[Show abstract][Hide abstract] ABSTRACT: Disclosure of potential conflict of interest: M. Gattorno has received consultancy fees, research support, and lecture fees from Novartis and SOBI. C. Rodríguez-Gallego and C. Hinze have received lecture fees from Novartis. J. A. Bernstein has received consultancy fees from McKesson, research support from the National Institutes of Health (NIH) and CIRM, and lecture fees from the University of Minnesota. J. A. Church has received research support from Pfizer and BioProducts Laboratory. R. Skinner has provided expert testimony for the Medical Defence Union and the NHS Health Service and has received research support from Newcastle upon Tyne Hospitals NHS Foundation Trust, Malawi-Royal Victoria Infirmary, the Children's Cancer Fund, and Children with Cancer UK. The rest of the authors declare that they have no relevant conflicts of interest.
The Journal of allergy and clinical immunology 05/2015; DOI:10.1016/j.jaci.2015.04.016 · 11.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: OBJECTIVES: Tumour necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS) is caused by TNFRSF1A mutations, known to induce intracellular retention of the TNFα receptor 1 (TNFR1) protein, defective TNFα-induced apoptosis, and production of reactive oxygen species. As downregulation of autophagy, the main cellular pathway involved in insoluble aggregate elimination, has been observed to increase the inflammatory response, we investigated whether it plays a role in TRAPS pathogenesis. METHODS: The possible link between TNFRSF1A mutations and inflammation in TRAPS was studied in HEK-293T cells, transfected with expression constructs for wild-type and mutant TNFR1 proteins, and in monocytes derived from patients with TRAPS, by investigating autophagy function, NF-κB activation and interleukin (IL)-1β secretion. RESULTS: We found that autophagy is responsible for clearance of wild-type TNFR1, but when TNFR1 is mutated, the autophagy process is defective, probably accounting for mutant TNFR1 accumulation as well as TRAPS-associated induction of NF-κB activity and excessive IL-1β secretion, leading to chronic inflammation. Autophagy inhibition due to TNFR1 mutant proteins can be reversed, as demonstrated by the effects of the antibiotic geldanamycin, which was found to rescue the membrane localisation of mutant TNFR1 proteins, reduce their accumulation and counteract the increased inflammation by decreasing IL-1β secretion. CONCLUSIONS: Autophagy appears to be an important mechanism in the pathogenesis of TRAPS, an observation that provides a rationale for the most effective therapy in this autoinflammatory disorder. Our findings also suggest that autophagy could be proposed as a novel therapeutic target for TRAPS and possibly other similar diseases.
Annals of the rheumatic diseases 10/2012; 72(6). DOI:10.1136/annrheumdis-2012-201952 · 10.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The immune system consists of 2 branches: innate and adaptive. The former represents the first line of host defense during infection and plays a key role in the early recognition and protection against invading pathogens. The latter orchestrates elimination of pathogens in the late phase of infection and leads to the generation of immunologic memory. Innate and adaptive immunity should not be considered separate compartments. Innate and adaptive immune responses represent an integrated system of host defense. The authors review the mechanisms driving the induction and perpetuation of the inflammatory responses observed during pathogen-associated, autoimmune, and autoinflammatory diseases.
Pediatric Clinics of North America 04/2012; 59(2):225-43. DOI:10.1016/j.pcl.2012.03.004 · 2.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dendritic cells (DC) are highly specialized antigen-presenting cells characterized by the ability to prime T-cell responses. Mesenchymal stem cells (MSC) are adult stromal progenitor cells displaying immunomodulatory activities including inhibition of DC maturation in vitro. However, the specific impact of MSC on DC functions, upon in vivo administration, has never been elucidated. Here we show that murine MSC impair Toll-like receptor-4 induced activation of DC resulting in the inhibition of cytokines secretion, down-regulation of molecules involved in the migration to the lymph nodes, antigen presentation to CD4(+) T cells, and cross-presentation to CD8(+) T cells. These effects are associated with the inhibition of phosphorylation of intracellular mitogen-activated protein kinases. Intravenous administration of MSC decreased the number of CCR7 and CD49dβ1 expressing CFSE-labeled DC in the draining lymph nodes and hindered local antigen priming of DO11.10 ovalbumin-specific CD4(+) T cells. Upon labeling of DC with technetium-99m hexamethylpropylene amine oxime to follow their in vivo biodistribution, we demonstrated that intravenous injection of MSC blocks, almost instantaneously, the migration of subcutaneously administered ovalbumin-pulsed DC to the draining lymph nodes. These findings indicate that MSC significantly affect DC ability to prime T cells in vivo because of their inability to home to the draining lymph nodes and further confirm MSC potentiality as therapy for immune-mediated diseases.
Proceedings of the National Academy of Sciences 09/2011; 108(42):17384-9. DOI:10.1073/pnas.1103650108 · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: T helper 17 cells (T(H)-17) represent a lineage of effector T cells critical in host defence and autoimmunity. In both mouse and human IL-1β has been indicated as a key cytokine for the commitment to T(H)-17 cells. Cryopyrin-associated periodic syndromes (CAPS) are a group of inflammatory diseases associated with mutations of the NLRP3 gene encoding the inflammasome component cryopyrin. In this work we asked whether the deregulated secretion of IL-1β secondary to mutations characterizing these patients could affect the IL-23/IL-17 axis.
A total of 11 CAPS, 26 systemic onset juvenile idiopathic arthritis (SoJIA) patients and 20 healthy controls were analyzed. Serum levels of IL-17 and IL-6 serum were assessed by ELISA assay. Frequency of T(H)17 cells was quantified upon staphylococcus enterotoxin B (SEB) stimulation. Secretion of IL-1β, IL-23 and IL-6 by monocyte derived dendritic cells (MoDCs), were quantified by ELISA assay. A total of 8 CAPS and 11 SoJIA patients were also analysed before and after treatment with IL-1β blockade. Untreated CAPS patients showed significantly increased IL-17 serum levels as well as a higher frequency of T(H)17 compared to control subjects. On the contrary, SoJIA patients displayed a frequency of T(H)17 similar to normal donors, but were found to have significantly increased serum level of IL-6 when compared to CAPS patients or healthy donors. Remarkably, decreased IL-17 serum levels and T(H)17 frequency were observed in CAPS patients following in vivo IL-1β blockade. On the same line, MoDCs from CAPS patients exhibited enhanced secretion of IL-1β and IL-23 upon TLRs stimulation, with a reduction after anti-IL-1 treatment.
These findings further support the central role of IL-1β in the differentiation of T(H)17 in human inflammatory conditions.
PLoS ONE 05/2011; 6(5):e20014. DOI:10.1371/journal.pone.0020014 · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: NLRP12 mutations have been described in patients affected with peculiar autoinflammatory symptoms. This study was undertaken to characterize NLRP12 mutations in patients with autoinflammatory syndromes, particularly a novel missense mutation, p.D294E, affecting a protein sequence crucial for ATP binding, which was identified in a Caucasian family with familial cold-induced autoinflammatory syndrome in some family members.
Fifty patients were tested for NLRP12 mutations. A Caucasian family with the p.D294E missense mutation of NLRP12 in some family members was clinically characterized. In vitro analysis of the effects of the mutation on NF-κB activity was performed in HEK 293 cells after cotransfection of the cells with a luciferase NF-κB-responsive element and mutant or wild-type (WT) NLRP12 expression plasmids. NF-κB activity was also evaluated 24 hours after stimulation with tumor necrosis factor α in monocytes from individual family members carrying the mutation. Furthermore, secretion of interleukin-1β (IL-1β), production of reactive oxygen species (ROS), and activation of antioxidant systems in patient and healthy donor monocytes, under resting conditions and after stimulation with pathogen-associated molecular patterns (PAMPs), were also assessed.
In the family assessed, the p.D294E mutation segregated in association with a particular sensitivity to cold exposure (especially arthralgias and myalgia), but not always with an inflammatory phenotype (e.g., urticarial rash or fever). In vitro, the mutant protein maintained the same inhibitory activity as that shown by WT NLRP12. Consistently, NLRP12-mutated monocytes showed neither increased levels of p65-induced NF-κB activity nor higher secretion of IL-1β. However, the kinetics of PAMP-induced IL-1β secretion were significantly accelerated, and high production of ROS and up-regulation of antioxidant systems were demonstrated.
Even with a variable range of associated manifestations, the extreme sensitivity to cold represents the main clinical hallmark in an individual carrying the p.D294E mutation of the NLRP12 gene. Although regulation of NF-κB activity is not affected in patients, redox alterations and accelerated secretion of IL-1β are associated with this mild autoinflammatory phenotype.
[Show abstract][Hide abstract] ABSTRACT: Recent reports have highlighted that adult stem cells are granted with yet poorly understood properties other than multipotentiality. In particular, mesenchymal stem cells (MSCs) represent a subset of adult stromal cells that can down-regulate several functions of the immune cells. In addition, MSCs may promote survival of damaged cells and tissues through paracrine mechanisms, possibly under the guidance of environmental cues. Thus, MSCs clinical application in autoimmune diseases seems an appealing opportunity and preclinical results in different experimental models of autoimmunity further support this strategy. Despite the absolute need for caution related to several clinical and technical issues, MSCs are now on the edge of a new era of clinical applications.
[Show abstract][Hide abstract] ABSTRACT: Several observations suggest a potential role of T-cell-mediated immunity in the control of neuroblastoma (NB). However, the generation of NB-specific cytotoxic T lymphocytes (CTL) on T-cell priming with tumor mRNA-transfected dendritic cells (DC) has never been investigated before. In the present study, the feasibility of this strategy has been analyzed, both in healthy donors and in NB patients. Monocyte-derived DC were raised from three human leukocyte antigen (HLA) A2+ NB patients and seven HLA-A1+ or HLA-A2+ healthy donors transfected with mRNA from four NB cell lines and cocultured with autologous CD8+ lymphocytes. Expanded CTL expressed an effector/memory phenotype and a T cytotoxic 1-like profile of cytokine secretion. CTL specificity was demonstrated by interferon-gamma release on incubation with HLA-matched NB cell lines. The latter cell lines, but not autologous T-cell blasts, were lysed by CTL in an HLA-restricted manner. Cytotoxicity was found to involve the release of granzyme B. When tested for reactivity against NB-associated antigens, CTL from normal individuals recognized anaplastic lymphoma-associated kinase (ALK) and preferentially expressed antigen of melanoma (PRAME) peptides only, whereas patients' CTL reacted also to survivin, telomerase, and tyrosine hydroxylase peptides. This study demonstrates that DC transfected with NB mRNA induce the generation of patients' CTL specific for different NB-associated antigens, supporting the feasibility of NB T-cell immunotherapy.
Neoplasia (New York, N.Y.) 11/2006; 8(10):833-42. DOI:10.1593/neo.06415 · 5.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to investigate T cell immunity to Toxoplasma gondii (Tg) in pregnant women with primary toxoplasmosis. This issue has never been addressed before in humans and available information derives from murine models. Peripheral blood mononuclear cells (PBMC) from pregnant women with primary Tg infection were stimulated with Tg tachyzoites, excretory-secretory antigens (ESA) or recombinant surface antigen-1 (rSAG-1), and tested for proliferation, immunophenotype, cytokine production and antigen specific cytotoxic activity. Pregnant women with primary toxoplasmosis displayed a significant decrease of the CD4/CD8 T cell ratio and a significant increase of circulating T cell receptor (TCR) gammadelta+ cells as compared to their uninfected counterparts. T cells from Tg infected pregnant women proliferated to Tg tachyzoites, ESA or rSAG-1. Most tachyzoite and ESA specific T cell blasts were CD4+, whereas SAG-1 specific blasts were CD4+ and CD8+. ESA and tachyzoite specific T cell blasts displayed a Th1 or Th0 cytokine profile with overexpression of IFN-gamma. This pattern was unchanged upon in vitro exposure of T cells to progesterone, tested at a concentration close to that reached in vivo at the maternal-fetal interface. Finally, tachyzoite or ESA specific T cell blasts lysed, through a granule exocytosis dependent mechanism, autologous lymphoblastoid cell lines presenting Tg antigens. In conclusion, pregnant women with primary toxoplasmosis mounted in vitro Tg-specific Th1/Th0 responses whose impact on neonatal infection warrants further investigation.
Microbes and Infection 03/2006; 8(2):552-60. DOI:10.1016/j.micinf.2005.08.008 · 2.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of the study was to characterise CCR7+ and CCR7- memory T cells infiltrating the inflamed joints of patients with juvenile idiopathic arthritis (JIA) and to investigate the functional and anatomical heterogeneity of these cell subsets in relation to the expression of the inflammatory chemokine receptors CXCR3 and CCR5. Memory T cells freshly isolated from the peripheral blood and synovial fluid (SF) of 25 patients with JIA were tested for the expression of CCR7, CCR5, CXCR3 and interferon-gamma by flow cytometry. The chemotactic activity of CD4 SF memory T cells from eight patients with JIA to inflammatory (CXCL11 and CCL3) and homeostatic (CCL19, CCL21) chemokines was also evaluated. Paired serum and SF samples from 28 patients with JIA were tested for CCL21 concentrations. CCR7, CXCR3, CCR5 and CCL21 expression in synovial tissue from six patients with JIA was investigated by immunohistochemistry. Enrichment of CD4+, CCR7- memory T cells was demonstrated in SF in comparison with paired blood from patients with JIA. SF CD4+CCR7- memory T cells were enriched for CCR5+ and interferon-gamma+ cells, whereas CD4+CCR7+ memory T cells showed higher coexpression of CXCR3. Expression of CCL21 was detected in both SF and synovial membranes. SF CD4+ memory T cells displayed significant migration to both inflammatory and homeostatic chemokines. CCR7+ T cells were detected in the synovial tissue in either diffuse perivascular lymphocytic infiltrates or organised lymphoid aggregates. In synovial tissue, a large fraction of CCR7+ cells co-localised with CXCR3, especially inside lymphoid aggregates, whereas CCR5+ cells were enriched in the sublining of the superficial subintima. In conclusion, CCR7 may have a role in the synovial recruitment of memory T cells in JIA, irrespective of the pattern of lymphoid organisation. Moreover, discrete patterns of chemokine receptor expression are detected in the synovial tissue.
[Show abstract][Hide abstract] ABSTRACT: It has been suggested that CD45RO+CD27+ T cells represent recently activated memory cells, whereas CD45RO+CD27- T cells are activated memory T cells in the process of differentiating into effector cells. We investigated (1) CCR7 and CCR5 expression and (2) modulation of cytokine expression in "early" (CD27+) and "differentiated" (CD27-) memory CD4+ T cells from peripheral blood and synovial fluid (SF) of patients with juvenile idiopathic arthritis (JIA).
SF CD4+CD45RO+CD27+ and CD27- memory T cells from 6 patients with JIA were tested by flow cytometry for intracellular interferon-gamma (IFN-gamma) and interleukin 4 (IL-4) after in vitro priming with CD3 and CD28 mAb in the presence of IL-4, and subsequent culture with IL-2.
SF CD4+CD45RO+CD27+ cells contained higher proportions of CCR7+ (median 46% vs 23%) and lower proportions of CCR5+ (73% vs 90%) cells than paired CD27- T cells. Both CD27+ and CD27- memory T helper cells from SF displayed a higher IFN-gamma/IL-4 ratio than their peripheral blood counterparts. No significant difference was observed in the percentage of IFN-gamma-expressing cells between CD27+ (32%, range 4-47%) and CD27- (29.4%, range 5-52%) memory T helper cells from SF.
Irrespective of their differentiation stage, both CD27+ and CD27- SF memory T helper cells were found to switch from a proinflammatory to an antiinflammatory pattern of cytokine production.
The Journal of Rheumatology 11/2004; 31(10):2048-54. · 3.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Peritoneal T cell responses can be polarized toward Th1 or Th2 in children on chronic peritoneal dialysis. Previous studies on the peritoneal immune system described the presence of activated T lymphocytes in peritoneal effluents from subjects on chronic peritoneal dialysis (CPD). Since Th1/Th2 polarized response can influence the outcome of specific infectious diseases, we investigated if activated Th1/Th2 cells can be detected in peritoneal effluents during peritoneal dialysis, in order to better understand the role of T cells in the mechanisms of peritoneal defense. We have studied 8 children (4 males, 4 females, mean age 5.8 +/- 5.7 years, range 0.3-13.4) on CPD. Peritoneal cells have been isolated from peritoneal effluents by centrifugation. Immunofluorescent staining of intracellular cytokines for flow cytometric analysis was used to detect the percentage of T cells producing either IFN-gamma (Th1) or IL-4 (Th2). In the initial study 3 months after CPD initiation, high percentages of IFN-gamma positive peritoneal T cells (38% and 63%) were detected in two subjects; this finding is consistent with a Th1 polarization of peritoneal T cells. In another subject, high percentages of IL-4 positive T cells (31%) were detected, suggesting a Th2 polarization of peritoneal T cell response. Small amounts of either Th1 or Th2 T cells (2-4%) were also detected in the other subjects. At the 1 year follow-up, Th1 polarization persisted in one subject (18% IFN-gamma positive peritoneal T cells), in another a shift from Th1 to Th2 was observed, and in the other subject a down regulation of both T cell subsets occurred. The finding that a predominance of T cells producing either IFN-gamma or IL-4 was found in 3 out of 8 children strongly suggests that peritoneal T cell responses can be polarized toward Th1 or Th2. The decrease of Th1 and/or Th2 polarized T cells in the peritoneum of 4 out of 6 subjects (after 1 year) suggests that CPD can play an immunosuppressive role on T cell peritoneal responses. Further studies are needed in order to define whether different T helper activation patterns are associated with a higher risk of peritoneal infection or of peritoneal damage.
[Show abstract][Hide abstract] ABSTRACT: Alendronate treatment for 12 months in pediatric patients with rheumatic diseases and secondary low bone mass was reported to result in a substantial increase in bone mineral density (BMD). In this study, we evaluated the changes in bone metabolism and disease activity markers in 45 patients ages 5 to 18 years (31 female, 14 male) with rheumatic diseases treated with alendronate for 12 months.
Variables analyzed included demographic and anthropometric data, biochemical markers of bone metabolism, disease activity indexes, and BMD values. For all variables, the differences between levels at baseline and at 12 months were calculated; correlations between the variables and between the BMD variation over 12 months and baseline levels of the different variables were also evaluated.
There was a statistically significant decrease of both bone resorption and bone formation markers over the 12 month treatment period. By contrast, no disease activity index changed significantly over one year. BMD Z score change over one year did not correlate with erythrocyte sedimentation rate, matrix metalloproteinase-3, interleukin 6, or C-reactive protein variations over the same period.
These results support the conclusion that alendronate treatment is accompanied by a reduction of bone turnover in pediatric patients and that the observed BMD increase is not secondary to a reduction of inflammatory activity.
The Journal of Rheumatology 09/2002; 29(8):1786-92. · 3.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is characterized by hypogammaglobulinemia and recurrent bacterial infections. Here we report a novel case of ICF syndrome with hypogammaglobulinemia and an inverted CD4/CD8 ratio. Cytogenetically abnormal cells,that were identified in both CD4+ and CD4- peripheral blood lymphocytes, retained their ability to proliferate in vitro following polyclonal stimulation. A primitive defect of B-cell differentiation was detected.
[Show abstract][Hide abstract] ABSTRACT: CD27 is a member of tumour necrosis factor receptor family. Its expression is predominantly confined to mature lymphocytes and is strongly enhanced after cell activation. Shedding of the CD27 from the surface of activated cells is related to their effector phase. The aim of the present study was to evaluate the levels of soluble CD27 in sera and synovial fluids, together with its expression on circulating and synovial fluid (SF) memory T cells, in children with JIA.
Sera from 40 patients with active JIA were studied for soluble CD27. Paired SF samples were available in 20 patients. Sera from 12 age-matched patients affected with various acute infectious diseases and 12 age-matched healthy subjects were used as controls. In 8 JIA patients freshly isolated circulating and SF lymphocytes were stained for CD27 in CD4+CD45 RO+ T cell subpopulation and analyzed by cytometry.
Soluble CD27 serum levels were significantly higher in patients with polyarticular JIA and acute systemic infectious diseases than in patients with active oligoarticular or healthy controls. Both polyarticular and oligoarticular JIA patients showed increased levels of soluble CD27 in SF when compared with paired serum samples (p = 0.01). In all the patients tested a significant enrichment of CD27- T cells was seen in the SF (median 39.5%, range 18-56%) when compared to paired CD4+CD45RO+ peripheral lymphocytes (median 19.5%, range 5-43%; p = 0.01).
A clear enrichment of CD4+ memory SF T cells with a CD27-phenotype is observed when compared to correspondent circulating T lymphocytes. This issue is conceivably related to re-activation and recruitment of memory T cells to the site of inflammation, and to the subsequent expansion of a subpopulation of "effector" memory T cells.
Clinical and experimental rheumatology 01/2002; 20(6):863-6. · 2.97 Impact Factor