Tommaso Marcucci

Is Total Parathyroidectomy the Treatment of Choice for Hyperparathyroidism in Multiple Endocrine Neoplasia Type 1

Objective:The aim of the present report is to describe the results obtained with total parathyroidectomy (TPTX) guided by rapid intraoperative parathyroid hormone (PTH) evaluation, followed by immediate parathyroid autograft with fresh tissue. Summary Background Data:Surgery for hyperparathyroidism (HPT) in multiple endocrine neoplasia type 1 (MEN1) is performed with various surgical approaches. Methods:We report our 16-year experience of surgical treatment of 51 MEN1-HPT patients using TPTX and thymectomy. Forty-five patients underwent TPTX as the first surgical procedure, whereas for 6 patients, a parathyroid operation was the second surgical procedure. PTH intraoperative values less than 10 pg/mL, at the end of the surgery, were indicative for reimplantation of a few fragments (∼7) of fresh parathyroid tissue in the brachioradial muscle of the forearm. Parathyroid autograft was performed in all patients, except 3 in whom the fourth parathyroid gland was not found. Results:Persistent hypoparathyroidism occurred in 13 patients (25%), with higher incidence in patients undergoing a second surgical revision for cervical recurrence than in patients submitted to the first surgery. At follow-up, 5 recurrences (∼10%) in the forearm were observed after a mean time of 7 ± 5 (M ± SD) years. No cervical recurrence was documented. The forearm recurrence was treated with removal of 1 or 2 enlarged fragments obtaining the resolution of HPT in all but 1 case. Conclusions:Based on the occurrence of complications in our experience, TPTX followed by autograft and guided by intraoperative PTH monitoring represents a better surgical option in MEN1-HPT compared with other surgical approaches.

Somatostatinoma: clinico-pathological features of three cases and literature reviewed.

Somatostatinoma is a rare endocrine tumor that comprises around 1% of all gastroenteropancreatic endocrine neoplasms. This paper gives an updated review on somatostatinoma and describes three sporadic cases of somatostatinoma located in the pancreas, duodenum, and jejunum. Approximately 200 case histories of somatostatinoma have been published, with the duodenum being the most frequent site, followed by the pancreas. Somatostatinomas may be sporadic or associated with neurofibromatosis type 1, Multiple Endocrine Neoplasia type 1, and Von Hippel–Lindau syndromes. Functional somatostatinomas release excessive amounts of somatostatin suppressing gallbladder motility and inhibiting the secretory activity of various endocrine and exocrine cell types. A triad of mild diabetes mellitus, cholelithiasis, and diarrhea/steatorrhoea characterizes the somatostatinoma or ‘inhibitory’ syndrome. Non‐functional somatostatinomas tend either to be asymptomatic or to present with obstructive symptoms. These tumors are often malignant and by the time they are detected, nearly two‐thirds have already metastasized to the regional lymph nodes or the liver. A comparison between our three cases and those in the literature provides useful insights into the clinical management of these patients. Interestingly, the jejunal somatostatinoma described here is the second case ever reported.

Oxidative state and IL‐6 production in intestinal myofibroblasts of Crohn's disease patients

Background: Intestinal subepithelial myofibroblasts (ISEMFs) produce inflammatory cytokines in response to certain stimuli. In the intestine of patients with Crohns disease (CD), cytokine synthesis is modified and an increased number of myofibroblasts has been observed. The intracellular redox state influences cytokine production and oxidative stress is present in the intestinal mucosa of CD patients. Methods: This study was performed in ISEMFs isolated from the colon of patients with active CD and in a myofibroblast cell line derived from human colonic mucosa: 18Co cells. Cellular glutathione (GSH) levels were modulated by treatment with buthionine sulfoximine, an inhibitor of GSH synthesis, or N‐acetylcysteine, a GSH precursor. GSH and oxidized glutathione (GSSG) levels were measured by high‐performance liquid chromatography (HPLC) methods. Interleukin (IL)‐6 production was detected by enzyme‐linked immunosorbent assay (ELISA). Results: ISEMFs of CD patients exhibited an increased oxidative state due to a decrease in the GSH/GSSG ratio, which is related to an increase in basal IL‐6 production or is stimulated by tumor necrosis factor alpha (TNF&agr;) or bacterial products. This relationship was also confirmed in 18Co cells. Phosphorylation and activation of ERK1/2 and p38 MAPK, which are signaling factors involved in the IL‐6 synthesis, were also increased when there is oxidative stress in ISEMFs. Conclusions: This study shows for the first time in ISEMFs of CD patients an increased production of IL‐6 synthesis related to the decrease in the GSH/GSSH ratio, suggesting redox regulation with the involvement of specific kinase activation. The present data shed light on the pathogenesis of inflammatory chronic processes and relapses that occur in this pathology. (Inflamm Bowel Dis 2010;)

Redox State and O2•- Production in Neutrophils of Crohn's Disease Patients:

The aim of this in vitro study was to evaluate the intracellular redox state and respiratory burst (RB) in neutrophils of patients with Crohns disease (CD). The intracellular redox state and RB in neutrophils was assessed by the superoxide anion (O2•-) production induced in these cells after stimulation by various factors related to the molecular mechanisms that, if altered, may be responsible for an abnormal immune response. This can, in part, cause the onset of inflammation and tissue damage seen in CD. This study demonstrated a decreased glutathione/glutathione disulfide (GSH/GSSG) ratio index of an increased oxidative state in CD patient neutrophils. Moreover, our findings showed a decrease in tumor necrosis factor (TNF-α)- or phorbol 12-myristate 13-acetate (PMA)–induced O2•- production in CD patient neutrophils adherent to fibronectin as compared with controls. A decreased adhesion was also demonstrated. For this reason, the involvement of altered mechanisms of protein kinase C (PKC) and β-integrin activation in CD patient neutrophils is suggested. These data also showed that the harmful effects of TNF-α cannot be caused by excessive reactive oxygen species (ROS) production Induced by neutrophils. Decreased cell viability after a prolonged time of adhesion (20 hrs) was also measured in CD patient neutrophils. The findings of this study demonstrate, for the first time, that granulocyte-macrophage colony–stimulating factor (GM-CSF), a compound recently used in CD therapy, is able to activate the RB for a prolonged time both in control and CD patient neutrophils. Increased viability of CD patient neutrophils caused by GM-CSF stimulation was also observed. In conclusion, our results indicate that decreased O2•- production and adhesion, caused, in part, by an anomalous response to TNF-α, together with low GSH level and low cell viability, may be responsible for the defective neutrophil function found in CD patients. This can contribute to the chronic inflammation and relapses that characterize this pathology. A possible role of GM-CSF in inducing O2•- production and in restoring the defensive role of neutrophils in CD patients is suggested.

Apoptosis and Bax, Bcl-2, Mcl-1 expression in neutrophils of Crohn's disease patients

Background: The etiology of Crohns disease (CD) remains unknown, and the defective function of neutrophils appears to be associated with this pathology. Neutrophils undergo spontaneous apoptosis which, if not tightly regulated, can induce the development of chronic inflammatory disease. The Bcl‐2 protein family is also involved in the regulation of neutrophil apoptosis. Methods: This study investigated the apoptosis and expression of some regulatory factors in CD patient and control polymorphonuclear neutrophils (PMN) in suspension and in adhesion on fibronectin, an extracellular matrix protein. These 2 conditions mimic circulating neutrophils before they are recruited at the intestinal levels, and their adhesion to tissue. Results: Apoptosis in CD patient PMN was delayed in suspension and accelerated in adhesion, which is the opposite of what happens in controls. Higher levels of Bax, Bcl‐2, and Mcl‐1 proteins were registered in freshly isolated CD patient PMN, in contrast to controls, in which Bcl‐2 protein was undetectable. Among the studied pro‐ and antiapoptotic factors, Bax levels seem to be mainly related to the difference in apoptosis between PMN of CD patients and controls. Conclusions: For the first time it has been demonstrated by direct experimental evidence that apoptosis in CD patient PMN is regulated differently from that of control PMN. Abnormal expression of regulating apoptosis proteins is shown in CD patient PMN. These data suggest that the defective functionality of neutrophils can be the early event responsible for the altered mucosal immune response in CD, and that neutrophil apoptosis may offer a new target for specific drugs and therapy tools.

Role of N-acetylcysteine and GSH redox system on total and active MMP-2 in intestinal myofibroblasts of Crohn's disease patients

PurposeIntestinal subepithelial myofibroblasts (ISEMFs)1 are the predominant source of matrix metalloproteinase-2 (MMP-2) in gut, and a decrease in glutathione/oxidized glutathione (GSH/GSSG) ratio, intracellular redox state index, occurs in the ISEMFs of patients with Crohn’s disease (CD). The aim of this study is to demonstrate a relationship between MMP-2 secretion and activation and changes of GSH/GSSG ratio in ISEMFs stimulated or not with tumor necrosis factor alpha (TNFα).MethodsISEMFs were isolated from ill and healthy colon mucosa of patients with active CD. Buthionine sulfoximine, GSH synthesis inhibitor, and N-acetylcysteine (NAC), precursor of GSH synthesis, were used to modulate GSH/GSSG ratio. GSH and GSSG were measured by HPLC and MMP-2 by ELISA Kit.ResultsIn cells, stimulated or not with TNFα, a significant increase in MMP-2 secretion and activation, related to increased oxidative stress, due to low GSH/GSSG ratio, was detected. NAC treatment, increasing this ratio, reduced MMP-2 secretion and exhibited a direct effect on the secreted MMP-2 activity. In NAC-treated and TNFα-stimulated ISEMFs of CD patients’ MMP-2 activity were restored to physiological value. The involvement of c-Jun N-terminal kinase pathway on redox regulation of MMP-2 secretion has been demonstrated.ConclusionFor the first time, in CD patient ISEMFs, a redox regulation of MMP-2 secretion and activation related to GSH/GSSG ratio and inflammatory state have been demonstrated. This study suggests that compounds able to maintain GSH/GSSG ratio to physiological values can be useful to restore normal MMP-2 levels reducing in CD patient intestine the dysfunction of epithelial barrier.

Redox regulation of MMP-3/TIMP-1 ratio in intestinal myofibroblasts: effect of N-acetylcysteine and curcumin.

Matrix metalloproteinases (MMPs) play a critical role in inflammation and ulcerations in gut of Crohn׳s disease (CD) patients. Intestinal subepithelial myofibroblasts (ISEMFs) secrete MMPs in response to inflammatory stimuli. Previous data showed in CD-ISEMFs increased oxidative status. The aim of this study was to investigate the role of ISEMFs in modulating the production of MMP-3 and TIMP-1, an inhibitor of MMPs activity. A relationship among oxidative stress, activity of antioxidants and MMP-3/TIMP-1 was also studied. ISEMFs isolated from CD patient colon and human colonic cell line of myofibroblasts (18Co) were used. Oxidative state was modulated by buthionine sulfoximine, an inhibitor of glutathione (GSH) synthesis, and N-acetylcysteine (NAC), GSH precursor. An up-regulation of MMP-3 due to increased oxidative state was found in CD-ISEMFs. Stimulation by tumor necrosis factor (TNF)α increased further MMP-3 levels. On the contrary, no change in TIMP-1 production was determined. NAC treatment decreased MMP-3 production in CD-ISMEFs and removed the enhancement due to TNFα. Similar effects were observed in 18Co cells treated with curcumin, antioxidant with anti-inflammatory properties. The involvement of MAPKs on MMP-3 redox regulation was also shown. This study demonstrates the involvement of ISEMFs and high oxidative state in the increased MMP-3 production found in intestinal mucosa of CD patients. NAC and curcumin normalize MMP-3 levels mainly in TNFα stimulated cells. A modulation of MMP-3 production by NAC and curcumin due to their direct action on transcriptional factors has been also suggested. Therefore, they could have a therapeutic use for the prevention and treatment of fistulaes in CD.

Tumor Necrosis Factor‐Alpha Up‐Regulates ICAM‐1 Expression and Release in Intestinal Myofibroblasts by Redox‐Dependent and –Independent Mechanisms

Intercellular adhesion molecule‐1 (ICAM‐1) is distributed and expressed on cell surface and is present in circulation as soluble form (sICAM‐1). Tumor necrosis factor‐alpha (TNFα) and radical oxygen species (ROS) up‐regulate the expression of ICAM‐1. This study demonstrates for the first time in 18 Co cells, a myofibroblast cell line derived from human colonic mucosa, an up‐regulation of ICAM‐1 expression and sICAM‐1 release induced by oxidative stress and TNFα stimulation. The intracellular redox state was modulated by L‐buthionine‐S,R‐sulfoximine (BSO) or N‐acetylcysteine (NAC), inhibitor and precursor respectively of GSH synthesis. ROS production increases in cells treated with BSO or TNFα, and this has been related to an up‐regulation of ICAM‐1 expression and sICAM‐1 release. The involvement of metalloproteinases in ICAM‐1 release has been demonstrated. Moreover, also expression and activation of A disintegrin and metalloproteinase 17, a membrane‐bound enzyme known as TNFα‐converting enzyme (TACE), have been related to ROS levels. This suggests the possible involvement of TACE in the cleavage of ICAM‐1 on cell surface in condition of oxidative stress. NAC down‐regulates the expression and release of ICAM‐1 as well as the expression and activation of TACE. However, in TNFα stimulated cells NAC treatment reduces only in part ICAM‐1 expression and sICAM‐1 release. Given this TNFα may also act on these events by a redox‐independent mechanism. J. Cell. Biochem. 117: 370–381, 2016.

MMPs, ADAMs and their natural inhibitors in inflammatory bowel disease: involvement of oxidative stress

Matrix metalloproteinases (MMPs) are enzymes involved in the degradation of extracellular matrix proteins and in several functions such as wound repair. The regulation of MMPs occurs at various levels including expression and inhibition by tissue specific inhibitors of MMPs (TIMPs). An alteration of intracellular redox state can modulate the activation and/or expression of MMPs. The increased expression and activity of MMPs are related to inflammatory state and pathogenesis of inflammatory bowel disease (IBD). In particular, an imbalance between MMPs and TIMPs is present at intestinal level in IBD, and this can be responsible of alterations of different processes such as repair of damaged mucosa, function and migration of immune cells and mucosal ulceration. A disintegrin and metalloproteinase 17 (ADAM17), belonging to ADAM family, plays an important role in the intestinal inflammation and increases in human colonic mucosa of IBD patients. In this review we report the effects of MMP, ADAM17 and TIMP altered levels on damage amplification caused by inflammation and oxidative stress in IBD. Moreover, the review evidences the role of the intracellular redox state and antioxidants on regulation of expression and activity of some MMPs in condition of oxidative stress in IBD.

Surgery in MEN 2A Patients Older Than 5 Years with Micro-MTC: Outcome at Long-term Follow-up

In multiple endocrine neoplasia syndrome type 2A (MEN 2A), early total thyroidectomy (TT; performed before the age of 5 years) is the best option to prevent medullary thyroid carcinoma (MTC) development, but the management of MEN 2A patients diagnosed after childhood is still under debate. Seventeen consecutive patients diagnosed with MEN 2A after the age of 5 years (mean age, 23.3 years) with a pathologic diagnosis of micro-MTC without nodal involvement were enrolled. All patients underwent TT with thymectomy and central compartment lymph node dissection. During surgery, parathyroid tissue removal occurred in 14 patients. No major postoperative complications nor persistent hypoparathyroidism was observed. After a mean follow-up of 16.6 years, no patient developed primary hyperparathyroidism or disease recurrence. Even if TT is recommended before the age of 5, when MEN 2A diagnosis is performed after this age in micro-MTC without nodal involvement, TT with thymectomy and central compartment lymphadenectomy can provide good oncologic and functional results.