Targeting IL-6 signalling in early rheumatoid arthritis is followed by Th1 and Th17 suppression and Th2 expansion

OBJECTIVES: To investigate the in vitro and ex-vivo effect of IL-6 inhibition on the balance between Th1, Th2, Th17 and Treg cells. METHODS: Ten consecutive adult patients with active early rheumatoid arthritis (ERA) and ten healthy volunteers were included in the study. The percentages of Th1, Th2, Th17 and Treg cells were analysed by flow cytometry in the peripheral blood mononuclear cells obtained from controls and from RA patients at the time of first evaluation and just before the third TCZ infusion. The in vitro effect of TCZ on the different subsets of CD4+ T cells and the expression levels of Th1, Th2, Th17 and Treg-related cytokines was also assessed. RESULTS: Treatment with TCZ, both ex vivo and in vitro, resulted in a significant reduction of the percentage of Th1, Th17 and Treg cells with a concomitant significant increase of Th2 cell subsets. The reduction of the different subsets of T lymphocytes was associated with an intense staining with Annexin V, suggesting an apoptotic-related cell reduction. A significant decrease of Th1, Th17 and Treg cytokines and a concomitant increase of IL-4 was also observed after TCZ treatment in PBMC isolated from RA patients. CONCLUSIONS: TCZ could modify the immune imbalance in RA inducing apoptosis of Th1, Th17 and Treg cells and promoting the appearance of a Th2 response

Invariant NKT cells are expanded in peripheral blood but are undetectable in salivary glands of patients with primary Sj\uf6gren's syndrome.

OBJECTIVES: Invariant NKT (iNKT) cells play a role in regulating the function of autoreactive B cells before their entry into germinal centres. Absence and/or reduction of iNKT cells have been demonstrated in patients with systemic lupus erythematosus (SLE) together with an increase of autoreactive B cell activity. Primary Sj\uf6gren's syndrome (pSS) is a systemic autoimmune disease in which lymphocyte infiltration and organisation in lymphoid structures of inflamed salivary glands occurs. The aim of the study was to investigate the percentage and function of iNKT in the salivary glands and peripheral blood of patients with pSS. METHODS: Minor salivary gland biopsies were obtained from patients with pSS and with non-specific chronic sialoadenitis (nSS). Flow cytometry analysis of CD1d/\u3b1-GalactosylCeramide (\u3b1-GalCer) tetramer positive cells, producing IFN-\u3b3 and IL-17, and quantitative gene expression analysis by TaqMan real-time PCR for V\u3b124 were performed on salivary glands biopsies and peripheral blood samples obtained from patients and controls. Flow cytometry and immunofluorescence analysis for autoreactive B lymphocytes and ELISA for anti-SSA antibodies (Ab) detection were also performed. RESULTS: An increase of iNKT was detected ex vivo in peripheral blood of pSS patients; after \u3b1-GalCer stimulation this subset produce IL-17 and IFN-iNKT were undetectable in the salivary glands of pSS patients and anti-SSA specific B cells were found in target tissue. Invariant NKT cells were able to inhibit autoantibody production by B cells obtained from salivary glands of pSS. CONCLUSIONS: Impaired iNKT migration to inflamed sites might induce the activation of autoreactive B cells specific for SSA-antigen in salivary glands of pSS 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

Macrophage phenotype in the subclinical gut inflammation of patients with ankylosing spondylitis

OBJECTIVE: Long-term evolution of subclinical gut inflammation to overt Crohn's disease (CD) has been described in AS patients. The aim of this study was to evaluate macrophage polarization occurring in the inflamed gut of patients with AS. METHODS: Twenty-seven HLA-B27(+) AS patients, 20 CD patients and 17 normal controls were consecutively enrolled. Classic M1 (iNOS(+)IL-10(-)), resolution phase (iNOS(+)IL-10(+)), M2 and CD14(+) macrophages were characterized by immunohistochemistry and flow cytometry. Quantitative gene expression analysis of IFN-γ, IL-4, IL-5, IL-33 and STAT6 was performed by real time PCR. RESULTS: Classic M1 macrophages were expanded in CD and AS, where resolution phase macrophages predominate. A large increase in CD163(+) (M2) macrophages was observed in AS strictly correlated with the expression of IL-33, a Th2 cytokine involved in M2 polarization. Unlike in CD, CD14(+) macrophages were virtually absent in the gut of AS patients and controls. CONCLUSION: The absence of CD14(+) macrophages together with the expansion of resolution phase and M2 macrophages is the immunological signature of subclinical ileal inflammation in AS

Detection rate for significant cancer at confirmatory biopsy in men enrolled in Active Surveillance protocol: 20 cores vs 30 cores vs MRI/TRUS fusion prostate biopsy

Introduction: The detection rate for significant prostate cancer of extended vs saturation vs mMRI/TRUS fusion biopsy was prospectively evaluated in men enrolled in active surveillance (AS) protocol. Mterials and methods: From May 2013 to September 2016 75 men aged 66 years (median) with very low risk PCa were enrolled in an AS protocol and elegible criteria were: life expectancy greater than 10 years, cT1C, PSA below 10 ng/ml, PSA density < 0.20, 2 < unilateral positive biopsy cores, Gleason score (GS) equal to 6, greatest percentage of cancer (GPC) in a core < 50%. All patients underwent 3.0 Tesla pelvic mpMRI before confirmatory transperineal extended (20 cores) or saturation biopsy (SPBx; 30 cores) combined with mpMRI/TRUS fusion targeted biopsy (4 cores) of suspicious lesions (PI-RADS 3-5). Results: 21/75 (28%) patients were reclassified by SPBx based on upgraded GS ≥ 7; mpMRI lesions PI-RADS 4-5 vs PI-RADS 3-5 diagnosed 9/21 (42.8%) vs 16/21 (76.2%) significant PCa with 2 false positives (6.5%). The detection rate for significant PCa was equal to 76.2% (mpMRI/TRUS fusion biopsy) vs 81% (extended) vs 100% (SPBx) (p = 0.001); mpMRI/TRUS targeted biopsy and extended biopsy missed 5/21 (23.8%) and 4/21 (19%) significant PCa which were found by SPBx (p = 0.001) being characterised by the presence of a single positive core of GS ≥ 7 with GPC < 10%. Conclusions: Although mpMRI improve the diagnosis of clinically significant PCa, SPBx is provided of the best detection rate for PCa in men enrolled in AS protocols who underwent confirmatory biopsy

Tailored treatment including radical prostatectomy and radiation therapy + androgen deprivation therapy versus exclusive radical prostatectomy in high-risk prostate cancer patients: results from a prospective study

Purpose To evaluate outcomes of patients with high risk prostate cancer (PCa) who underwent radical prostatectomy (RP) in a context of a multidisciplinary approach including adjuvant radiation (RT) + androgen deprivation therapy (ADT). Matherials and Methods 244 consecutive patients with high risk localized PCa underwent RP and bilateral extended pelvic lymph node dissection at our institution. Adjuvant RT + 24 months ADT was carried out in subjects with pathological stage ≥ T3N0 and/or positive surgical margins or in patients with local relapse. Results After a median follow-up was 54.17 months (range 5.4-117.16), 13 (5.3%) subjects had biochemical progression, 21 (8.6%) had clinical progression, 7 (2.9%) died due to prostate cancer and 15 (6.1%) died due to other causes. 136 (55.7%) patients did not receive any adjuvant treatment while 108 (44.3%) received respectively adjuvant or salvage RT+ADT. Multivariate Cox proportional hazard analysis showed that pre-operative PSA value at diagnosis is a significant predictive factor for BCR (HR: 1.04, p < 0.05) and that Gleason Score 8-10 (HR: 2.4; p<0.05) and PSMs (HR: 2.01; p < 0.01) were significant predictors for clinical progression. Radical prostatectomy group was associated with BPFS, CPFS, CSS and OS at 5-years of 97%, 90%, 95% and 86% respectively, while adjuvant radiation + androgen deprivation therapy group was associated with a BPFS, CPFS and CSS at 5-years of 91%, 83%, 95% and 88%, without any statistical difference. Conclusions Multimodality tailored treatment based on RP and adjuvant therapy with RT+ADT achieve similar results in terms of OS after 5-years of follow-up

Protective, Antioxidant and Antiproliferative Activity of Grapefruit IntegroPectin on SH-SY5Y Cells

Tested in vitro on SH-SY5Y neuroblastoma cells, grapefruit IntegroPectin is a powerful protective, antioxidant and antiproliferative agent. The strong antioxidant properties of this new citrus pectin, and its ability to preserve mitochondrial membrane potential and morphology, severely impaired in neurodegenerative disorders, make it an attractive therapeutic and preventive agent for the treatment of oxidative stress-associated brain disorders. Similarly, the ability of this pectic polymer rich in RG-I regions, as well as in naringin, linalool, linalool oxide and limonene adsorbed at the outer surface, to inhibit cell proliferation or even kill, at high doses, neoplastic cells may have opened up new therapeutic strategies in cancer research. In order to take full advantage of its vast therapeutic and preventive potential, detailed studies of the molecular mechanism involved in the antiproliferative and neuroprotective of this IntegroPectin are urgently needed

ABERRANT EXPRESSION OF IL-22RA1 ON HEMATOPOIETIC CELLS AS IMMUNOLOGICALLY SIGNATURE OF PRIMARY SJOGREN\u2019S SYNDROME AND SJOGREN-ASSOCIATED NON-HODGKIN LYMPHOMAS

Background: Interleukin (IL)-22 is a potent mediator of cellular inflammatory responses that has been recently reported to play a role in the pathogenesis of primary Sjogren's Syndrome (p-SS) (1, 2) and of T and B lymphomas. IL-22 biological activity is initiated by binding to a cell-surface complex composed of two subunits, IL-22R1 and IL-10R2 receptor chains, and further regulated by interactions with a soluble binding protein, IL-22BP. Unlike the IL-10R2, which is constitutively expressed in many human tissues, IL-22R1 is not detectable in immune cells. Objectives: Aim of this study was to better characterize the role of IL-22 axis in the pathogenesis of p-SS and p-SSassociated lymphomas. Methods: Minor salivary gland biopsies were obtained from 30 patients with p-SS and 20 with non-specific chronic sialoadenitis (n-SS) and evaluated by RT-PCR for IL-22, IL-22R1 and IL-22BP expression. The protein expression of IL-22, IL- 22R1 and p-STAT3 was evaluated by immunohistochemistry and IL-22R1-expressing cells were caharcterized by confocal microscopy. Flow cytometry analysis of IL-22R1 expression was also conducted on peripheral blood mononuclear cells (PBMCs) from p-SS (n=30), n-SS (n=20), SLE (n=20) and rheumatoid arthritis (n=20) patients and normal controls (n=30). PBMCs isolated from 10 p-SS and 10 controls were also cultured with (or without) recombinant IL- 22 and the modulation of the transcripts for pro-inflammatory cytokines was assessed by RT-PCR. Paraffin embedded sections of non-Hodgkin lymphomas from 5 p-SS patients were finally evaluated for the expression of IL-22R1. Results: IL-22, STAT3 and IL-22BP but not IL-22R1 transcript levels were significantly up-regulated in the inflamed salivary glands of p-SS but not in n-SS. Immunohistochemistry confirmed the increased salivary expression of IL-22 and p-STAT3 also demonstrating a significant increased protein levels of IL-22R1 in p-SS. Confocal microscopy analysis showed that IL-22R1 was aberrantly and strongly expressed in monocytes/macrophages infiltrating the p-SS salivary glands and these cells also co-expressed p-STAT3, suggesting the occurrence of autocrine activation of p-STAT3. CD68+ cells obtained from the peripheral blood also aberrantly expressed IL-22R1 in p-SS patients. IL-22R1 expression on cells of hematopoietic origin was never observed in control disease patients, n-SS and healthy controls. The stimulation with recombinant IL-22 of PBMCs from p-SS but not controls significantly up-regulated the expression of IL-17 and IL-22. Non-Hodgkin lymphoma tissues from p-SS patients were also characterized by the aberrant expression of IL- 22R1 on macrophages and neoplastic B cells. Conclusions: The aberrant expression of IL-22RA1 on cells of hematopoietic origin seems to be a specific immunological signature of patients with p-SS and p-SS associated lymphomas and suggests a fundamental role of IL-22 signaling in the pathogenesis of p-SS and its neoplastic evolution. Targeting of IL-22 pathway may represent a successful therapeutic strategy in p-SS patients. References: 1. Ciccia F, et al. Ann RheumDis 2012. 2. Ciccia F, et al. Ann Rheum Dis 2012

A test on Ellenberg indicator values in the Mediterranean evergreen woods (Quercetea ilicis)

The consistency and reliability of Ellenberg’s indicator values (Eiv) as ecological descriptors of the Mediterranean evergreen vegetation ascribed to the phytosociological class Quercetea ilicis have been checked on a set of 859 phytosociological relevés × 699 species. Diagnostic species were identified through a Twinspan analysis and their Eiv analyzed and related to the following independent variables: (1) annual mean temperatures, (2) annual rainfall. The results provided interesting insights to disentangle the current syntaxonomical framework at the alliance level demonstrating the usefulness of ecological indicator values to test the efficiency and predictivity of the phytosociological classification

Tailored treatment including radical prostatectomy and radiation therapy + androgen deprivation therapy versus exclusive radical prostatectomy in high-risk prostate cancer patients: results from a prospective study

Purpose To evaluate outcomes of patients with high risk prostate cancer (PCa) who underwent radical prostatectomy (RP) in a context of a multidisciplinary approach including adjuvant radiation (RT) + androgen deprivation therapy (ADT). Matherials and Methods 244 consecutive patients with high risk localized PCa underwent RP and bilateral extended pelvic lymph node dissection at our institution. Adjuvant RT + 24 months ADT was carried out in subjects with pathological stage ≥ T3N0 and/or positive surgical margins or in patients with local relapse. Results After a median follow-up was 54.17 months (range 5.4-117.16), 13 (5.3%) subjects had biochemical progression, 21 (8.6%) had clinical progression, 7 (2.9%) died due to prostate cancer and 15 (6.1%) died due to other causes. 136 (55.7%) patients did not receive any adjuvant treatment while 108 (44.3%) received respectively adjuvant or salvage RT+ADT. Multivariate Cox proportional hazard analysis showed that pre-operative PSA value at diagnosis is a significant predictive factor for BCR (HR: 1.04, p < 0.05) and that Gleason Score 8-10 (HR: 2.4; p<0.05) and PSMs (HR: 2.01; p < 0.01) were significant predictors for clinical progression. Radical prostatectomy group was associated with BPFS, CPFS, CSS and OS at 5-years of 97%, 90%, 95% and 86% respectively, while adjuvant radiation + androgen deprivation therapy group was associated with a BPFS, CPFS and CSS at 5-years of 91%, 83%, 95% and 88%, without any statistical difference. Conclusions Multimodality tailored treatment based on RP and adjuvant therapy with RT+ADT achieve similar results in terms of OS after 5-years of follow-up