Single-institution experience of intensity-modulated radiotherapy for malignant pleural mesothelioma at University of Catania.

AIM: The multimodal approach to malignant pleural mesothelioma is gradually becoming the standard of care for this disease in patients with good performance status. Materials & methods: We report our experience concerning eight cases treated with the use of static step-and-shoot intensity-modulated radiotherapy to the whole pleural cavity, in patients already undergoing surgical and/or antiblastic therapy. Results & conclusion: Results at a median follow-up of 16 months showed a median survival from the initial treatment of 29 months, with lung toxicity of grade II reported only in two patients.

Ectopic expression of CXCL13, BAFF, APRIL and LT-ß is associated with artery tertiary lymphoid organs in giant cell arteritis.

OBJECTIVES: To investigate whether artery tertiary lymphoid organs (ATLOs) are present in giant cell arteritis (GCA) and that their formation is associated with the ectopic expression of constitutive lymphoid tissue-homing chemokines. METHODS: Reverse transcriptase PCR, immunohistochemical and immunofluorescence analysis were used to determine the presence of ectopic ATLOs in GCA and the expression of chemokines/chemokine receptors and cytokines involved in lymphoneogenesis in the temporal artery samples obtained from 50 patients with GCA and 30 controls. The presence of lymphatic conduits, of follicular dendritic cells (FDCs) precursors and lymphoid tissue inducer cells was also investigated. Finally, expression of CXCL13, B cell activating factor (BAFF), a proliferation-inducing ligand (APRIL) and CCL21 by isolated myofibroblasts was evaluated before and after stimulation with Toll-like receptors (TLRs) agonists and cytokines. RESULTS: ATLOs were observed in the media layer of 60% of patients with GCA in close proximity to high endothelial venules and independently by the age of patients and the presence of atherosclerosis. ATLO formation was also accompanied by the expression of CXCL13, BAFF, a proliferation-inducing ligand (APRIL), lymphotoxin (LT)-ß, interleukin (IL)-17 and IL-7, the presence of FDC precursors and of lymphoid conduits. Stimulation of myofibroblasts with TLR agonists and cytokines resulted in the upregulation of BAFF and CXCL13. CONCLUSIONS: ATLOs occur in the inflamed arteries of patients with GCA possibly representing the immune sites where immune responses towards unknown arterial wall-derived antigens may be organised.

Type 3 innate lymphoid cells producing IL-17 and IL-22 are expanded in the gut, in the peripheral blood, synovial fluid and bone marrow of patients with ankylosing spondylitis.

BACKGROUND: The aim of the study was to better characterise the immunological origin and the behaviour of interleukin (IL)-23-responsive innate lymphoid cells (ILCs) in the gut, synovial fluid (SF) and bone marrow (BM) of patients with ankylosing spondylitis (AS). METHODS: ILC1, ILC2 and ILC3 cells were determined and characterised by confocal microscopy and flow cytometry in ileal and BM biopsies, in peripheral blood (PB) and SF mononuclear cells obtained from patients with AS and controls. Mucosal vascular addressin cell adhesion molecule 1 (MADCAM-1), IL-7, IL-15 and aggregates of lymphoid tissue inducer cells (LTi) were evaluated by immunohistochemistry. The in vitro ability of epithelial cells in driving the differentiation of ILC3 and the effect of tumour necrosis factor inhibitors (TNFi) on the frequency of ILC3 and the expression of MADCAM1 were also assessed. RESULTS: ILC3 characterised as Lyn(-)RORc(-)Tbet(+) NKp44(+) cells were significantly expanded in the gut, SF and BM of patients with AS compared with controls, produced high levels of IL-17 and IL-22 and expressed α4ß7. MADcAM1 was overexpressed in BM and ileal high endothelial venules. IL-7 was significantly increased in AS gut, especially in the context of Paneth cells, and accompanied by the presence of aggregates of c-kit/IL-7R(+) cells (LTi). In in vitro experiments, epithelial cells from patients with AS actively induced differentiation of ILC3 from LTi. TNFi efficacy was accompanied by a significant decrease in the percentage of intestinal and circulating ILC3 and in the expression of MADCAM1. CONCLUSIONS: Gut-derived IL-17(+) and IL-22(+)ILC3 are expanded in the peripheral blood, SF and inflamed BM of patients with AS, suggesting the presence of an active homing axis between the gut and the inflamed sacroiliac joints.

Difference in the expression of IL-9 and IL-17 correlates with different histological pattern of vascular wall injury in giant cell arteritis.

OBJECTIVE: GCA is a large- and medium-vessel arteritis characterized by a range of histological patterns of vascular wall injury. The aim of this study was to immunologically characterize the various histological patterns of GCA. METHODS: Thirty-five consecutive patients with biopsy-proven GCA and 15 normal controls were studied. IL-8, IL-9, IL-9R, IL-17, IL-4, TGF-ß and thymic stromal lymphopoietin expression was evaluated by RT-PCR and immunohistochemistry on artery biopsy specimens. Confocal microscopy was used to characterize the phenotypes of IL-9-producing and IL-9R-expressing cells. Five additional patients who had received prednisone when the temporal artery biopsy was performed were also enrolled to evaluate the effect of glucocorticoids on IL-9 and IL-17 expression. RESULTS: IL-17 overexpression was observed mainly in arteries with transmural inflammation and vasa vasorum vasculitis. IL-9 overexpression and Th9 polarization predominated in arteries with transmural inflammation and small-vessel vasculitis. The tissue expression of both IL-9 and IL-17 was correlated with the intensity of the systemic inflammatory response. IL-4, TGF-ß and thymic stromal lymphopoietin, which are involved in the differentiation of Th9 cells, were overexpressed in arteries with transmural inflammation and small-vessel vasculitis. IL-9R was also overexpressed in GCA arteries with transmural inflammation and was accompanied by increased expression of IL-8. CONCLUSION: Herein we provide the first evidence that distinct populations of potentially autoreactive T cells, expressing different cytokines (Th17 vs Th9), characterize patients with particular histological subsets of GCA and may thus contribute to the heterogeneity of tissue lesions observed in these patients.

Evidence that autophagy, but not the unfolded protein response, regulates the expression of IL-23 in the gut of patients with ankylosing spondylitis and subclinical gut inflammation.

OBJECTIVES: Interleukin (IL)-23 has been implicated in the pathogenesis of ankylosing spondylitis (AS). The aim of the study was to clarify the mechanisms underlying the increased IL-23 expression in the gut of AS patients. METHODS: Consecutive gut biopsies from 30 HLA-B27(+) AS patients, 15 Crohn's disease (CD) patients and 10 normal subjects were obtained. Evidence for HLA-B27 misfolding was studied. Unfolded protein response (UPR) and autophagy were assessed by RT-PCR and immunohistochemistry. The contribution of UPR and autophagy in the regulation of IL-23 expression was evaluated in in vitro experiments on isolated lamina propria mononuclear cells (LPMCs). RESULTS: Intracellular colocalisation of SYVN1 and FHCs but not a significant overexpression of UPR genes was observed in the gut of AS patients. Conversely, upregulation of the genes involved in the autophagy pathway was observed in the gut of AS and CD patients. Immunohistochemistry showed an increased expression of LC3II, ATG5 and ATG12 but not of SQSTM1 in the ileum of AS and CD patients. LC3II was expressed among infiltrating mononuclear cells and epithelial cells resembling Paneth cells (PC) and colocalised with ATG5 in AS and CD. Autophagy but not UPR was required to modulate the expression of IL-23 in isolated LPMCs of AS patients with chronic gut inflammation, CD patients and controls. CONCLUSIONS: Our data suggest that HLA-B27 misfolding occurs in the gut of AS patients and is accompanied by activation of autophagy rather than a UPR. Autophagy appears to be associated with intestinal modulation of IL-23 in AS.

Rituximab modulates IL-17 expression in the salivary glands of patients with primary Sjögren's syndrome.

OBJECTIVE: The aim of this study was to evaluate the role of rituximab (RTX) in modulating the expression of the IL-17/IL-23 pathway in the salivary glands (SGs) of patients with primary SS (pSS). METHODS: Consecutive SG biopsies were obtained from 15 patients with pSS before and after 1 year of RTX therapy. The SG expression of IL-17, IL-23p19 and p-STAT3 was evaluated by immunohistochemistry at baseline and after RTX therapy. The role of mast cells in pSS patients in modulating the Th17 response and the immunologic effect of RTX on mast cells were also studied in in vitro experiments. RESULTS: IL-17 was overexpressed in the SGs of patients with pSS mainly by infiltrating T cells and mast cells. After RTX therapy, the SG expression of IL-17, but not of IL-23p19 and p-STAT3, was significantly reduced and was accompanied by the depletion of tissue mast cells. In in vitro experiments with heterologous peripheral lymphocytes RTX significantly induced the apoptosis of isolated mast cells. Finally, mast cells isolated from peripheral blood mononuclear cells of pSS patients in vitro significantly increased Th17 lymphocytes. CONCLUSION: RTX acts on pSS patients by globally reducing the expression of IL-17 and specifically inducing a pronounced apoptotic depletion of mast cells.

Activated IL-22 pathway occurs in the muscle tissues of patients with polymyositis or dermatomyositis and is correlated with disease activity.

OBJECTIVE: The aim of this study was to assess the expression of IL-22, IL-22 receptor 1 (IL-22R1), IL-22 binding protein (IL-22BP) and p-STAT3 in muscle tissue from patients with PM and DM. METHODS: Levels of IL-22, IL-22R1, IL-22BP and STAT3 mRNA were quantified by RT-PCR. The expression of IL-22, IL-22R1, IL-22BP and p-STAT3 was also analysed using immunohistochemistry. RESULTS: Significant modulation of the IL-22 pathway was observed in inflammatory myopathic tissues. In particular, a significant overexpression of IL-22 at the protein but not the mRNA level was observed in PM/DM tissues and was correlated with myositis activity. IL-22R1 aberrant expression was also observed among infiltrating mononuclear cells and necrotic muscle cells. IL-22BP, which inhibits IL-22 signalling, was expressed only in some muscle fibres in PM/DM patients. CONCLUSION: Our findings indicate that the IL-22 pathway is activated in inflammatory myopathic tissues and may be involved in the induction of muscle inflammatory processes and muscle necrosis.

Could JC virus be linked to chronic idiopathic intestinal pseudo-obstruction?

JC virus is a member of the Polyomavirus family, infects humans worldwide, and 90% of the population carry antibodies to the virus by adult life. The initial infection is asymptomatic, but it may become persistent. JC virus DNA is frequently present in the upper and lower gastrointestinal tracts of healthy adults. Chronic idiopathic intestinal pseudo-obstruction, one of the most severe gastrointestinal motility disorders, is a condition characterized by a clinical picture mimicking small bowel occlusion with related symptoms and signs in the absence of demonstrable mechanical obstruction. Because of the known neuropathic capability of this virus, and its frequent presence in the gut, it has been proposed that JCV might be detectable in tissues of patients with chronic idiopathic intestinal pseudo-obstruction, and possibly be involved in the pathogenesis of this disease, because the virus may actively infect the enteroglial cells of the myenteric plexuses of the patients with chronic idiopathic intestinal pseudo-obstruction. We report two cases of upper idiopathic intestinal pseudo-obstruction associated with JCV infection.

Interleukin-9 Overexpression and Th9 Polarization Characterize the Inflamed Gut, the Synovial Tissue, and the Peripheral Blood of Patients With Psoriatic Arthritis.

OBJECTIVE: To investigate the expression and tissue distribution of Th9-related cytokines in patients with psoriatic arthritis (PsA). METHODS: Quantitative gene expression analysis of Th1, Th17, and Th9 cytokines was performed in intestinal biopsy samples obtained from patients with PsA, HLA-B27-positive patients with ankylosing spondylitis (AS), patients with Crohn's disease (CD), and healthy controls. Expression and tissue distribution of interleukin-23 (IL-23), IL-17, IL-22, IL-9, and IL-9 receptor (IL-9R) were evaluated by immunohistochemistry and confocal microscopy. Flow cytometry was used to study the frequency of Th9 cells among peripheral blood, lamina propria, and synovial fluid mononuclear cells. The functional relevance of IL-9R expression on epithelial cells was assessed in functional in vitro studies. Th9 cells in synovial tissue from patients with PsA were also studied. RESULTS: Subclinical gut inflammation in PsA patients was characterized by a clear Th17 and Th22, but not Th1, polarized immune response. Unlike AS and CD, a strong and significant up-regulation of IL-9 was observed in PsA gut, especially among infiltrating mononuclear cells, high endothelial venules, and Paneth cells. IL-9-positive mononuclear cells were demonstrated to be in large part Th9 cells. IL-9 overexpression was accompanied by significant Paneth cell hyperplasia. Paneth cells strongly overexpressed IL-9R, and stimulation of epithelial cells, isolated from PsA patients, with IL-9 resulted in overexpression of α-defensin 5 and IL-23p19. Peripheral and synovial expansion of α4ß7+ Th9 cells was also observed in patients with PsA. Increased expression of IL-9 and IL-9R was also found in synovial tissue. CONCLUSION: Strong IL-9/Th9 polarization seems to be the predominant immunologic signature in patients in PsA.

Once-Weekly Hypofractionated Whole-Breast Radiotherapy After Breast-Conserving Surgery in Older Patients: A Potential Alternative Treatment Schedule to Daily 3-Week Hypofractionation.

BACKGROUND: The purpose of this study was to retrospectively report clinical outcomes on a consecutive series of older early breast cancer patients treated with once-weekly adjuvant whole-breast radiation therapy (WBRT) after breast-conserving surgery (BCS). PATIENTS AND METHODS: A total of 291 patients (298 breasts) were treated with WBRT between 2007 and 2013. Patients were given 6 to 6.5 Gy in 5 weekly fractions (total dose, 32.5-30 Gy) over 5 weeks. Clinical end points were local control (LC), disease-free (DFS), cancer-specific (CSS), and overall survival (OS), and acute and late toxicity and cosmesis. Prognostic clinical variables were assessed with respect to DFS. RESULTS: Median follow-up was 46.5 months (range, 12-84 months). The 3- and 5-year LC rates were 99.5% (95% confidence interval [CI], 96.4-99.9) and 98% (95% CI, 91.1-99.6). The 3- and 5-year CSS and OS were 97.7% (95% CI, 94.5-99.1), 95.3% (95% CI, 90.5-97.7), 94.4% (95% CI, 90.4-96.7), and 83.6% (95% CI, 76.1-88.9), respectively. Maximum detected acute skin toxicity was Grade (G) 0 in 71.8% of patients, G1 in 22.6%, G2 in 4.8%, G3 in 1%, and G4 in 0.3%. Treatment interruption occurred in 2 patients because of severe skin reactions. Late skin toxicity consisted of G1 fibrosis in 31.5% of patients, G2 in 4.2%, and G3 in 3.5%. Grade 1 edema was observed in 7% of patients, G2 in 4.2%, and G3 in 1.4%. G1 telangiectasia occurred in 1.8% and G3 in 0.7%. G1 hyperpigmentation was found in 4.6% of patients, G2 in 2.4%, and G1 atrophy was detected in 2.1%. Pain was observed as G1 in 13%, G2 in 1.8%, and G3 in 0.4%. Cosmetic results were good to excellent in 86.4% and fair to poor in 13.6%. CONCLUSION: Once-weekly hypofractionated WBRT (30-32-5 Gy in 5 fractions), delivered with standard tangential fields with the patient in the supine position seems feasible and effective for a selected population of primarily old breast cancer patients with predominantly low-risk features. This schedule might allow fragile patients to receive adjuvant WBRT after BCS, increasing radiotherapy accessibility and utilization.

In vitro antitumor effects of the cold-water extracts of Mediterranean species of genus Pleurotus (higher Basidiomycetes) on human colon cancer cells.

The aim of this study was to evaluate whether the cold-water extracts of Pleurotus eryngii var. ferulae (CWE-Pef) and Pleurotus nebrodensis (CWE-Pn), 2 of the most prized wild and cultivated edible mushrooms, can affect the tumor phenotype of human colon cancer HCT116 cells. Our results showed that treatment with CWE-Pef and CWE-Pn resulted in a significant inhibition of the viability of HCT116 cells and promoted apoptosis, as also demonstrated by the increase of Bax-to-Bcl-2 messenger RNA ratio. Moreover, we observed that both extracts were able to inhibit cell migration and to affect homotypic and heterotypic cell-cell adhesion. It also was found that treatment with CWE-Pef and CWE-Pn negatively modulated the phosphorylation of the protein tyrosine as well as the phosphorylation levels of extracellular signal-regulated kinase 1/2. In conclusion, the in vitro antitumor effects of CWE-Pef and CWE-Pn indicate that they can be considered as possible sources for new alternative therapeutic agents for cancer treatment.

Familial hypobetalipoproteinemia due to apolipoprotein B R463W mutation causes intestinal fat accumulation and low postprandial lipemia.

OBJECTIVE: Familial hypobetalipoproteinemia (FHBL) is characterized by inherited low plasma levels of apolipoprotein B (apoB)-containing lipoproteins. In this paper we investigated whether the already described APOB R463W missense mutation, a FHBL mutation able to impair the activity of microsomal triglyceride transfer protein (MTP), may cause intestinal fat accumulation and reduced postprandial lipemia. METHODS: Four out of five probands harboring APOB R463W mutation were compared with six healthy controls and six patients with celiac disease (CD). An oral fat load supplemented with retinyl palmitate (RP) was administered and a gastro-duodenal endoscopy with biopsy was performed. RESULTS: Plasma triglyceride area under curves was significantly reduced in FHBL probands compared to controls and CD patients; the proportion of absorbed RP was similar to that of CD patients. Only the intestinal biopsies of FHBL patients showed lipids accumulating within the duodenal mucosa. CONCLUSIONS: FHBL due to R463W apoB mutation is a cause of intestinal fat accumulation and postprandial lipid absorption impairment.