Publications by authors named "Giusi Prencipe"

33 Publications

Monocytes From Patients With Macrophage Activation Syndrome and Secondary Hemophagocytic Lymphohistiocytosis Are Hyperresponsive to Interferon Gamma.

Front Immunol 2021 17;12:663329. Epub 2021 Mar 17.

Division of Rheumatology, IRCCS Ospedale Pediatrico Bambino Gesù, Rome, Italy.

Objective: To investigate the activation of the IFNγ signaling pathway in monocytes of patients with secondary hemophagocytic lymphohistiocytosis (sHLH)/macrophage activation syndrome (MAS) and to evaluate whether levels of phosphorylated STAT1 represent a biomarker for the identification of patients at early stages of the disease.

Methods: Fresh whole blood samples from pediatric patients with active sHLH/MAS, not receiving (n=10) and receiving glucocorticoids (n=14) at time of sampling, were prospectively collected. As disease control groups, patients with active systemic juvenile idiopathic arthritis (sJIA) without MAS, patients with sHLH/MAS in remission and patients with other rheumatic diseases were also sampled. Whole blood cells were left unstimulated or stimulated with increasing concentrations of IFNγ for 10 minutes and the intracellular Tyrosine (701)-phosphorylated STAT1 (pSTAT1) levels were evaluated in monocytes by flow cytometry.

Results: Monocytes from untreated sHLH/MAS patients showed significantly higher basal levels of pSTAT1 compared to those observed in monocytes from glucocorticoid-treated sHLH/MAS patients and from all the other disease controls. In addition, a significant increase in responsiveness to IFNγ, as assessed by increased levels of pSTAT1 following stimulation, was observed in monocytes from untreated sHLH/MAS patients. pSTAT1 levels in monocytes distinguished patients with sHLH/MAS not treated with glucocorticoids from patients with active sJIA or with other rheumatic diseases [AUC, 0.93; 95% confidence interval 0.85-1.00, p<0.001]. Statistically significant correlations between mRNA levels in whole blood cells, circulating IFNγ levels and pSTAT1 levels in sHLH/MAS monocytes were found.

Conclusion: Our data demonstrating higher basal levels of pSTAT1 as well as a hyperreactivity to IFNγ stimulation in monocytes from patients with sHLH/MAS point to perturbations in the activation of downstream IFNγ signaling pathway as a contributor to the hyperinflammation occurring in these patients. Finally, the observation that glucocorticoids affect pSTAT1 levels , makes it difficult to consider the measurement of pSTAT1 levels as a biomarker to identify patients at early stages of sHLH/MAS in clinical practice.
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http://dx.doi.org/10.3389/fimmu.2021.663329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010171PMC
March 2021

Early Treatment and IL1RN Single-Nucleotide Polymorphisms Affect Response to Anakinra in Systemic Juvenile Idiopathic Arthritis.

Arthritis Rheumatol 2021 Jun 2;73(6):1053-1061. Epub 2021 May 2.

Ospedale Pediatrico Bambino Gesù, IRCCS, Rome, Italy.

Objective: To evaluate the impact of early treatment and IL1RN genetic variants on the response to anakinra in systemic juvenile idiopathic arthritis (JIA).

Methods: Response to anakinra was defined as achievement of clinically inactive disease (CID) at 6 months without glucocorticoid treatment. Demographic, clinical, and laboratory characteristics of 56 patients were evaluated in univariate and multivariate analyses as predictors of response to treatment. Six single-nucleotide polymorphisms (SNPs) in the IL1RN gene, previously demonstrated to be associated with a poor response to anakinra, were genotyped by quantitative polymerase chain reaction (qPCR) or Sanger sequencing. Haplotype mapping was performed with Haploview software. IL1RN messenger RNA (mRNA) expression in whole blood from patients, prior to anakinra treatment initiation, was assessed by qPCR.

Results: After 6 months of anakinra treatment, 73.2% of patients met the criteria for CID without receiving glucocorticoids. In the univariate analysis, the variable most strongly related to the response was disease duration from onset to initiation of anakinra treatment, with an optimal cutoff at 3 months (area under the curve 84.1%). Patients who started anakinra treatment ≥3 months after disease onset had an 8-fold higher risk of nonresponse at 6 months of treatment. We confirmed that the 6 IL1RN SNPs were inherited as a common haplotype. We found that homozygosity for ≥1 high-expression SNP correlated with higher IL1RN mRNA levels and was associated with a 6-fold higher risk of nonresponse, independent of disease duration.

Conclusion: Our findings on patients with systemic JIA confirm the important role of early interleukin-1 inhibition and suggest that genetic IL1RN variants predict nonresponse to therapy with anakinra.
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http://dx.doi.org/10.1002/art.41612DOI Listing
June 2021

Deficiency Causing Dysregulation of NK Cell Functions and Presenting With Hemophagocytic Lymphohistiocytosis.

Front Genet 2020 18;11:937. Epub 2020 Sep 18.

Division of Rheumatology, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy.

We describe a 2 year old boy with two previously undescribed frameshift mutations in the interferon (IFN)α/β receptor 2 () gene presenting with hemophagocytic lymphohistiocytosis (HLH) following measles-mumps-rubella vaccination. Functional analyses show the absence of response to type I IFN in the patient's cells, as revealed by the lack of phosphorylation of STAT1 and the lack of induction of interferon-stimulated genes upon stimulation with IFNα. HLH has been reported in patients with inborn errors of type I IFN-mediated immune responses following vaccination with live-attenuated viruses. The relation between HLH and defective type I IFN-mediated responses is unclear. We show that in patient's natural killer (NK) cells stimulated with IFNα the expected increase in degranulation and inhibition of IFNγ production were affected. These data support a role for NK cell function dysregulation and lack of inhibition of IFNγ production as contributors to the development of HLH in patients with impaired type I IFN signaling.
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http://dx.doi.org/10.3389/fgene.2020.00937DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7531586PMC
September 2020

Mannose Binding Lectin, S100 B Protein, and Brain Injuries in Neonates With Perinatal Asphyxia.

Front Pediatr 2020 17;8:527. Epub 2020 Sep 17.

Unit of Epidemiology, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy.

Perinatal asphyxia triggers an acute inflammatory response in the injured brain. Complement activation and neuroinflammation worsen brain damage after a systemic ischemia/reperfusion insult. The increase of mannose binding lectin (MBL) during asphyxia may contribute to the brain damage, via activation of the complement lectin pathway. The possible role of gene variants in influencing the severity of post-asphyxia brain injuries is still unexplored. This retrospective study included 53 asphyxiated neonates: 42 underwent therapeutic hypothermia (TH) and 11 did not because they were admitted to the NICU later than 6 h after the hypoxic insult. Blood samples from TH-treated and untreated patients were genotyped for gene variants, and biomarker plasma levels (MBL and S100 B protein) were measured at different time points: during hypothermia, during rewarming, and at 7-10 days of life. The timing of blood sampling, except for the T1 sample, was the same in untreated infants. Highest (peak) levels of MBL and genotypes were correlated to neuroimaging brain damage or death and long-term neurodevelopmental delay. wild-type genotype was associated with the highest MBL levels and worst brain damage on MRI ( = 0.046) at 7-10 days after hypoxia. MBL increased in both groups and S100B decreased, slightly more in treated than in untreated neonates. The progressive increase of MBL ( = 0.08) and to be untreated with TH ( = 0.08) increased the risk of brain damage or death at 7-10 days of life, without affecting neurodevelopmental outcomes at 1 year. The effect of TH on MBL plasma profiles is uncertain.
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http://dx.doi.org/10.3389/fped.2020.00527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7527601PMC
September 2020

Blood-based test for diagnosis and functional subtyping of familial Mediterranean fever.

Ann Rheum Dis 2020 07 20;79(7):960-968. Epub 2020 Apr 20.

VIB Center for Inflammation Research, Zwijnaarde, Belgium

Background And Objective: Familial Mediterranean fever (FMF) is the most common monogenic autoinflammatory disease (AID) worldwide. The disease is caused by mutations in the gene encoding the inflammasome sensor Pyrin. Clinical diagnosis of FMF is complicated by overlap in symptoms with other diseases, and interpretation of genetic testing is confounded by the lack of a clear genotype-phenotype association for most of the 340 reported variants. In this study, the authors designed a functional assay and evaluated its potential in supporting FMF diagnosis.

Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from patients with Pyrin-associated autoinflammation with an FMF phenotype (n=43) or with autoinflammatory features not compatible with FMF (n=8), 10 asymptomatic carriers and 48 healthy donors. Sera were obtained from patients with distinct AIDs (n=10), and whole blood from a subset of patients and controls. The clinical, demographic, molecular genetic factors and other characteristics of the patient population were assessed for their impact on the diagnostic test read-out. Interleukin (IL)-1β and IL-18 levels were measured by Luminex assay.

Results: The ex vivo colchicine assay may be performed on whole blood or PBMC. The functional assay robustly segregated patients with FMF from healthy controls and patients with related clinical disorders. The diagnostic test distinguished patients with classical FMF mutations (M694V, M694I, M680I, R761H) from patients with other mutations and variants (K695R, P369S, R202Q, E148Q) that are considered benign or of uncertain clinical significance.

Conclusion: The ex vivo colchicine assay may support diagnosis of FMF and functional subtyping of Pyrin-associated autoinflammation.
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http://dx.doi.org/10.1136/annrheumdis-2019-216701DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7307214PMC
July 2020

Chitotriosidase as a Novel Biomarker for Therapeutic Monitoring of Nephropathic Cystinosis.

J Am Soc Nephrol 2020 05 9;31(5):1092-1106. Epub 2020 Apr 9.

Division of Pediatric Nephrology, Department of Pediatrics, University Hospitals Leuven, Leuven, Belgium

Background: Nephropathic cystinosis, a hereditary lysosomal storage disorder caused by dysfunction of the lysosomal cotransporter cystinosin, leads to cystine accumulation and cellular damage in various organs, particularly in the kidney. Close therapeutic monitoring of cysteamine, the only available disease-modifying treatment, is recommended. White blood cell cystine concentration is the current gold standard for therapeutic monitoring, but the assay is technically demanding and is available only on a limited basis. Because macrophage-mediated inflammation plays an important role in the pathogenesis of cystinosis, biomarkers of macrophage activation could have potential for the therapeutic monitoring of cystinosis.

Methods: We conducted a 2-year prospective, longitudinal study in which 61 patients with cystinosis who were receiving cysteamine therapy were recruited from three European reference centers. Each regular care visit included measuring four biomarkers of macrophage activation: IL-1, IL-6, IL-18, and chitotriosidase enzyme activity.

Results: A multivariate linear regression analysis of the longitudinal data for 57 analyzable patients found chitotriosidase enzyme activity and IL-6 to be significant independent predictors for white blood cell cystine levels in patients of all ages with cystinosis; a receiver operating characteristic analysis ranked chitotriosidase as superior to IL-6 in distinguishing good from poor therapeutic control (on the basis of white blood cell cystine levels of <2 nmol 1/2 cystine/mg protein or ≥2 nmol 1/2 cystine/mg protein, respectively). Moreover, in patients with at least one extrarenal complication, chitotriosidase significantly correlated with the number of extrarenal complications and was superior to white blood cell cystine levels in predicting the presence of multiple extrarenal complications.

Conclusions: Chitotriosidase enzyme activity holds promise as a biomarker for use in therapeutic monitoring of nephropathic cystinosis.
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http://dx.doi.org/10.1681/ASN.2019080774DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7217422PMC
May 2020

Is fibrodysplasia ossificans progressiva an interleukin-1 driven auto-inflammatory syndrome?

Pediatr Rheumatol Online J 2019 Dec 21;17(1):84. Epub 2019 Dec 21.

Pediatric Rheumatology Unit, Department of Pediatrics, Meir Medical Center, 59 Tchernichovsky St., 4428164, Kfar Saba, Israel.

Background: Fibrodysplasia ossificans progressiva (FOP) is the most catastrophic form of heterotopic ossification, due to ongoing intracellular signaling through the bone morphogenic protein pathway. The paroxysmal appearance of inflammatory lumps and elevated inflammatory markers during flares, suggest that FOP is an auto-inflammatory disease. Based on evidence, demonstrating a role for interleukin-1β (IL-1β) in other forms of heterotopic ossification, we hypothesized that treating FOP patients with anti-IL-1 agents could help lower the rate of FOP paroxysms and/or limit the symptoms and residual lesions.

Case Presentation: A 13.5-year-old Arab boy was diagnosed with FOP. Treatment with anti-inflammatory drugs did not change the disease course. New lumps appeared in a rate of approximately one every 8 days. Treatment with the anti-IL-1 agents anakinra and canakinumab resulted in significantly lower rate of paroxysms (every 22-25 days, of which almost all involved only 2 existing lumps), as well as shorter duration. High levels of IL-1β were found in the patient's plasma samples, collected during a paroxysm that appeared 8 weeks after the last canakinumab dose. In contrast, IL-1β plasma levels were undetectable in the previous three plasma samples, obtained while he was treated with anti-IL-1 agents.

Conclusions: Our data demonstrate the efficacy of anti-IL-1 agents in the treatment of a patient with FOP. Results showing the marked increase in IL-1β plasma levels during a paroxysm support a role for IL-1β in the pathogenesis of FOP and further provide the rationale for the use of anti-IL-1 agents in FOP treatment.
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http://dx.doi.org/10.1186/s12969-019-0386-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6925442PMC
December 2019

The interferon-gamma pathway is selectively up-regulated in the liver of patients with secondary hemophagocytic lymphohistiocytosis.

PLoS One 2019 17;14(12):e0226043. Epub 2019 Dec 17.

Division of Rheumatology, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy.

Aim of this study was to investigate the activation of the IFNγ pathway in the affected liver and in the blood of patients with secondary hemophagocytic lymphohistiocytosis (sHLH). To this purpose, the mRNA expression levels of IFNG and IFNγ-inducible genes as well as Tyrosine (701)-phosphorylated signal transducer and activator of transcription 1 (STAT1) protein levels were evaluated in the liver and in peripheral blood mononuclear cells (PBMCs) of three patients with sHLH with predominant liver involvement. The mRNA expression levels of IFNG and IFNγ-inducible genes were markedly higher in patient livers compared to control livers and to one disease control liver. Conversely, slight differences in the expression levels of Type I IFN-inducible genes and other classical inflammatory cytokine genes were found. Further supporting the activation of the IFNγ pathway, higher protein levels of phosphorylated and total STAT1 were detected in patient livers compared to control livers. When the expression of the same genes analysed in liver tissues was evaluated in PBMCs collected from 2 out of 3 patients before the liver biopsy, we found that mRNA levels of IFNγ-inducible genes were markedly increased. Accordingly, high circulating levels of IFNγ-inducible CXCL9 were observed in patients. Altogether, these data demonstrate the selective and marked up-regulation of the IFNγ pathway in the liver tissue and blood of patients with active sHLH. Finally, we show that measurement of circulating CXCL9 levels and evaluation of IFNγ-inducible gene expression levels in PBMCs may represent a new valid tool to better identify patients with suspected HLH with predominant liver involvement.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0226043PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6917341PMC
April 2020

NLRP2 Regulates Proinflammatory and Antiapoptotic Responses in Proximal Tubular Epithelial Cells.

Front Cell Dev Biol 2019 24;7:252. Epub 2019 Oct 24.

Department of Laboratories, Immuno-Rheumatology Research Area, Bambino Gesù Children's Hospital, Rome, Italy.

Nod-like Receptor Pyrin domain containing proteins (NLRPs) expressed by resident renal cells may contribute to the pathogenesis of multiple renal diseases. Cystinosis is a genetic disorder that affects kidney and particularly proximal tubular epithelial cells (PTEC). Here, we investigated the expression of NLRP family members in human control and cystinotic conditionally immortalized PTEC. Among all the NLRPs tested, we found that NLRP2 is highly expressed in cystinostic PTEC, but not in PTEC from healthy subjects. The NLRP2 overexpression was confirmed in primary PTEC and in kidney biopsies from cystinotic patients. In order to elucidate the role of NLRP2 in PTEC, we stably transfected control PTEC with an NLRP2-containing plasmid. We showed that NLRP2 markedly increases the production of several NF-κB regulated cytokines and chemokines. Accordingly, we demonstrated that NLRP2 interacts with IKKa and positively regulates the DNA-binding activity of p50 and p65 NF-κB, by modulating the p65 NF-κB phosphorylation status in Serine 536. Transcriptome analysis revealed that NLRP2 also upregulates the expression of profibrotic mediators and reduces that of several interferon-inducible genes. Finally, NLRP2 overexpression decreased the apoptotic cell rate. Consistently, silencing of NLRP2 by small-interfering RNA in cystinotic PTEC resulted in a significant decrease in cytokine and chemokine production as well as in an increase in the apoptosis rate. Altogether, our data reveals a previously unrecognized role for NLRP2 in regulating proinflammatory, profibrotic and antiapoptotic responses in PTEC, through NF-κB activation. Moreover, our findings unveil a novel potential mechanism involving NLRP2 overexpression in the pathogenesis of cystinosis.
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http://dx.doi.org/10.3389/fcell.2019.00252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6822264PMC
October 2019

A novel disorder involving dyshematopoiesis, inflammation, and HLH due to aberrant CDC42 function.

J Exp Med 2019 12 10;216(12):2778-2799. Epub 2019 Oct 10.

Baylor-Hopkins Center for Mendelian Genomics, Houston, TX.

Hemophagocytic lymphohistiocytosis (HLH) is characterized by immune dysregulation due to inadequate restraint of overactivated immune cells and is associated with a variable clinical spectrum having overlap with more common pathophysiologies. HLH is difficult to diagnose and can be part of inflammatory syndromes. Here, we identify a novel hematological/autoinflammatory condition (NOCARH syndrome) in four unrelated patients with superimposable features, including neonatal-onset cytopenia with dyshematopoiesis, autoinflammation, rash, and HLH. Patients shared the same de novo mutation (Chr1:22417990C>T, p.R186C) and altered hematopoietic compartment, immune dysregulation, and inflammation. mutations had been associated with syndromic neurodevelopmental disorders. In vitro and in vivo assays documented unique effects of p.R186C on CDC42 localization and function, correlating with the distinctiveness of the trait. Emapalumab was critical to the survival of one patient, who underwent successful bone marrow transplantation. Early recognition of the disorder and establishment of treatment followed by bone marrow transplant are important to survival.
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http://dx.doi.org/10.1084/jem.20190147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6888978PMC
December 2019

Interleukin-18 in pediatric rheumatic diseases.

Curr Opin Rheumatol 2019 09;31(5):421-427

Division of Rheumatology, IRCCS Bambino Gesù Children's Hospital, Rome, Italy.

Purpose Of Review: IL-18 is a pleiotropic cytokine involved in the regulation of innate and adaptive immune responses. IL-18 pro-inflammatory activities are finely regulated in vivo by the inhibitory effects of the soluble IL-18-binding protein (IL-18BP). The elevation of circulating levels of IL-18 has been described in children with systemic juvenile idiopathic arthritis (sJIA). In the recent years, the role of IL-18 in the pathogenesis of secondary haemophagocytic lymphohistiocytosis (sHLH), also referred to as macrophage activation syndrome (MAS), in the context of autoinflammatory diseases, including sJIA, is emerging.

Recent Findings: A large number of studies in patients and animal models pointed to the imbalance in IL-18/IL-18BP levels, causing increased systemic levels of free bioactive IL-18, as a predisposing factor in the development of MAS. Although the exact mechanisms involved in the development of MAS are not clearly understood, increasing evidence demonstrate the role of IL-18 in upregulating the production of interferon (IFN)-γ.

Summary: On the basis of the first emerging data on the possibility of blocking IL-18, we here discuss the scientific rationale for neutralizing the IL-18/IFNγ axis in the prevention and treatment of sHLH and MAS.
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http://dx.doi.org/10.1097/BOR.0000000000000634DOI Listing
September 2019

An unusual presentation of purine nucleoside phosphorylase deficiency mimicking systemic juvenile idiopathic arthritis complicated by macrophage activation syndrome.

Pediatr Rheumatol Online J 2019 May 22;17(1):25. Epub 2019 May 22.

Division of Rheumatology, IRCCS Ospedale Pediatrico Bambino Gesù, Piazza S. Onofrio 4, 00165, Rome, Italy.

Background: Systemic juvenile idiopathic arthritis (sJIA) is an inflammatory condition that presents with fever, rash and arthritis. At onset systemic features are predominant and the diagnosis may be a challenge. Secondary hemophagocytic lymphohistiocytosis (sHLH) forms may be associated with different disorders, including rheumatic diseases, and this form is called macrophage activation syndrome (MAS). CXCL9 levels, a chemokine induced by IFNγ, are significantly elevated in patients with sHLH or MAS and are correlated with laboratory features of disease activity. High levels of IL-18 have been reported in patients with MAS during sJIA, as well as in some patients with sHLH and IL-18 is indeed known to induce IFNγ production.

Findings: We report a patient with a clinical presentation highly suggestive for systemic juvenile idiopathic arthritis (sJIA) onset complicated by MAS, and was later diagnosed with purine nucleoside phosphorylase (PNP)-deficiency with HLH. Some unusual features appeared when HLH was controlled and further investigations provided the correct diagnosis. Serum CXCL9 and IL-18 levels were found markedly elevated at disease onset, during the active phase of MAS and decreased progressively during the course.

Conclusion: The reported case underlines the potential difficulties in discriminating sJIA from other causes of systemic inflammation. Furthermore, this supports the notion that especially in young children with a sJIA-like disease other mimicking conditions should be actively sought for. CXCL9 and IL-18 levels suggested that patients with PNP-deficiency may have a subclinical activation of the IFNγ pathway and indeed they are predisposed to develop sHLH.
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http://dx.doi.org/10.1186/s12969-019-0328-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6532153PMC
May 2019

ProNGF-p75NTR axis plays a proinflammatory role in inflamed joints: a novel pathogenic mechanism in chronic arthritis.

RMD Open 2017 12;3(2):e000441. Epub 2017 Sep 12.

Division of Rheumatology and Immuno-Rheumatology Research Laboratories, Bambino Gesù Children's Hospital, Rome, Italy.

Objective: To identify the role of mature nerve growth factor (mNGF), its immature form proNGF and their receptors in arthritis inflammation.

Methods: Real-time PCR, western blot and ELISA were performed to evaluate NGF, proNGF, their receptor and cytokine expression in synovial tissue and cells of patients with juvenile idiopathic arthritis (JIA) and rheumatoid arthritis (RA), and controls.

Results: proNGF and not mNGF is the prevalent form measured in synovial fluids of patients with JIA and RA with synovial fibroblasts as a major source of proNGF in the inflamed synoviae. p75NTR, the specific receptor for proNGF, is the NGF receptor most expressed in mononuclear cells of patients with JIA, while TrkA is the prevalent receptor in healthy donors. In ex vivo experiments the effects of proNGF differ from those of mNGF, suggesting that the balance of p75NTR and TrkA expression represents a critical factor in regulating mNGF/proNGF functions, determining which intracellular pathways and biological activities are triggered. Contrary to NGF, proNGF administration increased inflammatory cytokines but not interleukin (IL)-10 expression, inducing a stronger activation of p38 and JNK pathways. proNGF effects depend on its binding to p75NTR, as inhibition of p75NTR with neutralising antibodies or LM11A-31 abolished proNGF-induced production of IL-6 in patients' mononuclear cells, while inhibition of TrkA did not. There is a correlation in patients with arthritis between high p75NTR levels and severity of clinical symptoms.

Conclusions: Our data suggest that an active proNGF-p75NTR axis promotes proinflammatory mechanisms contributing to chronic tissue inflammation, and that the use of p75NTR inhibitors may represent a new therapeutic approach in chronic arthritis.
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http://dx.doi.org/10.1136/rmdopen-2017-000441DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5604749PMC
September 2017

Neutralization of IFN-γ reverts clinical and laboratory features in a mouse model of macrophage activation syndrome.

J Allergy Clin Immunol 2018 04 12;141(4):1439-1449. Epub 2017 Aug 12.

Division of Rheumatology, Bambino Gesù Children's Hospital IRCCS, Rome, Italy.

Background: The pathogenesis of macrophage activation syndrome (MAS) is not clearly understood: a large body of evidence supports the involvement of mechanisms similar to those implicated in the setting of primary hemophagocytic lymphohistiocytosis.

Objective: We sought to investigate the pathogenic role of IFN-γ and the therapeutic efficacy of IFN-γ neutralization in an animal model of MAS.

Methods: We used an MAS model established in mice transgenic for human IL-6 (IL-6TG mice) challenged with LPS (MAS mice). Levels of IFN-γ and IFN-γ-inducible chemokines were evaluated by using real-time PCR in the liver and spleen and by means of ELISA in plasma. IFN-γ neutralization was achieved by using the anti-IFN-γ antibody XMG1.2 in vivo.

Results: Mice with MAS showed a significant upregulation of the IFN-γ pathway, as demonstrated by increased mRNA levels of Ifng and higher levels of phospho-signal transducer and activator of transcription 1 in the liver and spleen and increased expression of the IFN-γ-inducible chemokines Cxcl9 and Cxcl10 in the liver and spleen, as well as in plasma. A marked increase in Il12a and Il12b expression was also found in livers and spleens of mice with MAS. In addition, mice with MAS had a significant increase in numbers of liver CD68 macrophages. Mice with MAS treated with an anti-IFN-γ antibody showed a significant improvement in survival and body weight recovery associated with a significant amelioration of ferritin, fibrinogen, and alanine aminotransferase levels. In mice with MAS, treatment with the anti-IFN-γ antibody significantly decreased circulating levels of CXCL9, CXCL10, and downstream proinflammatory cytokines. The decrease in CXCL9 and CXCL10 levels paralleled the decrease in serum levels of proinflammatory cytokines and ferritin.

Conclusion: These results provide evidence for a pathogenic role of IFN-γ in the setting of MAS.
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http://dx.doi.org/10.1016/j.jaci.2017.07.021DOI Listing
April 2018

Inflammatory events during food protein-induced enterocolitis syndrome reactions.

Pediatr Allergy Immunol 2017 08 22;28(5):464-470. Epub 2017 Jun 22.

Division of Allergy, IRCCS Ospedale Pediatrico Bambino Gesù, Rome, Italy.

Background: An increasing number of infants are diagnosed with food protein-induced enterocolitis syndrome (FPIES), a non-IgE-mediated food allergy. Until now, T-cell, food-specific mechanisms have been hypothesized.

Methods: Sixteen children (11M, 5F), affected by FPIES from cow's milk, wheat, fruit, rice, and others, experienced 25 acute episodes managed at our emergency department (ED) and eight FPIES reactions during oral food challenges (OFC). We compared the laboratory data in resting conditions, in the absence of infectious diseases, with data collected during the 25 acute ED episodes (blood samples drawn at 2-12 hours) and the eight positive OFCs (three samples at 2, 6, and 12 hours). The onset of symptoms was used as a reference time point.

Results: In basal conditions, total IgE, WBC, neutrophil and eosinophil count, CRP, and SGPT were found normal. LDH and SGOT values were high (627.81±97.88 and 45.75±10.26 UI/L, respectively). During ED reactions, LDH and SGOT increased to 794.21±247.28 (P=.028) and 51.08±16.99 UI/L (P=.14) and neutrophils count and CRP to 8.44±3.82×10 /μL (P=.0009) and 3.27±5.73 mg/dL (P=.0014), respectively. During positive OFC, LDH and SGOT did not vary significantly; CRP increased from 0.14±0.18 to 2.49±3.65 mg/dL (P=.00189) and neutrophil count from 2.79±1.42 to 7.10±3.98×10 /μL (P=.00096).

Conclusions: FPIES reactions are characterized by neutrophilia and by a time-dependent, significant increase in CRP, indicating that inflammatory mechanisms are in place. This suggests new directions for research on FPIES pathogenesis.
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http://dx.doi.org/10.1111/pai.12723DOI Listing
August 2017

Mannose-Binding Lectin: Biologic Characteristics and Role in the Susceptibility to Infections and Ischemia-Reperfusion Related Injury in Critically Ill Neonates.

J Immunol Res 2017 26;2017:7045630. Epub 2017 Jan 26.

Department of Laboratories, Laboratory of Chemical Chemistry, Bambino Gesù Children's Hospital (IRCCS), Piazza S. Onofrio 4, 00165 Rome, Italy.

The mannose-binding lectin (MBL) is a member of the collectin family, belonging to the innate immunity system. Genetic, biologic, and clinical properties of MBL have been widely investigated throughout the last decades, although some interesting aspects of its potential clinical relevance are still poorly understood. Low circulating concentrations of MBL have been associated with increased risk of infection and poor neurologic outcome in neonates. On the other hand, an excessive and uncontrolled inflammatory response by the neonatal intestine after the exposure to luminal bacteria, leading to an increased production of MBL, may be involved in the onset of necrotizing enterocolitis. The purpose of the present review is to summarize the current knowledge about genetic and biologic characteristics of MBL and its role in the susceptibility to infections and to ischemia-reperfusion related tissue injuries to better explore its clinical relevance during the perinatal period and the possible future therapeutic applications.
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http://dx.doi.org/10.1155/2017/7045630DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5299167PMC
March 2017

Macrophage Activation Syndrome: different mechanisms leading to a one clinical syndrome.

Pediatr Rheumatol Online J 2017 Jan 17;15(1). Epub 2017 Jan 17.

Division of Rheumatology, Ospedale Pediatrico Bambino Gesù IRCCS, Piazza S. Onofrio 4, 00165, Rome, Italy.

Background: Macrophage activation syndrome (MAS) is a severe complication of rheumatic disease in childhood, particularly in systemic Juvenile Idiopathic Arthritis (sJIA). It is characterize by an uncontrolled activation and proliferation of T lymphocytes and macrophages.

Main Content: MAS is currently classified among the secondary or acquired forms of haemophagocytic lymphohistiocytosis (sHLH). The reason is that MAS shares clinical and laboratory features with primary genetic HLH (pHLH). In this context is conceivable that some of the pathogenic mechanisms of pHLH may be involved in other forms of HLH. Heterozygosity for mutations of genes involved in pHLH may lead to a cytotoxic defect and to a development of clinical overt disease. But other different contributors might be involved to the development of MAS such as infections or underlying inflammation. In MAS, the inflammatory status of the patient is a major contributor of the disease. Indeed, the majority of the MAS episodes occurs during active disease phases or at disease onset. In addition, recent evidence in animals and humans suggest that genetics may also play a major role in contributing to hyperinflammation and particularly to macrophages hyper-responses.

Conclusions: We hypothesize that HLH may be one unique clinical syndrome, to whose generation different mechanisms may contribute, and maintained by one final effector mechanism.
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http://dx.doi.org/10.1186/s12969-016-0130-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5240371PMC
January 2017

Familial Mediterranean fever mutations lift the obligatory requirement for microtubules in Pyrin inflammasome activation.

Proc Natl Acad Sci U S A 2016 12 22;113(50):14384-14389. Epub 2016 Nov 22.

Inflammation Research Center, VIB, Zwijnaarde, B-9052, Belgium;

Familial Mediterranean fever (FMF) is the most common monogenic autoinflammatory disease worldwide. It is caused by mutations in the inflammasome adaptor Pyrin, but how FMF mutations alter signaling in FMF patients is unknown. Herein, we establish Clostridium difficile and its enterotoxin A (TcdA) as Pyrin-activating agents and show that wild-type and FMF Pyrin are differentially controlled by microtubules. Diverse microtubule assembly inhibitors prevented Pyrin-mediated caspase-1 activation and secretion of IL-1β and IL-18 from mouse macrophages and human peripheral blood mononuclear cells (PBMCs). Remarkably, Pyrin inflammasome activation persisted upon microtubule disassembly in PBMCs of FMF patients but not in cells of patients afflicted with other autoinflammatory diseases. We further demonstrate that microtubules control Pyrin activation downstream of Pyrin dephosphorylation and that FMF mutations enable microtubule-independent assembly of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) micrometer-sized perinuclear structures (specks). The discovery that Pyrin mutations remove the obligatory requirement for microtubules in inflammasome activation provides a conceptual framework for understanding FMF and enables immunological screening of FMF mutations.
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http://dx.doi.org/10.1073/pnas.1613156113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5167202PMC
December 2016

Elevated circulating levels of interferon-γ and interferon-γ-induced chemokines characterise patients with macrophage activation syndrome complicating systemic juvenile idiopathic arthritis.

Ann Rheum Dis 2017 Jan 13;76(1):166-172. Epub 2016 Jun 13.

Division of Rheumatology, IRCCS Ospedale Pediatrico Bambino Gesù, Rome, Italy.

Objectives: Interferon-γ (IFNγ) is the pivotal mediator in murine models of primary haemophagocytic lymphohistiocytosis (pHLH). Given the similarities between primary and secondary HLH (sec-HLH), including macrophage activation syndrome (MAS), we investigate the involvement of the IFNγ pathway in MAS by evaluating levels of IFNγ and of the induced chemokines, and their relation with laboratory parameters of MAS in systemic juvenile idiopathic arthritis (sJIA) patients with MAS and in a murine MAS model.

Methods: The Luminex multiplexing assay was used to assess serum levels of interleukin (IL)-1β, IL-6, IFNγ and of the IFNγ-induced chemokines CXCL9, CXCL10 and CXCL11 in patients with sec-HLH (n=11) and in patients with sJIA (n=54), of whom 20 had active MAS at sampling. Expression of IFNγ-induced chemokines was assessed in IL-6 transgenic mice in which MAS is induced by TLR4 stimulation with lipopolysaccharide.

Results: Levels of IFNγ and of IFNγ-induced chemokines were markedly elevated during active MAS and sec-HLH and were significantly higher in patients with MAS compared with active sJIA without MAS. Levels in patients with active sJIA without MAS were comparable to those of patients with clinically inactive sJIA. During MAS, ferritin and alanine transferase levels and neutrophil and platelet counts were significantly correlated with serum levels of IFNγ and CXCL9. In murine MAS, serum levels of ferritin were significantly correlated with mRNA levels of Cxcl9 in liver and spleen.

Conclusions: The high levels of IFNγ and of IFNγ-induced chemokines and their correlation with the severity of laboratory abnormalities of MAS suggest a pivotal role of IFNγ in MAS.
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http://dx.doi.org/10.1136/annrheumdis-2015-209020DOI Listing
January 2017

A Heterozygous RAB27A Mutation Associated with Delayed Cytolytic Granule Polarization and Hemophagocytic Lymphohistiocytosis.

J Immunol 2016 Mar 15;196(6):2492-503. Epub 2016 Feb 15.

Division of Pediatric Rheumatology, University of Alabama at Birmingham, Birmingham, AL 35233; Division of Clinical Immunology and Rheumatology, University of Alabama at Birmingham, Birmingham, AL 35294;

Frequently fatal, primary hemophagocytic lymphohistiocytosis (HLH) occurs in infancy resulting from homozygous mutations in NK and CD8 T cell cytolytic pathway genes. Secondary HLH presents after infancy and may be associated with heterozygous mutations in HLH genes. We report two unrelated teenagers with HLH and an identical heterozygous RAB27A mutation (c.259G→C). We explore the contribution of this Rab27A missense (p.A87P) mutation on NK cell cytolytic function by cloning it into a lentiviral expression vector prior to introduction into the human NK-92 cell line. NK cell degranulation (CD107a expression), target cell conjugation, and K562 target cell lysis was compared between mutant- and wild-type-transduced NK-92 cells. Polarization of granzyme B to the immunologic synapse and interaction of mutant Rab27A (p.A87P) with Munc13-4 were explored by confocal microscopy and proximity ligation assay, respectively. Overexpression of the RAB27A mutation had no effect on cell conjugate formation between the NK and target cells but decreased NK cell cytolytic activity and degranulation. Moreover, the mutant Rab27A protein decreased binding to Munc13-4 and delayed granzyme B polarization toward the immunologic synapse. This heterozygous RAB27A mutation blurs the genetic distinction between primary and secondary HLH by contributing to HLH via a partial dominant-negative effect.
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http://dx.doi.org/10.4049/jimmunol.1501284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4779709PMC
March 2016

Inhibition of natural killer cell cytotoxicity by interleukin-6: implications for the pathogenesis of macrophage activation syndrome.

Arthritis Rheumatol 2015 Nov;67(11):3037-46

IRCCS Bambino Gesú Children's Hospital and Sapienza University of Rome, Rome, Italy.

Objective: Systemic juvenile idiopathic arthritis (JIA) is associated with high levels of interleukin-6 (IL-6) in the serum and synovial fluid, and impairment of natural killer (NK) cell function is often observed. This study was undertaken to evaluate a possible link between these 2 biologic findings and whether they may be associated with the development of macrophage activation syndrome, a condition frequently observed in systemic JIA.

Methods: Splenocytes from wild-type (WT) or IL-6-transgenic (Tg) mice were evaluated for NK cell cytotoxicity using a (51) Cr-release assay. Numbers of NK cells and expression of perforin, granzyme B, CD69, and CD107a were evaluated by flow cytometry. Human peripheral blood mononuclear cells (PBMCs) isolated from healthy donors were treated with IL-6 and cultured in the presence or absence of tocilizumab (TCZ), an IL-6 receptor blocker. Human polyclonal NK cells from healthy donor PBMCs were evaluated for cell cytotoxicity and expression of perforin, granzyme B, and CD107a. PBMCs harvested from patients with systemic JIA during periods of active or inactive disease were left untreated or treated with IL-6 in combination with soluble IL-6 receptor and analyzed for the expression of perforin and granzyme B.

Results: Splenic NK cell cytotoxicity was reduced in IL-6-Tg mice compared to WT mice. Levels of CD69 and CD107a showed no significant differences, whereas expression of perforin and granzyme B was impaired in NK cells from IL-6-Tg mice. Exposure of human peripheral blood NK cells to IL-6 led to reduced expression of perforin and granzyme B. Culturing human polyclonal NK cells in the presence of TCZ significantly increased cell cytotoxicity, and also increased expression of perforin and granzyme B. In patients with systemic JIA, a reduction in IL-6 plasma levels during disease remission correlated with the rescue of perforin and granzyme B expression in NK cells from these patients.

Conclusion: In both mice and humans, IL-6 down-modulated the cytotoxic activity of NK cells. This decrease was associated with reduced perforin and granzyme B levels in the absence of altered granule exocytosis.
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http://dx.doi.org/10.1002/art.39295DOI Listing
November 2015

Use of early biomarkers in neonatal brain damage and sepsis: state of the art and future perspectives.

Biomed Res Int 2015 18;2015:253520. Epub 2015 Jan 18.

Neonatal Intensive Care Unit, Department of Medical and Surgical Neonatology, Bambino Gesù Children's Hospital, IRCCS, Piazza Sant'Onofrio 4, Rome, Italy.

The identification of early noninvasive biochemical markers of disease is a crucial issue of the current scientific research, particularly during the first period of life, since it could provide useful and precocious diagnostic information when clinical and radiological signs are still silent. The ideal biomarker should be practical and sensitive in the precocious identification of at risk patients. An earlier diagnosis may lead to a larger therapeutic window and improve neonatal outcome. Brain damage and sepsis are common causes of severe morbidity with poor outcome and mortality during the perinatal period. A large number of potential biomarkers, including neuroproteins, calcium binding proteins, enzymes, oxidative stress markers, vasoactive agents, and inflammatory mediators, have been so far investigated. The aim of the present review was to provide a brief overview of some of the more commonly investigated biomarkers used in case of neonatal brain damage and sepsis.
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http://dx.doi.org/10.1155/2015/253520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4313065PMC
November 2015

IL-6 amplifies TLR mediated cytokine and chemokine production: implications for the pathogenesis of rheumatic inflammatory diseases.

PLoS One 2014 1;9(10):e107886. Epub 2014 Oct 1.

Division of Rheumatology, Bambino Gesù Children's Hospital, Rome, Italy; Department of Cellular Biotechnologies and Haematology, Sapienza University of Rome, Rome, Italy.

The role of Interleukin(IL)-6 in the pathogenesis of joint and systemic inflammation in rheumatoid arthritis (RA) and systemic juvenile idiopathic arthritis (s-JIA) has been clearly demonstrated. However, the mechanisms by which IL-6 contributes to the pathogenesis are not completely understood. This study investigates whether IL-6 affects, alone or upon toll like receptor (TLR) ligand stimulation, the production of inflammatory cytokines and chemokines in human peripheral blood mononuclear cells (PBMCs), synovial fluid mononuclear cells from JIA patients (SFMCs) and fibroblast-like synoviocytes from rheumatoid arthritis patients (RA synoviocytes) and signalling pathways involved. PBMCs were pre-treated with IL-6 and soluble IL-6 Receptor (sIL-6R). SFMCs and RA synoviocytes were pre-treated with IL-6/sIL-6R or sIL-6R, alone or in combination with Tocilizumab (TCZ). Cells were stimulated with LPS, S100A8-9, poly(I-C), CpG, Pam2CSK4, MDP, IL-1β. Treatment of PBMCs with IL-6 induced production of TNF-α, CXCL8, and CCL2, but not IL-1β. Addition of IL-6 to the same cells after stimulation with poly(I-C), CpG, Pam2CSK4, and MDP induced a significant increase in IL-1β and CXCL8, but not TNF-α production compared with TLR ligands alone. This enhanced production of IL-1β and CXCL8 paralleled increased p65 NF-κB activation. In contrast, addition of IL-6 to PBMCs stimulated with LPS or S100A8-9 (TLR-4 ligands) led to reduction of IL-1β, TNF-α and CXCL8 with reduced p65 NF-κB activation. IL-6/IL-1β co-stimulation increased CXCL8, CCL2 and IL-6 production. Addition of IL-6 to SFMCs stimulated with LPS or S100A8 increased CXCL8, CCL2 and IL-1β production. Treatment of RA synoviocytes with sIL-6R increased IL-6, CXCL8 and CCL2 production, with increased STAT3 and p65 NF-κB phosphorylation. Our results suggest that IL-6 amplifies TLR-induced inflammatory response. This effect may be relevant in the presence of high IL-6 and sIL-6R levels, such as in arthritic joints in the context of stimulation by endogenous TLR ligands.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0107886PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4182736PMC
June 2015

MBL2 gene polymorphisms increase the risk of adverse neurological outcome in preterm infants: a preliminary prospective study.

Pediatr Res 2014 Nov 13;76(5):464-9. Epub 2014 Aug 13.

Epidemiology Unit, Bambino Gesù Children's Hospital (IRCCS), Rome, Italy.

Background: As described in animal models, the lectin-complement pathway is central to the propagation of ischemia-reperfusion injuries in many tissues, including the brain. Similarly, it might affect the genesis of brain damage in preterm infants. MBL2 gene single-nucleotide polymorphisms (SNPs), regulating mannose-binding lectin (MBL) serum levels, could predict the risk of adverse neurological outcome in these infants.

Methods: To evaluate the association between SNPs of the MBL2 gene and long-term neurological outcomes in preterm infants, 75 infants (gestational age (GA) ≤ 32 wk) were observed in a prospective longitudinal study and assessed by clinical and instrumental exams at 12 and 24 mo of corrected age (CA). They were genotyped for the promoter polymorphism -221 and for the exon-1 variant alleles (at codons 52, 54, and 57) of the MBL2 gene.

Results: The MBL2 exon-1 OO genotype was more frequent in children with an adverse neurological outcome (5/35; 7%) than in controls (0/40; 0%), P = 0.045. The risk of intraventricular hemorrhage in carriers of the genotype OO was marked, without reaching statistical significance (odds ratio: 8.67; 95% confidence interval: 0.87-86.06; P = 0.07).

Conclusion: Preterm infants who are carriers of MBL2 exon-1 OO genotype are exposed to an increased risk of adverse neurological outcomes.
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http://dx.doi.org/10.1038/pr.2014.118DOI Listing
November 2014

Deregulation of the IL-1β axis in chronic recurrent multifocal osteomyelitis.

Pediatr Rheumatol Online J 2014 17;12:30. Epub 2014 Jul 17.

Rheumatology Unit, Bambino Gesù Children's Hospital, Rome, Italy.

Background: This study aims to investigate the inflammasome response in peripheral blood mononuclear cells (PBMCs) and the expression of inflammasome components in bone biopsies from patients with chronic recurrent multifocal osteomyelitis (CRMO).

Methods: The expression of inflammasome components mRNAs was evaluated in PBMCs isolated from 15 CRMO patients and 13 healthy controls by quantitative real-time PCR. The Interleukin (IL)-1β released in the medium of PBMC cultures after treatment with lipopolysaccharides (LPS) alone or LPS and ATP was measured by ELISA. Immunohistochemical staining for Apoptosis-associated Speck-like protein (ASC), caspase-1 (CASP-1), Nod-like receptor protein-3 (NLRP3) and IL-1β expression was performed in bone biopsies from CRMO patients.

Results: mRNA levels of ASC, CASP-1 and IL-1β were significantly higher in freshly isolated PBMCs from CRMO patients in active disease than in healthy controls. CASP-1 and IL-1β transcript levels were significantly higher also in PBMCs from CRMO patients in remission compared to healthy controls. PBMCs from CRMO patients in active disease stimulated in vitro with LPS showed a significant increase in IL-1β release compared to healthy control cells. Immunohistochemistry staining of bone tissue revealed the expression of inflammasome components in CRMO osteoclasts.

Conclusions: Our data suggest that an abnormal regulation of IL-1β axis may be involved in CRMO pathogenesis.
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http://dx.doi.org/10.1186/1546-0096-12-30DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4109750PMC
November 2015

Nerve growth factor downregulates inflammatory response in human monocytes through TrkA.

J Immunol 2014 Apr 28;192(7):3345-54. Epub 2014 Feb 28.

Research Laboratories, Unit of Rheumatology, Bambino Gesù Children's Hospital, 00165 Rome, Italy;

Nerve growth factor (NGF) levels are highly increased in inflamed tissues, but their role is unclear. We show that NGF is part of a regulatory loop in monocytes: inflammatory stimuli, while activating a proinflammatory response through TLRs, upregulate the expression of the NGF receptor TrkA. In turn, NGF, by binding to TrkA, interferes with TLR responses. In TLR-activated monocytes, NGF reduces inflammatory cytokine production (IL-1β, TNF-α, IL-6, and IL-8) while inducing the release of anti-inflammatory mediators (IL-10 and IL-1 receptor antagonist). NGF binding to TrkA affects TLR signaling, favoring pathways that mediate inhibition of inflammatory responses: it increases Akt phosphorylation, inhibits glycogen synthase kinase 3 activity, reduces IκB phosphorylation and p65 NF-κB translocation, and increases nuclear p50 NF-κB binding activity. Use of TrkA inhibitors in TLR-activated monocytes abolishes the effects of NGF on the activation of anti-inflammatory signaling pathways, thus increasing NF-κB pathway activation and inflammatory cytokine production while reducing IL-10 production. PBMC and mononuclear cells obtained from the synovial fluid of patients with juvenile idiopathic arthritis show marked downregulation of TrkA expression. In ex vivo experiments, the addition of NGF to LPS-activated juvenile idiopathic arthritis to both mononuclear cells from synovial fluid and PBMC fails to reduce the production of IL-6 that, in contrast, is observed in healthy donors. This suggests that defective TrkA expression may facilitate proinflammatory mechanisms, contributing to chronic tissue inflammation and damage. In conclusion, this study identifies a novel regulatory mechanism of inflammatory responses through NGF and its receptor TrkA, for which abnormality may have pathogenic implications for chronic inflammatory diseases.
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http://dx.doi.org/10.4049/jimmunol.1300825DOI Listing
April 2014

Inflammasome activation by cystine crystals: implications for the pathogenesis of cystinosis.

J Am Soc Nephrol 2014 Jun 13;25(6):1163-9. Epub 2014 Feb 13.

Division of Rheumatology.

Intralysosomal cystine crystal accumulation, due to mutations in the CTNS gene, is a hallmark of nephropathic cystinosis, but the role of these crystals in disease pathogenesis remains unclear. We hypothesized that, similar to other host-derived crystalline moieties, cystine crystals can induce IL-1β production through inflammasome activation. Thus, we investigated the proinflammatory effects of cystine crystals in primary human PBMCs. LPS-primed PBMCs stimulated with cystine crystals secreted IL-1β in a dose-dependent manner. Similarly to IL-1β secretion induced by other crystalline inflammasome activators, cystine crystal-induced IL-1β secretion required activation of caspase-1. Additionally, exogenous cystine crystals were internalized by monocytes, and inhibition of phagocytosis, cathepsin B leakage, generation of reactive oxygen species, and potassium efflux reduced cystine crystal-induced IL-1β secretion. Patients with cystinosis had higher levels of circulating IL-1β and IL-18 compared with controls. Analysis of inflammasome-related gene expression in PBMCs from patients with cystinosis revealed a significant increase in IL-1β and CASP-1 transcript levels compared with controls. Moreover, knockout of cystinosin in mice led to significant increases in serum IL-18 levels and kidney expression of inflammasome-related genes (Casp-1, Pycard, Il-18, Il18r1, Il1r1, and Il1rl2). Taken together, these data demonstrate that cystine crystals are endogenous inflammasome-activating stimuli, suggesting a novel role for cystine crystals in the pathogenesis of nephropathic cystinosis.
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http://dx.doi.org/10.1681/ASN.2013060653DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4033370PMC
June 2014

A novel mutation in the CIAS1/NLRP3 gene associated with an unexpected phenotype of cryopyrin-associated periodic syndromes.

Clin Exp Rheumatol 2014 Jan-Feb;32(1):123-5. Epub 2013 Oct 11.

Division of Rheumatology, Department of Pediatric Medicine, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy.

Objectives: Cryopyrin-associated periodic syndromes (CAPS) comprise a spectrum of distinct, rare, autosomal dominant autoinflammatory disorders of increasing severity caused by NLRP3 gene mutations.

Methods: We describe a 13-year-old female who presented, in the initial phase of the disease, recurrent episodes of high fever, pericarditis, arthralgia, arthritis of the knees, abdominal pain and marked increase in inflammatory markers. In the subsequent months she developed recurrent episodes of chest pain, skin rash and swelling of the subcutaneous tissue, without fever, and with spontaneous resolution.

Results: Molecular analysis of the CIAS1 gene revealed the presence of the Q703K variant and also a c.1105C>A mutation in the heterozygous state, that predicts a L369M amino acid substitution. The latter variant has never been reported. The L369M mutation was predicted to significantly affect protein structure (scoring as 'dangerous' and 'deleterious') by the Variant Effect Predictor tool. Therapy with anakinra was started with rapid disappearance of clinical symptoms and normalization of CRP levels in 24 hours.

Conclusions: The rapid response to IL-1 inhibition suggests that the disease of this patient is driven by IL-1 and supports the conclusion that this novel mutation is pathogenic and may be associated with a new CAPS phenotype. The role played by the concomitant presence of the mutation Q703K remains to be clarified.
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October 2014

The macrophage migration inhibitory factor -173G/C polymorphism is not significantly associated with necrotizing enterocolitis in preterm infants.

J Pediatr Surg 2013 Jul;48(7):1499-502

Laboratory of Rheumatology, Bambino Gesù Children's Hospital, Roma, Italy.

Background And Purpose: Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality among premature infants. Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that has been implicated in the pathophysiology of inflammatory bowel diseases. The MIF promoter contains a functionally relevant single nucleotide polymorphism (SNP) G→C at position -173, with the MIF -173*C allele being associated with higher MIF expression in vitro and with higher MIF levels in vivo. The aim of this study was to investigate whether the G/C polymorphism at -173 of the MIF promoter is associated with the development of NEC.

Methods: In this retrospective cohort study, 107 preterm infants (GA ≤ 32 weeks), of whom 41 had NEC (NEC Stage I n = 20, Stage II n = 3, Stage III n = 18) and 66 were not affected, were genotyped for the MIF -173 SNP. MIF genotyping was carried out by PCR and DHPLC.

Results: We did not find significant differences in the prevalence of the -173G/C polymorphism and in the distribution of the -173 MIF genotype in infants with NEC compared to controls. Moreover, we did not observe an association between the polymorphism and mortality.

Conclusions: The polymorphism -173G/C of the MIF promoter does not appear to be of major importance in the pathophysiology of NEC in preterm infants.
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http://dx.doi.org/10.1016/j.jpedsurg.2013.01.004DOI Listing
July 2013

Procalcitonin in detecting neonatal nosocomial sepsis.

Arch Dis Child Fetal Neonatal Ed 2012 Sep;97(5):F368-70

Neonatal Intensive Care Unit, Bambino Gesù Children's Hospital, Piazza S Onofrio, 4-00165 Rome, Italy.

Objective: To investigate the accuracy of procalcitonin (PCT) as a diagnostic marker of nosocomial sepsis (NS) and define the most accurate cut-off to distinguish infected from uninfected neonates.

Setting: Six neonatal intensive care units (NICUs).

Patients: 762 neonates admitted to six NICUs during a 28-month observational study for whom at least one serum sample was taken on admission.

Main Outcome Measures: Positive and negative predictive values at different PCT cut-off levels.

Results: The overall probability of an NS was doubled or more if PCT was >0.5 ng/ml. In very-low-birth-weight (VLBW) infants, a cut-off of >2.4 ng/ml gave a positive predictive value of NS near to 50% with a probability of a false-positive diagnosis of NS in about 10% of the patients.

Conclusions: In VLBW neonates, a serum PCT value >2.4 ng/ml prompts early empirical antibiotic therapy, while in normal-birth-weight infants, a PCT value ≤2.4 ng/ml carries a low risk of missing an NS.
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http://dx.doi.org/10.1136/fetalneonatal-2010-194100DOI Listing
September 2012