Giusi Prencipe

Procalcitonin in detecting neonatal nosocomial sepsis

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.

Nerve Growth Factor Downregulates Inflammatory Response in Human Monocytes through TrkA

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.

Inhibition of Natural Killer Cell Cytotoxicity by Interleukin-6: Implications for the Pathogenesis of Macrophage Activation Syndrome

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.

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

Significance Familial Mediterranean fever (FMF) is an autoinflammatory disease caused by more than 310 mutations in the gene MEFV, which encodes Pyrin. Pyrin recently was shown to trigger inflammasome activation in response to Rho GTPase-modifying bacterial toxins. Here we report that Clostridium difficile infection and intoxication with its enterotoxin TcdA engage the Pyrin inflammasome. Moreover, activation of the Pyrin inflammasome, but not other inflammasomes, was hampered by microtubule-depolymerizing drugs in mouse and humans. Unexpectedly, we found that FMF mutations render Pyrin activation independent of microtubules. Thus, our findings provide a conceptual framework for understanding Pyrin signaling and enable functional diagnosis of FMF. 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.

Inflammasome Activation by Cystine Crystals: Implications for the Pathogenesis of Cystinosis

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.

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

BackgroundThis 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).MethodsThe 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.ResultsmRNA 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.ConclusionsOur data suggest that an abnormal regulation of IL-1β axis may be involved in CRMO pathogenesis.

Role of mannose-binding lectin in nosocomial sepsis in critically ill neonates.

We investigated the association of mannose-binding lectin (MBL) serum levels with nosocomial sepsis (NS), their changes overtime during infection, their relation with pathogens, with the MBL2 genotype and their relationship with mortality. In a prospective observational study, we included 365 critically ill neonates: 261 had no infection and 104 had at least 1 septic event. The median MBL serum concentration was significantly lower in infected than in noninfected neonates (p < 0.001). Low MBL levels on admission increased the risk of infection, independently from gestational age and invasive procedures. The median peak MBL level during infection was higher than the median level on admission (p < 0.001) and was correlated with it (r(2) = 0.83, p < 0.001). Moreover, MBL levels on admission were not associated with death (OR = 0.80, 95% CI = 0.56-1.14, p = 0.21). Similarly, no association was found between MBL peak levels during infection and death among infected neonates (OR = 1.10, 95% CI = 0.78-1.57, p = 0.57). In 127 neonates (42 infected) genotyped for exon-1 and -221 promoter MBL2 variants, we did not find significant difference in the frequencies of MBL2 genotypes between infected and noninfected neonates. Moreover, no association was found between MBL2 genotypes and death.

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

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.

A polymorphism in the macrophage migration inhibitory factor promoter is associated with bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a common adverse outcome of prematurity, causing severe morbidity and mortality. The cytokine macrophage migration inhibitory factor (MIF) has been recently shown to favor murine fetal lung development. In this prospective study, we evaluate the expression of MIF in the lung and in the serum of preterm infants (n = 50) and investigate whether the −173 G/C MIF promoter polymorphism is associated with the risk of BPD (n = 103). MIF was highly expressed in lung tissue from preterm infants. Serum MIF levels were measured by ELISA at d 1 after birth. MIF levels were increased [median (interquartile range), 71.01 (44.9–162.3) ng/mL], particularly in those infants with RDS [110.4 (59.4–239.2) ng/mL] compared with healthy adults [2.4 (1.2–5.0) ng/mL], (p < 0.001). The MIF −173*C allele, which predisposes to higher MIF production, was associated with a lower incidence of BPD (OR, 0.2; 95% CI, 0.04–0.93), independently from mechanical ventilation and oxygen exposure (p = 0.03). In conclusion, these data show that MIF expression is increased in lung and serum of preterm infants and suggest that the high producing MIF −173*C allele may be a protective factor for BPD.

Association between mannose-binding lectin gene polymorphisms and necrotizing enterocolitis in preterm infants.

Objectives: The aim of the present study was to evaluate whether polymorphisms of the mannose-binding lectin (MBL-2) gene and MBL serum levels on admission to neonatal intensive care unit are associated with necrotizing enterocolitis (NEC) in preterm infants and to verify MBL expression in NEC bowels. Methods: In this retrospective cohort study, 107 neonates (41 with NEC and 66 controls) were included. MBL-2 genotyping for the promoter polymorphism −221 and for the exon 1 variant alleles at codons 52, 54, and 57 was performed. MBL levels were determined by enzyme-linked immunosorbent assay in 55 infants. Immunohistochemical staining for MBL expression was performed on bowel specimens. The main study outcome was severe NEC (Bell stages II/III). Results: The −221 Y allele and the MBL-2 YY genotype were more frequent in neonates with severe NEC than in controls (P = 0.04 and P = 0.004, respectively). In the multivariate analysis, the MBL-2 YA/YA genotype was associated with NEC (odds ratio = 3.03, 95% confidence interval 1.13%–8.13%, P = 0.024). Neonates with NEC had MBL level on admission >400 ng/mL more frequently than controls (P = 0.043). Among neonates with severe NEC, the deceased neonates were carriers of high or intermediate producing MBL-2 genotypes (P = 0.035). Finally, MBL was highly expressed in intestinal tissue from infants with NEC. Conclusions: MBL-2 genotypes associated with high MBL serum levels represent a risk factor for NEC. This finding, together with the MBL expression in bowel tissue, supports a role for MBL in the pathogenesis of NEC.