditions, such as transplant rejection, tumoral angiogenesis, and septic shock, where fibrinolysis is activated.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1783006PMC
http://dx.doi.org/10.1046/j.1365-2567.2003.01692.xDOI Listing
August 2003

Human leukocyte interferon-alpha treatment for chronic HCV-related hepatitis in hemophilic patients previously intolerant to other interferons-alpha.

Dig Dis Sci 2002 Feb;47(2):427-31

Department of Clinical Physiopathology, University of Turin, Italy.

Thirty patients affected by hemophilia A or B or von-Willebrand's disease and chronic posttransfusional active HCV hepatitis who developed major side effects during the course of a previous treatment with recombinant interferon-alpha (IFN-alpha) were studied. In all patients IFN-alpha therapy had to be discontinued and those who achieved a primary serologic and viral response to HCV relapsed within a few months. After a washout period, patients were retreated with human leukocyte IFN-alpha, 6 MU thrice weekly for 12 months. In about 90% of patients, a primary response, with normal AST and GGT values and undetectable HCV-RNA, was achieved within the third month of treatment and for the entire duration of treatment none of the patients had to discontinue therapy because of severe adverse reactions. During posttherapy follow-up only one patient relapsed. The human leukocyte IFN-alpha regimen looks to be very effective and safe for carriers of inherited clotting disorders who developed major side effects with recombinant IFN-alpha therapy for HCV-related chronic hepatitis.
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http://dx.doi.org/10.1023/a:1013798812144DOI Listing
February 2002