Elena Dal Cin

PTX3 in small‐vessel vasculitides: An independent indicator of disease activity produced at sites of inflammation

OBJECTIVE To verify whether the prototypical long pentraxin PTX3 represents an indicator of the activity of small-vessel vasculitis. METHODS Concentrations of PTX3, a pentraxin induced in endothelium by cytokines, were measured by enzyme-linked immunosorbent assay in the sera of 43 patients with Churg-Strauss syndrome, Wegeners granulomatosis, and microscopic polyangiitis. PTX3 was also measured in the sera of 28 patients with systemic lupus erythematosus (SLE), 22 with rheumatoid arthritis, and 16 with CREST syndrome (calcinosis, Raynauds phenomenon, esophageal dysmotility, sclerodactyly, telangiectasias). Serum concentrations of C-reactive protein (CRP) were measured by immunoturbidimetry. The cells involved in PTX3 production in vivo were identified in skin biopsy samples. RESULTS Patients with active vasculitis had significantly higher concentrations of PTX3 than did those with quiescent disease (P < 0.001). PTX3 levels in the latter group were similar to those in healthy controls. PTX3 levels were higher in patients with untreated vasculitis and lower in patients who underwent immunosuppressive treatments (P < 0.005). In contrast, patients with active SLE had negligible levels of the pentraxin. PTX3 levels did not correlate with CRP levels in vasculitis patients. Endothelial cells produced PTX3 in active skin lesions. CONCLUSION PTX3 represents a novel acute-phase reactant produced at sites of active vasculitis.

Cutting Edge: Dissociation Between Autoimmune Response and Clinical Disease After Vaccination with Dendritic Cells

Autoimmunity represents a caveat to the use of dendritic cells (DCs) as adjuvant for human vaccines. We derived DCs from normal BALB/c mice or from mice prone to autoimmunity (NZB × NZW) F1. We allowed DCs to phagocytose apoptotic thymocytes and vaccinated syngeneic animals. All mice developed anti-nuclear and anti-dsDNA Abs. Autoantibodies in normal mice were transient, without clinical or histological features of autoimmunity or tissue involvement. In contrast, autoimmunity was maintained in susceptible mice, which underwent renal failure and precociously died. The data suggest that DC vaccination consistently triggers autoimmune responses. However, clinical autoimmunity develops in susceptible subjects only.

Antinociceptive effect of a new P2Z/P2X7 antagonist, oxidized ATP, in arthritic rats

The neurotransmitter adenosine triphosphate (ATP) is released from sensory nerve endings during inflammation and acts at the level of P2X receptors. We used the irreversible inhibitor of P2z/P2X7 receptor, designated oxidized ATP (oATP), to test its possible antinociceptive activity in arthritic rats. We induced unilateral inflammation of the rat hind paw by local injection of Freunds complete adjuvant. Administration of the adjuvant resulted in a significant reduction of paw pressure threshold (PPT). Injection of oATP into inflamed paws significantly increased, in a dose-dependent manner, PPT values to levels comparable with or higher than those evaluated in control uninflamed paws. The data indicate that the P2z/P2X7 receptor system exerts a role in nociception and that oATP, by inhibiting such a receptor, reduces the nociceptive signal in the course of peripheral inflammation.

Plasma and tissue expression of the long pentraxin 3 during normal pregnancy and preeclampsia

OBJECTIVE: Cell death normally occurs during pregnancy and is critical during its common complication, preeclampsia. The long pentraxin 3 (PTX3) gene is generated in tissues that cope with excessive or deregulated cell death and inhibits the cross-presentation of cell-associated antigens. We examined whether PTX3 is expressed during pregnancy and possibly involved in the development of preeclampsia. METHODS: Women with preeclampsia (n = 30), women with uncomplicated pregnancies (n = 66), age-matched healthy women (n = 50), women who developed acute bacterial infections (n = 20), and women with rheumatoid arthritis (n = 20) were studied. The concentrations of PTX3 were measured in the blood by a sandwich enzyme-linked immunosorbent assay (ELISA) and in placentas by immunohistochemistry. The concentrations of PTX3 and C-reactive protein in the various groups were compared by nonparametric tests (the Mann-Whitney U and the Kruskal-Wallis tests). The odds of developing preeclampsia were assessed using logistic regression. RESULTS: PTX3 was expressed in amniotic epithelium and chorionic mesoderm, trophoblast terminal villi, and perivascular stroma in placentas from pregnancies of uncomplicated subjects. Circulating levels steadily rose during normal gestation and peaked during labor. Serum levels of PTX3 were strikingly higher in preeclampsia compared with normal control pregnancies (5.08 ± 1.34 and 0.59 ± 0.07 ng/mL, respectively, P < .001). Sites of higher expression in the placentas from preeclamptic patients include infarcts and fibrinoid zones. CONCLUSION: Defects in the homeostatic response to cell death/remodeling events, revealed by enhanced levels of PTX3, could be implicated in preeclampsia. LEVEL OF EVIDENCE: II-2

Chlamydia Infection and Lymphomas: Association Beyond Ocular Adnexal Lymphomas Highlighted by Multiple Detection Methods

Purpose:Chlamydia psittaci (Cp) has been associated to ocular adnexal lymphomas (OAL) with variable geographic distribution. Herein, we used multiple Chlamydia detection tools to identify Cp elementary bodies–containing cell and to assess Cp prevalence in both nodal and extranodal lymphomas. Experimental Design: TETR-PCR, immunohistochemistry, immunofluorescence, electron microscopy, and laser-capture microdissection were done in 35 OALs to define their effect in Chlamydia detection and, moreover, to identify the Cp cellular carrier. Cp prevalence was screened by TETR-PCR in 205 extraorbital lymphomas and 135 nonneoplastic controls. Results: Twenty-six (74%) OALs were associated with Cp infection: immunohistochemistry, immunofluorescence, and laser-capture microdissection-assisted PCR showed that monocytes/macrophages were the Cp carriers; electron microscopy showed the presence of intact Cp elementary bodies into these cells. Immunohistochemistry and TETR-PCR showed a 70% concordance rate (P = 0.001). Cp DNA was equally prevalent in non-OAL, nodal, and extranodal lymphomas: among the latter, it was more common in diffuse large B-cell lymphomas of the skin (P = 0.03) and Waldeyers ring. Conclusions: This multiparametric approach shows, for the first time, that monocytes/macrophages are the carriers of Cp, Cp seems preferentially associated with lymphomas arising in organs primarily exposed to antigens. The clinical implications of these findings deserve to be prospectively investigated.

Mucin expression pattern in pancreatic diseases: findings from EUS-guided fine-needle aspiration biopsies.

OBJECTIVES:Alterations in mucin (MUC) glycosylation and expression have been described in cancer. Endoscopic ultrasound-guided fine-needle aspiration (EUS–FNA) can provide material for molecular biology analysis. This study assessed the feasibility of evaluating MUC expression from material obtained by EUS–FNA and studied the profile of MUC expression in benign and malignant pancreatic lesions.METHODS:A total of 90 patients with solid or cystic pancreatic lesions underwent FNA. The aspirated material was used for cytological analysis and RNA extraction to assess the expression pattern of MUCs by reverse transcription-PCR with primers specific for the MUC1, MUC2, MUC3, MUC4, MUC5A, MUC5B, MUC6, and MUC7 genes.RESULTS:RNA extraction was successful in 81% of the biopsies. The prevalences of MUC1, MUC2, MUC4, and MUC7 in ductal adenocarcinoma were 57.7, 51.4, 18.9, and 73.0%, respectively. Fifty percent of benign lesions and neuroendocrine tumors (NETs), and 63% of intraductal papillary mucinous neoplasms (IPMNs) were positive for MUC1. Twenty-five percent of benign lesions, 86% of NETs, and 47% of IPMNs were positive for MUC2. Of NETs, 50% were positive for MUC1, and 14% were positive for MUC7. None of the benign lesions or NETs expressed MUC4. MUC7 expression was highly significant for adenocarcinoma (P=0.007) and borderline for IPMN (P=0.05). MUC7 was expressed in 37.5% of chronic pancreatitis cases.CONCLUSIONS:RNA can be extracted from samples obtained under EUS–FNA. MUC7 could serve as a potential biological marker to identify malignant lesions, especially pancreatic adenocarcinoma.

Characterization of t(6;11)(p21;q12) in a renal‐cell carcinoma of an adult patient

Renal‐cell carcinoma (RCC) constitutes a heterogeneous group of tumors with specific chromosome aberrations. Recently, a new small group of RCC, occurring in children and young adults, has been described as characterized by t(6;11)(p21;q12). It has been shown that this translocation results in the fusion of the 5′ portion of the ALPHA gene (11q12) with the transcription factor gene TFEB (6p21). Herewith, we report the first complete cytogenetic and molecular characterization of a t(6;11)‐positive RCC of an adult patient, a 54‐year‐old woman. The tumor was histologically defined as RCC with peculiar features and it was negative for epithelial markers and positive for melanocytic markers. Chromosome QFQ banding analysis of short‐term cultured cells from the RCC showed t(6;11)(p21;q12) as the sole cytogenetic abnormality. The translocation was confirmed by FISH analysis. RT‐PCR analysis, performed on total RNA isolated from both neoplastic and normal tissue samples, revealed an ALPHA–TFEB chimeric transcript in the tumor sample; sequencing of the RT‐PCR product defined a novel TFEB gene breakpoint cluster region, broader than the one reported thus far. Western blot analysis showed a band at the expected size of wild‐type TFEB in the neoplastic tissue compared to the normal sample, supporting that the fusion gene does not encode for a chimeric protein but it causes an upregulation of the wild‐type TFEB. Our data contribute to define better this rare RCC type, which is typical not only of childhood but can also be found in adulthood.

Inhibition of chemokine expression in rat inflamed paws by systemic use of the antihyperalgesic oxidized ATP

BackgroundWe previously showed that local use of periodate oxidized ATP (oATP, a selective inhibitor of P2X7 receptors for ATP) in rat paw treated with Freunds adjuvant induced a significant reduction of hyperalgesia Herein we investigate the role of oATP, in the rat paws inflamed by carrageenan, which mimics acute inflammation in humans.ResultsLocal, oral or intravenous administration of a single dose of oATP significantly reduced thermal hyperalgesia in hind paws of rats for 24 hours, and such effect was greater than that induced by diclofenac or indomethacin. Following oATP treatment, the expression of the pro-inflammatory chemokines interferon-gamma-inducible protein-10 (IP-10), mon ocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) within the inflamed tissues markedly decreased on vessels and infiltrated cells. In parallel, the immunohistochemical findings showed an impairment, with respect to the untreated rats, in P2X7 expression, mainly on nerves and vessels close to the site of inflammation. Finally, oATP treatment significantly reduced the presence of infiltrating inflammatory macrophages in the paw tissue.ConclusionTaken together these results clearly show that oATP reduces carrageenan-induced inflammation in rats.

Central nervous system marginal zone B-cell lymphoma associated with Chlamydophila psittaci infection

Extranodal marginal zone B-cell lymphomas are linked to bacterial infections that vary according to the anatomical site. The occurrence of these lymphomas in the central nervous system is a very rare event, and the identification of specific bacteria in this setting has not been previously addressed. Herein, we report for the first time a case of primary central nervous system marginal zone B-cell lymphoma involving the choroid plexus associated with Chlamydophila psittaci infection. No concomitant ocular involvement was detected. C psittaci was identified with 3 independent methods, and through immunohistochemistry, it was visualized in the cytoplasm of monocytes/macrophages present within lymphomatous tissues. This observation points toward the opportunity to investigate the prevalence of C psittaci infection in central nervous system lymphomas, particularly in those with low-grade histologic features.

Constitutive overexpression of CDC25A in primary human mammary epithelial cells results in both defective DNA damage response and chromosomal breaks at fragile sites

CDC25A phosphatase, an essential component of the cell cycle machinery, is also a key player in integrating the specific signals of checkpoint control in response to DNA damage. There are several lines of evidence that indicate a role for CDC25A in cancer development, consistent with the fact that its overexpression is detected in human cancers. In particular we previously reported that CDC25A is overexpressed also in early breast carcinoma. Recent data suggest that oncogene activation during early stages of tumor development causes DNA replication stress resulting in the induction of DNA damage response (DDR) and that the selection of cells defecting in their DDR could lead to malignant progression. To address how CDC25A overexpression contributes to breast cancer development we established a cell model in which CDC25A was constitutively overexpressed in hTERT‐immortalized primary human mammary epithelial cells. At the earliest passages following CDC25A transduction we observed DDR signs associated with unscheduled DNA replication origins. In the latest passages DDR was significantly impaired and, even after ionizing radiation exposition, cells failed to induce G1 and G2 checkpoints; moreover DNA replication stress conditions, such as aphidicolin treatment, highlighted increased fragile site breakages and destabilized chromosomes just in these latest passages cells. Our data suggest that CDC25A overexpression, pushing the cell through the cell cycle transitions, induces DDR alterations that might enhance genomic instability.