Walter Siquini

Human Dedifferentiated Adipocytes Show Similar Properties to Bone Marrow‐Derived Mesenchymal Stem Cells

Mature adipocytes are generally considered terminally differentiated because they have lost their proliferative abilities. Here, we studied the gene expression and functional properties of mature adipocytes isolated from human omental and subcutaneous fat tissues. We also focused on dedifferentiated adipocytes in culture and their morphologies and functional changes with respect to mature adipocytes, stromal‐vascular fraction (SVF)‐derived mesenchymal stem cells (MSCs) and bone marrow (BM)‐derived MSCs. Isolated mature adipocytes expressed stem cell and reprogramming genes. They replicated in culture after assuming a fibroblast‐like shape and expanded similarly to SVF‐ and BM‐derived MSCs. During the dedifferentiation process, mature adipocytes lost their lineage gene expression profile, assumed the typical mesenchymal morphology and immunophenotype, expressed stem cell genes and differentiated into multilineage cells. Moreover, during the dedifferentiation process, we showed changes in the epigenetic status of mature adipocytes, which led dedifferentiated adipocytes to display a similar DNA methylation condition to BM‐derived MSCs. Like SVF‐ and BM‐derived MSCs, dedifferentiated adipocytes were able to inhibit the proliferation of stimulated lymphocytes in coculture while mature adipocytes stimulated their growth. Furthermore, dedifferentiated adipocytes maintained the survival and complete differentiation characteristic of hematopoietic stem cells. This is the first study that in addition to characterizing isolated and dedifferentiated adipocytes also reports on the immunoregulatory and hematopoietic supporting functions of these cells. This structural and functional characterization might have clinical applications of both mature and dedifferentiated adipocytes in such fields, as regenerative medicine. STEM CELLS 2012;30:965–974

Nebivolol induces, via β3 adrenergic receptor, lipolysis, uncoupling protein 1, and reduction of lipid droplet size in human adipocytes.

Objectives: Most &bgr;-blockers may induce weight gain, dysglycemia, and dyslipidemia. Nebivolol is a third-generation &bgr;1-blocker with vasodilating properties mediated by &bgr;3 adrenergic receptors (&bgr;3AR). We investigated whether nebivolol is able to induce &bgr;3AR-mediated lipolysis, uncoupling protein 1 (UCP1), and size-reduction in human adipocytes. Methods: Human visceral (n = 28) and subcutaneous adipose tissue (n = 26) samples were used to obtain differentiated subcutaneous and visceral preadipocytes. Adipocytes were used to verify the effects of nebivolol onlipolysis, uncoupling protein 1 (UCP1) and other genes of the thermogenic program. Results: Lipolysis was induced by isoproterenol and specific &bgr;3AR agonist, as expected,and also by nebivolol at 100 nmol/l and by its L-enantiomer at 10 nmol/l (P < 0.01). Nebivolol-mediated lipolysis was blocked by SR59230A, a specific &bgr;3AR antagonist, suggesting that nebivolol acts through &bgr;3AR in human adipocytes. Interestingly, in human adipocytes, nebivolol activated UCP1, PPAR&ggr; coactivator-1&agr; (PGC-1&agr;) and cytochrome c (CYCS) gene expression in a p38 MAPK–dependent manner. Using propranolol (&bgr;1 and &bgr;2 antagonist) together with nebivolol we showed that the induction of these genes was still present suggesting again &bgr;3AR activation. Moreover, nebivolol significantly reduced the diameter of lipid droplets in cultured adipocytes. Conclusion: In summary, nebivolol, through &bgr;3AR, is able to induce lipolysis and promote thermogenic and mitochondrial genes. The induction of lipolysis and the thermogenic program could explain the reduction of lipid droplets size. In conclusion, the lower dysmetabolic effects of nebivolol in humans may depend on its &bgr;3 agonist activity and the consequent induction of thermogenic program in human adipocytes.

Vulvo-perineal reconstruction with a reverse sensitive rectus abdominis salvage flap in a multirecurrent anal carcinoma

Reconstructive options after vulvectomy is preferably performed using fasciocutaneous flaps. If the defect is very large, the use of vertical rectus abdominis myocutaneous (VRAM) flap is recommended. We report a case of a patient affected by multirecurrent anal carcinoma, treated by chemotherapy, radiotherapy and surgery several times, until an extended abdominoperineal resection of Miles was performed. Since other surgical options were no more available, a primary reverse VRAM flap reconstruction was harvested, together with an end-to-end nerve anastomosis between the cutaneous ramus of the 8th intercostal nerve and the superior branch of the pudendal nerve to achieve sensibility. Encouraging results, without actual recurrence of the disease, were obtained.

Nuclear factor-κB predicts outcome in locally advanced rectal cancer patients receiving neoadjuvant radio-chemotherapy

BACKGROUND NF-κB expression has been shown to be responsible for resistance to antineoplastic agents. AIMS The aim of our study was to investigate the importance of NF-κB expression as prognostic factor in locally advanced rectal cancer patients receiving neoadjuvant radiochemotherapy. METHODS We retrospectively analysed the immunoreactivity for NF-κB in patients with locally advanced rectal cancer who underwent neoadjuvant treatment (chemotherapy and/or radiotherapy) in our Institution between March 2003 and June 2006. RESULTS Seventy-four consecutive patients were enrolled into this study. Immunohistochemistry analysis for NF-κB was performed both in biopsies and in primary tumour samples. NF-κB was considered positive when at least 1% of the tumour cells showed nuclear positivity. A significant correlation between a positive NF-κB nuclear expression, both in biopsies and in tumour samples, and a worse overall survival was observed. Moreover, median time to progression was significantly shorter in the NF-κB-positive subgroup of patients. CONCLUSION Globally, our findings seem to suggest that NF-κB could represent an important parameter able to predict the outcome in patients receiving neoadjuvant treatment for rectal cancer. It also could be useful in order to select patients to receive adjuvant chemotherapy, intensifying the adjuvant therapy and, in the next future, obviating the use of drugs involving NF-κB system in their mechanism of action in NF-κB-positive patients.

The timing of surgery for resectable metachronous liver metastases from colorectal cancer: Better sooner than later? A retrospective analysis

BACKGROUND The benefit of preoperative chemotherapy in patients with initially resectable liver metastases from colorectal cancer is still a matter of debate. AIMS We aim to evaluate the role of neoadjuvant chemotherapy on the outcome of patients with colorectal cancer metachronous liver metastases undergoing potentially curative liver resection. METHODS One-hundred four patients were available for analysis. Tested variables included age, sex, primary tumour TNM stage, location and grading, the number of liver metastases, monolobar or bilobar location, interval time between liver metastases diagnosis and liver resection, Fong Clinical Risk Score (CRS). Neoadjuvant chemotherapy was administered according to the FOLFOX4 regimen. RESULTS Forty-four patients underwent liver resection without receiving neoadjuvant chemotherapy (group A); 60 patients received neoadjuvant chemotherapy (group B). At univariate analysis, only the time of liver resection seemed to affect overall survival: patients in group A showed a median survival time significantly superior to that of patients in group B (48 vs. 31 months; p=0.0358). CONCLUSIONS Our findings suggest that, when feasible, resection of liver metastases should be considered as an initial approach in this setting. Further studies are needed to better delineate innovative therapeutic strategies that may lead to an improved outcome for colorectal cancer patients with surgically resectable liver metastases.

Successful closure of anastomotic dehiscence after colon–rectal cancer resection using the Apollo overstitch suturing system

Anastomotic dehiscence is themost common cause of postoperative morbidity andmortality. The Overstitch endoscopic suturing system (Apollo Endosurgery, Austin, Texas, USA) is a disposable, single-use suturing device that is mounted onto a double-channel therapeutic endoscope and allows placement of either running or interrupted full-thickness sutures. Several reports have described endoscopic suturing as a treatment option for management of gastrointestinal defects and for stent anchorage [1–5]. To our knowledge, there is no report on the closure of colonic anastomotic dehiscence with this device. We describe the case of a 70-year-old man who underwent left colectomy for colon cancer. On the seventh postoperative day, fecal material appeared from his abdominal drain, but he had no signs of fever or peritonitis. A computed tomography (CT) scan of his abdomen confirmed the clinical suspicion of anastomotic dehiscence. We then performed an endoscopic evaluation, which confirmed the presence of a large anastomotic dehiscence (▶Fig. 1). We decided to treat the lesion with the Apollo Overstitch device. The procedure was carried out using CO2 insufflation, with the patient under conscious sedation. Initial tissue preparation prior to closure was done by creating a surgical surface using argon plasma coagulation at the immediate perimeter of the leak; subsequently, running sutures were placed with a distal to proximal technique, attempting to create a full-thickness suture (▶Video1). The procedure was successful with no complications occurring over the following days. However, because radiological follow-up performed a week later showed the persistence of a minimal leak at the site of the dehiscence, we decided to repeat the procedure with the Apollo device. At 1 month after the second procedure, no complications or symptoms were reported; moreover, no further fecal material and no contrast leakage on radiological follow-up had been observed. Repeat endoscopic evaluation at 6 months showed complete closure of the dehiscence (▶Fig. 2). This clinical report demonstrates the effectiveness of an endoscopic suturing device for closure of postoperative colorectal leakage.

Adenocarcinoma arising from ileoanal J-pouch mucosa: an announced event?

To the Editor: We describe a case of adenocarcinoma arising from ileal pouch mucosa 20 years after restorative proctocolectomy (RPC) performed for a severe hemorrhagic ulcerative colitis (UC), focusing on some histopathological features (acute and chronic inflammation, atrophy of the intestinal villi, proliferative activity, and the presence of dysplasia) evaluated in the several endoscopic biopsies obtained during the long follow-up period prior to cancer onset. A 40-year-old man underwent RPC with mucosectomy and handsewn 18 cm J-ileal pouch–anal anastomosis (IPAA) for a severe hemorrhagic UC refractory to medical treatment. Early on he experienced a severe acute and chronic inflammation of the ileal pouch mucosa which was evident even 1 year after ileostomy closure. Pouchitis became clinically manifest 4 years after intervention, with rectal bleeding and the occurrence of substenosis of the ileal pouch inlet and of the IPAA. In the following years the patient underwent a regular endoscopic follow-up with multiple biopsies. A typical picture of severe diffuse acute and chronic mucosal inflammation with edema, granularity, and multiple ulcerations was confirmed at each endoscopy and biopsy control. In January 2005 (17 years after operation) a lowgrade adenomatous dysplasia was diagnosed in an endoscopic biopsy of the ileal pouch. Because of recurrent perianal fistulas unresponsive to medical treatment, a new ileostomy was constructed in 2005. During the following year, the patient underwent multiple endoscopic dilatations of the recurrent ileal pouch inlet stenosis. In September 2008 a pouch endoscopy revealed the presence of a flat and irregular solid lesion in the posterior wall of the inflamed ileal pouch, 3–4 cm above the IPAA; the endoscopic biopsy showed the presence of ‘‘islets of adenocarcinoma within the ileal mucosa.’’ In order to achieve a definitive diagnosis of malignancy, we performed a transanal biopsy, which documented the lesion being an adenocarcinoma infiltrating the muscular tissue of the ileal pouch. A subsequent staging thoracic and abdominal computed tomography revealed no metastases. We therefore performed a total transabdominal excision of the ileal pouch with removal of the small rectal muscular cap; a new permanent ileostomy was constructed distally to the former one. The specimen showed a diffuse mucosal alteration typical of a chronic inflammatory process with wide ulcers; in the posterior wall of the distal portion of the pouch there was a 1.5 cm diameter solid flat area. Histology revealed this area to be a G2 adenocarcinoma invading the ileal wall and the perivisceral fat tissue, without any metastasis in the removed lymph nodes. The patient recovered uneventfully and he was discharged 8 days after surgical intervention. Then he was submitted to adjuvant chemotherapy. Eighteen biopsies of the ileal pouch obtained from the patient during the follow-up period from 1993 to 2008 were histologically reviewed by a gastrointestinal pathologist (M.A.). For each mucosal biopsy these histological parameters were recorded: acute and chronic inflammation, villous atrophy, proliferative activity assessed with Mib-1 immunostaining, and dysplasia. The results showed that chronic inflammation and villous atrophy developed at an early stage and they were always present during follow-up. Acute inflammation was not always present, suggesting that it was related to a response of the mucosa to some potential inflammatory agents. In our case, the ileal pouch mucosa predominantly showed type C pattern of histological adaptation (according to the classification of Veress et al) which evolved into dysplasia and cancer 17 and 20 years after surgical intervention, respectively. Proliferative activity showed an increasing trend over time with evidence of a high index of Mib-1 positivity in the biopsies that preceded the appearance of dysplasia and cancer. It is known that Mib-1 immunostaining helps to distinguish dysplastic areas from regenerative ones. In the regenerative mucosa Mib-1 immunoreactivity is more localized at the basis of the crypts. On the contrary, in dysplastic areas immunoreactivity is present in the cells of the superficial mucosa as well as in the crypts, suggesting a complete deregulation of normal cell proliferation. Similarly, in our case Mib-1 immunostaining was diffusely distributed in the superficial epithelium of the mucosal biopsies performed before the onset of dysplasia and cancer (Fig. 1). Few true cases of carcinoma arising in the ileal pouch mucosa following IPAA for UC have been reported in the literature so far. The peculiarity of our case is the 240month period before the occurrence of malignancy in the ileal pouch. Thanks to the strict clinical and endoscopic follow-up with multiple biopsies, we had the advantage of studying a possible pattern of evolution from acute and chronic inflammation to dysplasia and finally to cancer. Moreover, we documented a real evolution from dysplasia to cancer in an ileal pouch excluded from stools thanks to the diverting ileostomy during a period of 3 years. This fact underlines the importance of a strict endoscopic follow-up with multiple biopsies in these patients with a severe acute and chronic pouchitis, even in the presence of an excluded Copyright VC 2011 Crohn’s & Colitis Foundation of America, Inc. DOI 10.1002/ibd.21711 Published online 6 April 2011 inWiley Online Library (wileyonlinelibrary.com).

Role of lymph nodal dissection for gastric cancer in the elderly

Background Surgeons are being increasingly asked to treat older patients with gastric cancer due to longer life expectancy. D2 lymph node dissection is considered a standard procedure [1]. However, there is no consensus on the extent of lymphadenectomy in the elderly [2]. The aim of this study was to investigate the safety and the efficacy of D2 lymph node dissection in patients aged 75 or over, compared to younger ones.

Steps Shared by Total and Subtotal Gastrectomy

There is no substitute for a well-planned and conducted operation on the stomach to provide the best possible surgical outcome. Correct and stable positioning of the patient is the first step for a successful operation, as well as a careful monitoring of the vital parameters by the anesthetist and a great attention on prevention and control of intraoperative hypothermia and of postoperative pain and malnutrition. Incision should be tailored on the surgical procedure and type of patient in order to provide the best exposure of the operating field, minimizing the risk of operation site infection. Exploration of the abdominal cavity and intraoperative cytology of peritoneal fluid or lavage are of paramount importance for tumor staging and prognosis. Then operation goes on through a gentle approach toward any anatomical structure and following precise steps of the surgical procedure. Detachment of the greater omentum from the transverse colon is followed by the division of the right gastroepiploic pedicle and of the pyloric vessels. The duodenum is then closed and resected. Finally, the left gastric vein and artery are divided.

Position of the Patient

The patient is placed supine with the left arm abducted, as required by the anesthetist, and the right arm adducted to leave sufficient room for the surgeon and the assistant standing on the surgeon’s left. A heating mat is placed between the patient and the surgical table.