Francesco Tonelli

Phenotypic and functional features of human Th17 cells

T helper (Th) 17 cells represent a novel subset of CD4+ T cells that are protective against extracellular microbes, but are responsible for autoimmune disorders in mice. However, their properties in humans are only partially known. We demonstrate the presence of Th17 cells, some of which produce both interleukin (IL)-17 and interferon (IFN)-γ (Th17/Th1), in the gut of patients with Crohns disease. Both Th17 and Th17/Th1 clones showed selective expression of IL-23R, CCR6, and the transcription factor RORγt, and they exhibited similar functional features, such as the ability to help B cells, low cytotoxicity, and poor susceptibility to regulation by autologous regulatory T cells. Interestingly, these subsets also expressed the Th1-transcription factor T-bet, and stimulation of these cells in the presence of IL-12 down-regulated the expression of RORγt and the production of IL-17, but induced IFN-γ. These effects were partially inhibited in presence of IL-23. Similar receptor expression and functional capabilities were observed in freshly derived IL-17–producing peripheral blood and tonsillar CD4+ T cells. The demonstration of selective markers for human Th17 cells may help us to understand their pathogenic role. Moreover, the identification of a subset of cells sharing features of both Th1 and Th17, which can arise from the modulation of Th17 cells by IL-12, may raise new issues concerning developmental and/or functional relationships between Th17 and Th1.

Human interleukin 17–producing cells originate from a CD161+CD4+ T cell precursor

We demonstrate that CD161 is a highly up-regulated gene in human interleukin (IL) 17 T helper cell (Th17) clones and that all IL-17–producing cells are contained in the CD161+ fraction of CD4+ T cells present in the circulation or in inflamed tissues, although they are not CD1-restricted natural killer T cells. More importantly, we show that all IL-17–producing cells originate from CD161+ naive CD4+ T cells of umbilical cord blood, as well as of the postnatal thymus, in response to the combined activity of IL-1β and IL-23. These findings implicate CD161 as a novel surface marker for human Th17 cells and demonstrate the exclusive origin of these cells from a CD161+CD4+ T cell progenitor.

Clinical practice guidelines for multiple endocrine neoplasia type 1 (MEN1).

OBJECTIVE The aim was to provide guidelines for evaluation, treatment, and genetic testing for multiple endocrine neoplasia type 1 (MEN1). PARTICIPANTS The group, which comprised 10 experts, including physicians, surgeons, and geneticists from international centers, received no corporate funding or remuneration. PROCESS Guidelines were developed by reviews of peer-reviewed publications; a draft was prepared, reviewed, and rigorously revised at several stages; and agreed-upon revisions were incorporated. CONCLUSIONS MEN1 is an autosomal dominant disorder that is due to mutations in the tumor suppressor gene MEN1, which encodes a 610-amino acid protein, menin. Thus, the finding of MEN1 in a patient has important implications for family members because first-degree relatives have a 50% risk of developing the disease and can often be identified by MEN1 mutational analysis. MEN1 is characterized by the occurrence of parathyroid, pancreatic islet, and anterior pituitary tumors. Some patients may also develop carcinoid tumors, adrenocortical tumors, meningiomas, facial angiofibromas, collagenomas, and lipomas. Patients with MEN1 have a decreased life expectancy, and the outcomes of current treatments, which are generally similar to those for the respective tumors occurring in non-MEN1 patients, are not as successful because of multiple tumors, which may be larger, more aggressive, and resistant to treatment, and the concurrence of metastases. The prognosis for MEN1 patients might be improved by presymptomatic tumor detection and undertaking treatment specific for MEN1 tumors. Thus, it is recommended that MEN1 patients and their families should be cared for by multidisciplinary teams comprising relevant specialists with experience in the diagnosis and treatment of patients with endocrine tumors.

A clinical outcome-based prospective study on venous thromboembolism after cancer surgery: The @RISTOS Project

Summary Background Data:The epidemiology of venous thromboembolism (VTE) after cancer surgery is based on clinical trials on VTE prophylaxis that used venography to screen deep vein thrombosis (DVT). However, the clinical relevance of asymptomatic venography-detected DVT is unclear, and the population of these clinical trials is not necessarily representative of the overall cancer surgery population. Objective:The aim of this study was to evaluate the incidence of clinically overt VTE in a wide spectrum of consecutive patients undergoing surgery for cancer and to identify risk factors for VTE. Methods:@RISTOS was a prospective observational study in patients undergoing general, urologic, or gynecologic surgery. Patients were assessed for clinically overt VTE occurring up to 30 ± 5 days after surgery or more if the hospital stay was longer than 35 days. All outcome events were evaluated by an independent Adjudication Committee. Results:A total of 2373 patients were included in the study: 1238 (52%) undergoing general, 685 (29%) urologic, and 450 (19%) gynecologic surgery. In-hospital prophylaxis was given in 81.6% and postdischarge prophylaxis in 30.7% of the patients. Fifty patients (2.1%) were adjudicated as affected by clinically overt VTE (DVT, 0.42%; nonfatal pulmonary embolism, 0.88%; death 0.80%). The incidence of VTE was 2.83% in general surgery, 2.0% in gynecologic surgery, and 0.87% in urologic surgery. Forty percent of the events occurred later than 21 days from surgery. The overall death rate was 1.72%; in 46.3% of the cases, death was caused by VTE. In a multivariable analysis, 5 risk factors were identified: age above 60 years (2.63, 95% confidence interval, 1.21–5.71), previous VTE (5.98, 2.13–16.80), advanced cancer (2.68, 1.37–5.24), anesthesia lasting more than 2 hours (4.50, 1.06–19.04), and bed rest longer than 3 days (4.37, 2.45–7.78). Conclusions:VTE remains a common complication of cancer surgery, with a remarkable proportion of events occurring late after surgery. In patients undergoing cancer surgery, VTE is the most common cause of death at 30 days after surgery.

Smoking habits and recurrence in Crohn's disease

BACKGROUND/AIMS Smoking may be a risk factor for surgical recurrence of Crohns disease. However, other variables associated with recurrence could be confounding factors for smoking. The aim of this study was to evaluate the role of smoking as an independent predictor of clinical, surgical, and endoscopic recurrence. METHODS In a series of 182 patients who underwent surgery for Crohns disease, a multivariate analysis was performed that included all of the significant variables associated with recurrence: sex, age at diagnosis, time between onset of symptoms and surgery, site of disease, indication for surgery, extent of disease, extraintestinal manifestation, and smoking habit. RESULTS Independent predictors of clinical recurrence by the Cox proportional hazard model were smoking (hazard ratio, 1.46; 95% confidence interval [CI], 1.1-1.8), extraintestinal manifestations (hazard ratio, 1.61; 95% CI, 1.0-2.5), and extent of disease (hazard ratio, 1.57; 95% CI, 1.0-2.4). Smoking was the only significant predictor of surgical recurrence (hazard ratio, 2.0; 95% CI, 1.2-2.3). For endoscopic recurrence, logistic regression showed that smoking (odds ratio, 2.2; 95% CI, 1.2-3.8) and extent of disease (odds ratio, 2.6; 95% CI, 1.0-6.7) were predictive factors of recurrence. CONCLUSIONS Smoking is an independent risk factor for clinical, surgical, and endoscopic recurrence in Crohns disease.

Substance P (Neurokinin-1) and Neurokinin A (Neurokinin-2) Receptor Gene and Protein Expression in the Healthy and Inflamed Human Intestine

Increasing evidence suggests that tachykinins are involved in the control of pathophysiological states, such as inflammation. The precise localization of tachykinin receptors is of paramount importance in the search for their possible physiological and pathological role; in this study, therefore, we attempted to define cellular sites of substance P (NK-1R) and neurokinin A (NK-2R) receptor expression in the healthy and the inflamed human intestine by in situ hybridization and immunohistochemistry. In the normal ileum and colon, NK-1R and NK-2R were localized to smooth muscle cells of the muscularis mucosae and propria and a few inflammatory cells of the lamina propria; NK-1R expression was also found in the muscular wall of submucosal blood vessels, enteric neurons and, to a lesser degree, in surface epithelial cells. Patients with Crohns disease and ulcerative colitis showed a dramatic increase in NK-1R density relative to controls, in both the inflamed and the uninvolved mucosa. Up-regulation of NK-1R was particularly evident on epithelial cells lining the mucosal surface and crypts, as well as on endothelial cells of capillaries and venules. Also, a marked increase in NK-2R expression was found in both groups of patients on inflammatory cells of the lamina propria, especially eosinophils. Our findings demonstrate that in the normal human intestine NK-1R and NK-2R are expressed in multiple cell types, which are endowed with different physiological functions; in addition, they demonstrate that both NK-1R and NK-2R are up-regulated in patients with Crohns disease and ulcerative colitis. Taken together, these observations may have important physiological and pathophysiological implications, and provide the rationale for the use of NK-1R and NK-2R antagonists in the treatment of inflammatory bowel disease.

What affects continence after anterior resection of the rectum

Functional results after anterior rectal resections are commonly considered satisfactory but variable percentages of postoperative incontinence are often reported. Continence was evaluated after 20 low anterior resections (LAR) and 13 high anterior resections (HAR) by means of clinical assessment, anorectal manometry, and evacuation proctography. Whereas all HAR patients had perfect continence, 10 patients (50 percent of the LAR group had occasional episodes of soiling from liquid feces, 5 patients (25 percent had frequent soiling or occasional incontinence for solid feces, and 1 patient (5 percent had frequent solid stool loss requiring surgical treatment. Anal canal resting pressure at 3 and 4 cm from the anal verge was significantly lower in the LAR group (P<0.02 andP<0.05, respectively) than in the HAR group. However, the maximum voluntary contraction did not differ between the two groups. Rectoanal inhibitory reflex was found to be present in 17 of the 20 patients with LAR and in all patients with HAR. The volume at which the anal sphincter is continuously inhibited was significantly reduced in the LAR group (P<0.001). Also, the conscious rectal sensibility volumes were found to be significantly reduced for threshold, constant, and maximum tolerated volume. Threshold volume for internal sphincter relaxation was lower than the threshold volume for rectal sensation in some patients with LAR. This could allow postoperative fecal soiling. Rectal compliance was decreased (P<0.001) in the LAR group. Evacuation proctography, performed in six LAR patients affected by major soiling or solid stool loss, revealed an abnormal obtuse anorectal angle and pathologic lowering of the perineum at rest and during defecation. The concomitance of internal anal sphincter impairment, reduction in rectal compliance, and previous pelvic floor muscle damage are postulated as cause affecting continence in patients who underwent LAR.

Transforming growth factor beta signalling and matrix metalloproteinases in the mucosa overlying Crohn's disease strictures.

Background and Aims: In addition to its crucial role in dampening tissue-damaging immune responses in the gut, transforming growth factor β (TGFβ) is a potent profibrogenic agent inducing collagen synthesis and regulating the balance between matrix-degrading matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). TGFβ signalling was investigated by analysis of Smad proteins and MMPs/TIMPs in the mucosa overlying strictures in patients with Crohn’s disease (CD). Methods: Specimens were collected from macroscopically normal mucosa overlying strictured and non-strictured gut of patients with fibrostenosing CD. Isolated myofibroblasts were cultured with anti-TGFβ blocking antibody or TGFβ1. TGFβ transcripts were analysed by quantitative reverse transcription-PCR (RT-PCR). Smad proteins and MMPs were determined by immunoblotting. MMP-12 activity was measured by a real-time MMP-12 activity assay. An in vitro wound-healing scratch assay was used to assess myofibroblast migration. Results: TGFβ transcripts, phosphorylated Smad2–Smad3 (pSmad2–3) and TIMP-1 proteins were higher in mucosa overlying strictures than in mucosa overlying non-strictured areas. In contrast, mucosa overlying strictured gut had lower expression of Smad7, MMP-12 and MMP-3. Myofibroblasts from mucosa overlying strictured gut showed higher TGFβ transcripts, a greater pSmad2–3 response to TGFβ, increased TIMP-1, lower Smad7, increased collagen production and reduced migration ability compared with myofibroblasts from mucosa overlying non-strictured gut. TGFβ blockade increased myofibroblast MMP-12 production and migration, more obviously in myofibroblasts isolated from mucosa overlying non-strictured compared with strictured gut. Conclusions: Changes in TGF-β signalling and MMP production were identified in the mucosa overlying strictures in CD which may give a window into the process of fibrosis.

Rectal proliferation and polyp occurrence in patients with familial adenomatous polyposis after sulindac treatment

BACKGROUND/AIMS Sulindac, a nonsteroidal anti-inflammatory drug (NSAID), decreases the occurrence of polyps in patients with familial adenomatous polyposis (FAP). The effects of colectomy with ileorectal anastomosis (IRA) and sulindac treatment on rectal mucosa proliferation and polyp occurrence were examined in patients with FAP. METHODS The number and size of rectal polyps were measured with colonoscopy. The labeling index, the percentage of labeled cells per crypt compartment, was assessed in rectal biopsy specimens with [3H]thymidine incorporation and autoradiography in 6 non-IRA and 14 IRA patients before and after treatment with 200 mg of sulindac/day for 60 days. RESULTS The IRA patients had a lower labeling index and a decrease in the percentage of labeled cells in the upper compartment of the crypt (P < 0.01) relative to non-IRA subjects. Sulindac did not influence the labeling index and the distribution of labeled cells along the crypt. On the contrary, a dramatic decrease in the size and number of polyps was observed after sulindac treatment (P < 0.001). CONCLUSIONS The persistence of a abnormal mucosal proliferation after sulindac therapy, in spite of the reduction of polyp number, suggests caution in assuming a lower risk of rectal cancer in patients with FAP.

Prognostic factors for postoperative recurrence of Crohn's disease

Prognostic factors for postoperative recurrence of Crohns disease (CD) have been widely investigated but not yet clearly identified. PURPOSE: Aim of this study was, therefore, to analyze the association between demographic, clinical, laboratory, and surgical characteristics of patients and the cumulative probability of endoscopic postoperative recurrence. METHODS: The study was performed in 110 patients who were enrolled in the Italian multicenter, randomized, controlled trial on the effectiveness of 5-aminosalicylic acid (5-ASA) in the prevention of postoperative recurrence in CD. Patients had undergone their first intestinal resection for CD of the terminal ileum with or without involvement of cecum ascending colon. Recurrence was defined on the basis of endoscopy. The following variables were evaluated as potential prognostic factors: gender, age, years since diagnosis, clinical course (perforative and nonperforative), Crohns Disease Activity Index score, white blood count, erythrocyte sedimentation rate, C-reactive protein, and orosomucoids assessed before the operation. Timing of operation (elective or urgent), type of anastomosis (end-to-end, end-to-side, side-to-end, side-to-side), and prophylactic treatment were also evaluated. Colon ileoscopy was performed at 6, 12, 24, and 36 months after operation. The association between variables and the cumulative proportion of recurrence was analyzed both by univariate analysis (life table method, log-rank test) and multivariate regression analysis (Coxs model, stepwise procedure). RESULTS: Results of this study indicate that, of the features considered before surgery, only leukocytosis (white blood count, >9,000 ml) was significantly associated with an increased risk of recurrence (P<0.05) at univariate analysis. This finding was not confirmed by multivariate analysis. A trend toward a higher risk of recurrence for patients who have had a resection with end-to-end anastomosis compared with those who have had a resection and other types of anastomosis was also observed. This trend reached significancy in the group of patients submitted to treatment with 5-ASA. The multivariate analysis showed that 5-ASA-treated patients with end-to-end anastomosis had a risk of recurrence more than threefold higher than those with other types of anastomosis (relative risk, 3.40; 95 percent confidence interval, 1.00–11.96;P<0.03). CONCLUSIONS: From a practical point of view, it has been estimated that the combination of intestinal resection plus side-to-side or end-to-side anastomosis with oral 5-ASA treatment reduces by 64 percent the postoperative recurrence rate in CD at three years follow-up.