Surgery for ulcerative colitis complicated with colorectal cancer: when ileal pouch-anal anastomosis is the right choice.

Patients with ulcerative colitis (UC) are at risk of developing a colorectal cancer. The aim of this study was to examine our experience in the treatment of ulcerative Colitis Cancer (CC), the role of the ileal pouch-anal anastomosis (IPAA), and the clinical outcome of the operated patients. Data from 417 patients operated on for ulcerative colitis were reviewed. Fifty-two (12%) were found to have carcinoma of the colon (n = 43) or the rectum (n = 9). The indication to surgery, the histopathological type, the cancer stage, the type of surgery, the oncologic outcome, and the functional result of IPAA were examined. The majority of the patients had a mucinous or signet-ring carcinoma. An advanced stage (III or IV) was present in 28% of the patients. Early (stage I or II) CC was found in all except one patient submitted to surgery for high-grade dysplasia, low-grade dysplasia, or refractory colitis. Thirty-nine (75%) of the 52 patients underwent IPAA, 10 patients were treated with a total abdominal proctocolectomy with terminal ileostomy. IPAA was possible in 6/9 rectal CC. Cumulative survival rate 5 and 10 years after surgery was 61% and 53%, respectively. The survival rate was significantly lower for mucinous or signet-ring carcinomas than for other adenocarcinoma. No significant differences of the functional results and quality of life were observed between IPAA patients aged less than or more than 65 years. Failure of the pouch occurred in 5 of 39 (12.8%) patients for cancer of the pouch (2 pts) or for tumoral recurrence at the pelvic or peritoneal level. Early surgery must be considered every time dysplasia is discovered in patients affected by UC. The advanced tumoral stage and the mucous or signet-ring hystotype influence negatively the response to therapy and the survival after surgery. IPAA can be proposed in the majority of the patients with a functional result similar to that of UC patients not affected by CC. Failures of IPAA for peritoneal recurrence or metachronous cancer of the pouch can be observed when CC is advanced, moucinous, localized in the distal rectum, or is associated with primary sclerosing cholangitis.

New perspectives on the long-term outcome of segmental colectomy for Crohn's colitis: an observational study on 200 patients.

BACKGROUND AND AIMS: Surgical management of Crohn's colitis represents one of the most complex situations in colorectal surgery. Segmental colectomy (SC) and total abdominal colectomy with ileorectal anastomosis (TAC-IRA) are the most common procedures, but there are few available data on their long-term outcome. The aim of the present study was to analyze the long-term outcome of patients who underwent segmental colectomy for Crohn's colitis, with regard to the risk for total abdominal colectomy. METHODS: In this observational, monocentric, retrospective analysis, we analyzed patients who received a segmental colectomy for Crohn's colitis at our institution. The database was updated by asking patients to complete a questionnaire by telephone or at the outpatient clinic. Only patients followed up at our Hospital were included. Patients were followed up by a specialized multidisciplinary team (IBD Unit). The primary endpoint was the interval between segmental colectomy and, when performed, total abdominal colectomy. RESULTS: Between 1973 and 2014, 200 patients underwent segmental colectomy for Crohn's colitis. The median follow-up was 13.5 years (interquartile range [IQR] 7.8-21.5). Overall, 62 patients (31%) had a surgical recurrence, of these, 42 (21%) received total abdominal colectomy. At multivariate analysis, the presence of ≥ 3 sites (HR = 2.47; 95% CI 1.22-5.00; p = 0.018) and perianal disease (HR = 3.23; 95% CI 1.29-8.07; p = 0.006) proved to be risk factors for total abdominal colectomy. CONCLUSIONS: The risk for surgical recurrence after SC for Crohn's colitis is acceptable. We recommend a bowel-sparing policy for the treatment of Crohn's colitis in any case in which the extent of the disease at the moment of surgery makes the conservative approach achievable.

Rationalisation of the surgical technique for minimally invasive laparoscopic ileal pouch-anal anastomosis after previous total colectomy for ulcerative colitis.

INTRODUCTION: No previous study clearly focuses on laparoscopic technique to perform the second stage surgery (proctectomy with ileal pouch-anal anastomosis [IPAA]) after total colectomy for acute/severe ulcerative colitis (UC). We describe the procedural steps for a simple and rational minimally invasive second stage surgery, reporting intra- and short-term post-operative results. PATIENTS AND METHODS: During the period December 2014-December 2015, 10 consecutive patients (8 males and 2 females) with mean age of 48 years underwent laparoscopic proctectomy and IPAA adopting our novel approach. They were operated 3 months after the previous total colectomy which has been performed, respectively, for acute (three patients) or severe (seven patients) UC. Intraoperative data and post-operative complications, divided as minor and major, were recorded and analysed. A body image questionnaire was administered to all patients to evaluate the cosmetic results of the procedure. RESULTS: Overall mean surgical time was 235 ± 49 min. During the post-operative course, three patients required morphine for >48 h, no patient needed blood transfusion and bowel movements recovery happened as mean during the 2nd day. No early major complications happened. Two patients (20%) developed peri-ileostomic wound infection at the right flank. Only one patient (10%) suffered from ileal-anal anastomotic dehiscence, conservatively treated till resolution. The average length of hospital stay was 8 ± 2 days. The body image questionnaire showed in all patients an extreme satisfaction about the results obtained (mean value = 59/64 points). CONCLUSIONS: Through three standardised surgical steps easily reproducible, we describe an almost scar-less procedure able to optimise the intraoperative time with good post-operative results in terms of complications and cosmesis.

Results of Surgical Salvage Treatment for Anal Canal Cancer: A Retrospective Analysis with Overview of the Literature.

BACKGROUND AND AIM: Chemoradiotherapy (CRT) is the gold standard treatment for anal cancer, which permits the maintenance of the anal function. However, about 30-40% of patients develop local disease progression, for which surgery represents a good salvage therapy. The aim of this study is to evaluate survival and morbidity rate in patients who undergo salvage surgery in our single institution, with an overview of the literature. METHODS: A retrospective study was carried out on patients who underwent surgical treatment of anal canal cancer after failure of CRT. We evaluated overall survival at 1, 3, and 5 years and postoperative morbidity rate. RESULTS: Twenty patients who underwent radical surgery with abdominoperineal resection were included in the study. The survival rates at 1, 3, and 5 years were 75, 60, and 37.4%; with a disease-free survival of 67, 53, and 35%, respectively. There was no postoperative mortality. The morbidity rate was 35%. CONCLUSION: Surgery represents the recommended therapy for persistent or recurrent anal canal cancer after CRT, with a good survival rate and an acceptable morbidity.

Results of surgical treatment for jejunal Crohn's disease: choice between resection, strictureplasty, and combined treatment.

PURPOSE: The jejunum is a relatively uncommon localization of Crohn's disease (CD) compared to the terminal ileum or the large bowel. The type of surgery and the short and long-term results after surgery have not been extensively investigated. A review of patients who underwent surgery for jejunal CD in our center was examined. METHODS: Between 1986 and 2011, 110 patients underwent surgery for jejunal CD. Thirty patients (27.3 %) were surgically treated with resection, 29 patients (26.4 %) with one or more strictureplasties, and 51 patients (46.3 %) with both a resection and one or more strictureplasties. RESULTS: There was no statistical difference in terms of short-term postoperative complications between patients operated with three different options (p = 0.72). Patients were followed up for a period ranging from 2 to 18 years (mean 11 years). During this period, 42 patients had no CD recurrence, 11 patients had medical recurrences only, while 57 patients had surgical recurrences, too. There was no statistical difference in terms of medical and surgical recurrence between the three types of surgical procedures employed (p = 0.24) and between smokers and non-smokers. The recurrent CD was prevalently treated with strictureplasty. CONCLUSIONS: The most frequently employed surgery for the treatment of jejunal CD is a combined type of treatment, i.e., resection of the most affected tract and strictureplasty of the residual strictures. This approach does not imply an increased risk of postoperative complications and recurrence and can reduce the risk of the short bowel syndrome.

Could Total Colectomy with Ileorectal Anastomosis Be an Alternative to Total Proctocolectomy with Ileal Pouch-Anal Anastomosis in Selected Ulcerative Colitis Patients?

Purpose. To evaluate ileorectal anastomosis (IRA) in selected ulcerative colitis patients. Methods. Early and late complications after IRA and IPAA were investigated. Bowel function and quality of life were assessed. Functional and QoL studies were performed as a matched pair analysis, comparing 98 patients who underwent IRA versus 98 patients who underwent IPAA. Results. In IRA group, 2 patients (1.6%) developed anastomotic l dysplasia (HGD) developed in 3 patients dysplasia (HGD) developed in 3 patients eakage, 1 patient (0.8%) had intestinal obstruction, and 2 patients (1.6%) had abdominal hematoma. Mean follow-up was 11.5 (range: 2-24.3) years. Failure of IRA occurred in 19 patients (15.1%); in 12 patients (9.5%), failure was related to severe proctitis, in 3 patients (2.4%), it was related to the development of high-grade dysplasia, and in 4 patients (3.2%), it was related to the development of rectal cancer. About functional results, stool consistency [liquid (6.7% of IRA patients versus 29% of IPAA patients; p = 0.003)], daily soiling (0% versus 6%; p = 0.01), and nocturnal soiling (6% versus 25.5%; p = 0.03) were statistically different. Only 1% of IRA patients versus 11% of IPAA patients had episodes of perianal inflammation (p = 0.007). CGQoL was 0.72 (±0.14, SD) in IRA patients and 0.75 (±0.11, SD) in IPAA patients (p = ns). Conclusion. In selected patients, IRA is an appropriate surgical option, with low morbidity, comparable quality of life, and better functional results than IPAA.

Outcome after ileal pouch-anal anastomosis in ulcerative colitis patients: experience during a 27-year period.

BACKGROUND: No previous study describes the postoperative outcome and functional results after ileal pouch-anal anastomosis (IPAA), performed in ulcerative colitis by the same surgical team with the different anastomotic techniques adopted in a 27-year period. METHODS: Prospectively, consecutive 333 ulcerative colitis patients operated adopting different IPAA techniques during the open surgery period 1984-2011 were enrolled. IPAA was performed using single stapling (SS) technique in 38 patients, double stapling (DS) technique in 235 patients (TIA stapler 42 patients, Endo-GIA 131 patients, Contour 62 patients) and handsewn IPAA in 60 patients. RESULTS: Statistically different early and late complications were recorded among the different IPAA techniques. A lower frequency of daily and nocturnal defecations and a higher level of continence were observed in the DS-IPAA compared to handsewn IPAA. The distance between the anastomotic line and the anal verge was significantly lower in DS Endo-GIA or DS Contour groups than in the DS TIA and SS. In SS IPAA group, 31.6% developed cuffitis compared to 14.4% belonging to DS group, at a mean follow-up of 140.4 months. CONCLUSIONS: Technical improvements changed the IPAA technique. Stapled IPAA is characterized by better functional outcome than handsewn IPAA. DS Endo-GIA and Contour IPAA are followed by lower risk of cuffitis.

Effect of butyrate enemas on gene expression profiles and endoscopic/histopathological scores of diverted colorectal mucosa: A randomized trial.

BACKGROUND: A temporary stoma is often created to protect a distal anastomosis in colorectal surgery. Short-chain fatty acids, mainly butyrate, are the major fuel source for the epithelium and their absence in the diverted tract may produce mucosal atrophy and inflammation. AIMS: To investigate whether the administration of sodium butyrate enemas (Naburen(©), Promefarm, Italy) could prevent mucosal inflammation and atrophy and affect gene expression profiles after ileo/colostomy. METHODS: We performed a randomized, double-blind, placebo-controlled clinical trial, in patients with enterostomy performed for inflammatory bowel disease, colorectal cancer or diverticulitis. Twenty patients were randomly allocated to receive 30ml of sodium butyrate 600mmol/L (group A) or saline (group B), b.i.d. for 30 days. RESULTS: In group A endoscopic scores were significantly improved (p<0.01) while mucosal atrophy was reduced or unchanged; in group B mucosal atrophy was increased in 42.8% of patients. Despite the high dose of butyrate used, no short-chain fatty acids were detectable by gas chromatography-mass spectrometry in colorectal biopsies. Group A patients showed up-regulation of genes associated with mucosal repair such as Wnt signalling, cytoskeleton regulation and bone morphogenetic protein-antagonists. CONCLUSION: Butyrate enemas may prevent the atrophy of the diverted colon/rectum, thus improving the recovery of tissue integrity.

Surgical treatment of chagasic megacolon with Duhamel-Habr-Gama technique modulated by frozen-section examination.

BACKGROUND: Migration from Latin American countries has increased the number of cases of chagasic megacolon in Western countries. Megacolon is a late complication of Chagas disease, resulting from irreversible destruction of the intramural intestinal nervous system with extensive loss of neurons, ganglionitis, and myositis at the sites of the myenteric and submucosal plexuses. Several surgical procedures involving partial or total resection of the dilated colon have been proposed for treating chagasic megacolon, but intra-operative evaluation of neuronal degeneration in the residual colon has not been commonly done. METHODS: Case report and literature review. CASE REPORT: We describe a case of chagasic megacolon treated successfully with a modified Habr-Gama technique, with the intra-operative examination of frozen sections of the residual segments of the colon for the presence of neurons. CONCLUSIONS: The recurrence of constipation after surgical treatment can result from the progression of chagasic neuronal degeneration in the preserved colon, and may be preventable by intra-operative evaluation of whether signs of neuronal degeneration or inflammation are absent in the anastomosed colonic tract.

A multidisciplinary approach to desmoid tumors. When intra-abdominal fibromatosis degenerates into an abscess, which is the right treatment?

INTRODUCTION: Desmoid tumors are rare benign tumors that originates in the fibrous sheath or musculo-aponeurotic structure. Histologically benign, they tends to invade locally and to be recurrent. PRESENTATION OF CASE: We report a rare case of an intra-abdominal desmoid tumor in a patient affected by familial adenomatous polyposis, which degenerated into abscess. Male, 38 years, was hospitalized for abdominal pain, bowel obstruction and fever. The computed tomography showed a big dishomogeneous mass occupying the whole mesentery with internal massive liquefaction. The mass extended from the epigastrium for 13cm up to L3. On the right mesogastric side a solid, thick mass of about 2cm, with a length of 4.5cm, was identified; it was not cleavable from the wall and from some of the loops. We decided to perform a computed tomography-guided percutaneous drainage. Two hundred ml of purulent necrotic material was aspirated, and washing with antibiotic solution was carried out. Cytological examination of fluid drainage showed histiocytes and neutrophils. At follow-up, the patient's clinical condition had improved. An abdominal ultrasound showed a substantial reduction in the diameter of the mass. DISCUSSION: Diagnosis and treatment of desmoids tumor in patients with familial adenomatous polyposis may be difficult, especially when desmoids are located intra-abdominally and in the mesentery. Seldom will desmoid tumors be complicated by abscess formation. CONCLUSION: The management of desmoids tumors is not easy and the choice of the best treatment may be difficult due to the different possible anatomical presentations.

Colorectal cancer and Crohn's colitis: clinical implications from 313 surgical patients.

BACKGROUND: The relation between Crohn's colitis (CC) and colorectal cancer is still controversial. Several case reports and retrospective studies have shown that patients with Crohn's disease (CD) have a 6- to 20-fold higher risk to develop CRC than does the normal population. The extent of disease (extensive colitis), presence of anal fistula, age > 40 years, strictures, and length of disease >10 years may be important determinants for increasing risk. Despite this evidence, other population-based studies have shown no increased risk of colon or rectal cancer. The aim of this study was to investigate retrospectively factors that may predict the development of cancer. METHODS: We searched the histopathologic database of the Digestive Surgery Unit at Careggi University Hospital for CC patients (January 1987 to September 2011) and identified 313 patients with CC who underwent surgery. RESULTS: There are 11 (3.5 %) of adenocarcinomas. Multivariate analysis showed disease duration (p = 0.001), age at CD diagnosis (p = 0.002), distal localization (p = 0.045), and penetrating disease (p = 0.041) to be risk factors. Multivariate analysis showed that 40 patients who had undergone previous immunosuppressive therapy had a significant risk of developing CRC (p = 0.026). CONCLUSIONS: Crohn's colitis patients who require surgery are at higher risk for developing CRC, particularly those whose disease duration is >10 years, have distal localization, age at diagnosis was <40 years, and have penetrating disease. Previous immunosuppressive therapy should be better investigated. We recommend surgery for any patient presenting with colonic strictures.

Dermal fibroblasts display similar phenotypic and differentiation capacity to fat-derived mesenchymal stem cells, but differ in anti-inflammatory and angiogenic potential.

BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent stem cells able to differentiate into different cell lineages. However, MSCs represent a subpopulation of a more complex cell composition of stroma cells contained in mesenchymal tissue. Due to a lack of specific markers, it is difficult to distinguish MSCs from other more mature stromal cells such as fibroblasts, which, conversely, are abundant in mesenchymal tissue. In order to find more distinguishing features between MSCs and fibroblasts, we studied the phenotypic and functional features of human adipose-derived MSCs (AD-MSCs) side by side with normal human dermal fibroblasts (HNDFs) in vitro METHODS: AD-MSCs and HNDFs were cultured, expanded and phenotypically characterized by flow cytometry (FC). Immunofluorescence was used to investigate cell differentiation. ELISA assay was used to quantify angiogenic factors and chemokines release. Cultures of endothelial cells (ECs) and a monocyte cell line, U937, were used to test angiogenic and anti-inflammatory properties. RESULTS: Cultured AD-MSCs and HNDFs display similar morphological appearance, growth rate, and phenotypic profile. They both expressed typical mesenchymal markers-CD90, CD29, CD44, CD105 and to a minor extent, the adhesion molecules CD54, CD56, CD106 and CD166. They were negative for the stem cell markers CD34, CD146, CD133, CD117. Only aldehyde dehydrogenase (ALDH) was expressed. Neither AD-MSCs nor HNDFs differed in their multi-lineage differentiation capacity; they both differentiated into osteoblast, adipocyte, and also into cardiomyocyte-like cells. In contrast, AD-MSCs, but not HNDFs, displayed strong angiogenic and anti-inflammatory activity. AD-MSCs released significant amounts of VEGF, HGF and Angiopoietins and their conditioned medium (CM) stimulated ECs proliferation and tube formations. In addition, CM-derived AD-MSCs (AD-MSCs-CM) inhibited adhesion molecules expression on U937 and release of RANTES and MCP-1. Finally, after priming with TNFα, AD-MSCs enhanced their anti-inflammatory potential; while HNDFs acquired pro-inflammatory activity. CONCLUSIONS: AD-MSCs cannot be distinguished from HNDFs in vitro by evaluating their phenotypic profile or differentiation potential, but only through the analysis of their anti-inflammatory and angiogenic properties. These results underline the importance of evaluating the angiogenic and anti-inflammatory features of MSCs preparation. Their priming with inflammatory cytokines prior to transplantation may improve their efficacy in cell-based therapies for tissue regeneration.

Ileocecal strictureplasty for Crohn's disease: long-term results and comparison with ileocecal resection.

BACKGROUND: The aim of the present study was to evaluate strictureplasty as the first choice for surgical treatment for Crohn's terminal ileitis. METHODS: Between 1996 and 2000 we performed Finney-shaped ileocecal strictureplasty (ICS), doubling up the diseased terminal ileum, in 14 patients affected by Crohn's disease (group A). We compared the postoperative and long-term outcomes of these patients with those of 14 similar patients who underwent ileocecal resection with ileocolonic anastomosis during the same period (group B). RESULTS: No postoperative morbidity or mortality was recorded in group A, whereas two patients of group B had a pelvic hematoma and cholestatic hepatopathy, respectively. The mean hospital stay after surgery was 9.9 days (range 7-13 days) in group A and 7.4 days (range 6-10 days) in group B. After a median follow-up of 120 months (range 103-147 months), five patients of group A had a symptomatic recurrence: A stricture at the site of the ICS was present in four of them, but only one required surgery; symptoms were controlled by medical therapy in the other three. The fifth symptomatic patient was reoperated for multiple jejunoileal recurrence of the disease above the ICS. Group B patients have been followed for a mean of 108 months (range 90-140 months). Four of the patients had a preanastomotic recurrence, with subocclusive symptoms and mild malnutrition treated with medical therapy. CONCLUSIONS: Our results indicate that there are no significant differences between ICS and resection in terms of outcome and clinical relapse of Crohn's terminal ileitis.

Susceptibilidad in vitro de candida spp a caspofungina y anfotericina B por los métodos de microdilución en caldo (CLSI), etest y el sistema vitek2. Estudio Comparativo / In vitro susceptibility of candida spp to caspofungin and amphotericin B by broth microdilution methods (CLSI), etest and the vitek2 system. Comparative study

El objetivo de este estudio fue evaluar la susceptibilidad de Candida spp., a caspofungina (CS) y anfotericina B (AMB) por los métodos de microdilución en caldo (CLSI), Etest y sistema automatizado Vitek 2. Se determinó la Concentración Mínima Inhibitoria (CMI) de 208 aislados de Candida spp., obtenidos de pacientes con candidemia, a caspofungina por microdilución en caldo (documento M27-A3 del CLSI) e Etest y a anfotericina B por Etest y Vitek2. Las CMI para Etest y CLSI se leyeron visualmente tras 24 h de incubación a 35°C. El punto final para Vitek 2 fue determinado por el equipo. El punto final de CMI para CS se determinó como la menor concentración que produjo disminución significativa (≥50%) de crecimiento con respecto al control. El punto final para AMB se leyó como la menor concentración que produjo inhibición total (100%) de crecimiento. Se utilizaron las cepas C.parapsilosis ATCC 22019 y C.krusei ATCC 6258 como control de calidad, de acuerdo a las recomendaciones del CLSI. Los métodos Etest y Vitek 2 fueron capaces de detectar aislados susceptibles en todas las cepas evaluadas. Las CMI estuvieron dentro de los valores esperados. Los resultados mostraron buena concordancia esencial (98%) entre microdilución en caldo y Etest y entre Etest y Vitek (65%), Por lo tanto se recomienda su uso en la evaluación de susceptibilidad in vitro a caspofungina y anfotericina B como método alternativo para los laboratorios asistenciales. Se requieren estudios adicionales para verificar la habilidad de Vitek 2 en la identificación de cepas resistentes a AMB y CS

The aim of the study was to test the susceptibility of Candida spp., against caspofungina and amphotericin B with CLSI broth microdilution (BMD), Etest and an automated system Vitek2. We determined the Minimal Inhibitory Concentration (MIC) from 208 Candida spp. isolates recovered from candidemia patients, to caspofungina and amphotericin B, with broth microdilution (M27-A3 CLSI document), Etest and Vitek. BMD and Etest MICs were read visually after 24 h incubation at 35 ºC. Vitek2 endpoints were determined spectrophotometrically by automated components of this equipment. Caspofungina MIC endpoint was determined as the lowest concentration that caused a significant diminution ≥50% of growth below the growth control. Amphotericin B MIC endpoint was read as the lowest concentration that produced the complete inhibition (100%) of growth. Quality control was performed by testing CLSI recommended strains C. parapsilosis ATCC 22019 and C. krusei ATCC 6258. Etest and Vitek methods were able to detect susceptible isolates in all strains tested. MICs were within expected values. Values of essential agreement were good: 98% between CLSI and Etest and 65% between Vitek and Etest. Therefore, they are recommended to test in vitro susceptibility to caspofungina and amphotericin B as a reliable alternative method for using in clinical laboratories. More studies are necessary to verify Vitek´s 2 ability to identify resistance isolates to amphotericin B.

Omentum-derived stromal cells improve myocardial regeneration in pig post-infarcted heart through a potent paracrine mechanism.

Cell-based therapy could be a valid option to treat myocardial infarct (MI). Adipose-derived stromal cells (ADStCs) have demonstrated tissue regenerative potential including cardiomyogenesis. Omentum is an extremely rich source of visceral fat and its accumulation seems to correlate with cardiovascular diseases. We investigated the capacity of human fat Omentum-derived StCs (FOStCs) to affect heart function upon acute infarct in pigs induced by permanent ligation of the anterior interventricular artery (IVA). We demonstrated for the first time that the local injection of 50x10(6) of FOStCs ameliorates the functional parameters of post-infarct heart. Most importantly, histology of FOStCs treated hearts demonstrated a substantial improvement of cardiomyogenesis. In culture, FOStCs produced an impressive number and amount of angiogenic factors and cytokines. Moreover, the conditioned medium of FOStCs (FOStCs-CM) stimulates in vitro cardiac endothelial cells (ECs) proliferation and vascular morphogenesis and inhibits monocytes, EC activation and cardiomyocyte apoptosis. Since FOStCs in vivo did not trans-differentiate into cardiomyocyte-like cells, we conclude that FOStCs efficacy was presumably mediated by a potent paracrine mechanism involving molecules that concomitantly improved angiogenesis, reduced inflammation and prevented cardiomyocytes death. Our results highlight for the first time the important role that human FOStCs may have in cardiac regeneration.

Vitamin E-modified filters modulate Jun N-terminal kinase activation in peripheral blood mononuclear cells.

BACKGROUND: The generation during hemodialysis of activated complement fragments and reactive oxygen species, including nitric oxide (NO), may affect peripheral blood mononuclear cell (PBMC) function. Currently, little is known about signal transduction pathways involved in PBMC activation. Jun N-terminal kinase (JNK) is a novel mitogen-activated protein (MAP) kinase phosphorylated and activated in response to oxidative stress and directly involved in cell activation. METHODS: The present study evaluated the activation of JNK in PBMCs isolated from eight uremic patients undergoing, in a randomized manner, three month-subsequent periods of hemodialysis with a low-flux cellulose acetate (CA) and a vitamin E-modified cellulose membrane (CL-E). After each period of treatment, PBMCs were harvested before (T0), during (T15) and after three hours (T180) of dialysis. At the indicated time points, plasma C5b-9 generation by ELISA and inducible NO synthase (iNOS) gene expression by in situ hybridization were evaluated also. The activation of JNK was studied by Western blotting using a specific monoclonal anti-phospho-JNK antibody, which recognizes the activated form of JNK. RESULTS: At T0, a significant increase in plasma C5b-9 levels was found in CA patients compared to CL-E-treated patients. During hemodialysis, C5b-9 levels rose more significantly in CA patients than in CL-E patients and returned to baseline values only in CL-E patients. At the same time, in CA patients an increased iNOS gene expression was observed at T180 together with a striking activation of JNK. By contrast, PBMC from CL-E-treated patients showed undetectable levels of phospho-JNK and a significant reduction in iNOS expression. Interestingly, incubation of PBMCs with normal human plasma (10%), activated by contact with a cellulosic membrane, induced a time-dependent increase in JNK phosphorylation that was completely inhibited by blocking complement cascade activation. CONCLUSION: Our data suggest that JNK phosphorylation is strikingly increased in PBMCs obtained from CA-treated patients and may represent a key cellular event in PBMC activation during dialysis with bioincompatible membranes. The activation of this signaling enzyme, mediated by active complement fragments and PBMC-dialyzer interaction, can be significantly reduced by the use of vitamin E-coated membrane.

Activated coagulation factor X: a novel mitogenic stimulus for human mesangial cells.

Intraglomerular activation of the coagulation cascade is a common feature of mesangioproliferative glomerulonephritis. Besides thrombin, very little is known about the cellular effects of other components of the coagulation system. This study investigated the effect of activated factor X (FXa) on cultured human mesangial cells. This serine protease induced a significant and dose-dependent increase in DNA synthesis. In addition to its mitogenic effect, FXa caused a striking upregulation of platelet-derived growth factor (PDGF) A and B chain gene expression. Next, the intracellular mitogenic signaling pathways activated by FXa were investigated. FXa induced a rapid spike in cytosolic calcium concentration followed by a sustained plateau. This response was not influenced by the downregulation of thrombin receptors. In addition, FXa stimulated a significant upregulation of different tyrosine-phosphorylated proteins. One of these phosphorylated cellular proteins was represented by the c-jun N-terminal kinase, a member of the mitogen-activated protein kinase family. To evaluate the role of FXa enzymatic activity and of PDGF autocrine secretion, FXa-induced DNA synthesis was studied in the presence of leupeptin, a specific serine protease inhibitor, and neutralizing anti-PDGF antibody. To investigate the role of tyrosine kinase (TK) activation on FXa mitogenic effect, FXa-stimulated thymidine uptake was evaluated in the presence of genistein and herbimycin A, two powerful and specific TK inhibitors. FXa-elicited DNA synthesis was also examined after protein kinase C (PKC) downregulation by prolonged incubation with phorbol-12-myristate-13-acetate to study the influence of the phospholipase C-PKC axis. The proliferative effect of FXa required its proteolytic activity, and the activation of TK was only partially dependent on PKC activation while it was PDGF independent. Finally, it was shown by reverse transcription-PCR that mesangial cells do not express the signaling splicing variant of the putative FXa receptor, effector protease receptor-1. In conclusion, the present study demonstrated that FXa is a powerful mitogenic factor for human mesangial cells, and it induces its cellular effect not through effector protease receptor-1, but most likely by binding a protease-activated receptor and activating phospholipase C-PKC and TK signaling pathways.

Regenerative and proinflammatory effects of thrombin on human proximal tubular cells.

Interstitial fibrin deposition is a common histologic feature of tubulointerstitial diseases, which suggests that the coagulation system is activated. Thrombin, generated during the activation of the coagulation cascade, is a powerful activating factor for different cell types. Although proximal tubular cells are potential targets for this coagulation factor, no information is available on the effect of thrombin on these cells. Thus, the expression of protease-activated receptor-1 (PAR-1), the main thrombin receptor, was investigated in human proximal tubular cells (hPTC) in vivo and in vitro. A diffuse expression of PAR-1 was observed by immunohistochemistry along the basolateral membrane of PTC in normal human kidney. This observation was confirmed in vitro in cultured hPTC. Because tubular damage and monocyte infiltration are two hallmarks of tubulointerstitial injury, the effect of thrombin on DNA synthesis and monocyte chemotactic peptide-1 (MCP-1) gene and protein expression was evaluated in cultured hPTC. Thrombin induced a significant and dose-dependent increase in thymidine uptake and a striking upregulation of MCP-1 mRNA expression and protein release into the supernatant. Although PAR-1 is a G protein-coupled receptor, its activation in hPTC, as in other cell systems, resulted in a transient increase in cellular levels of tyrosine-phosphorylated proteins. An increased level of tyrosine-phosphorylated c-src suggested the activation of this cytoplasmic tyrosine kinase in response to thrombin and its potential role in thrombin-induced protein-tyrosine phosphorylation. Interestingly, thrombin-induced DNA synthesis and MCP-1 gene expression were completely blocked by genistein, a specific tyrosine kinase inhibitor, but not by its inactive analogue daidzein, demonstrating a central role for tyrosine kinase activation in the thrombin effects on hPTC. Moreover, the specific src inhibitor PP1 abolished the thrombin effect on DNA synthesis. In conclusion, thrombin might represent a powerful regenerative and proinflammatory stimulus for hPTC in acute and chronic tubulointerstitial diseases.