Similar Metabolic, Innate Immunity, and Adipokine Profiles in Adult and Pediatric Sepsis Versus Systemic Inflammatory Response Syndrome-A Pilot Study.

OBJECTIVES: To examine whether the septic profiles of heat shock protein 72, heat shock protein 90α, resistin, adiponectin, oxygen consumption, CO2 production, energy expenditure, and metabolic pattern, along with illness severity, nutritional, and inflammatory indices, differ between adult and pediatric patients compared with systemic inflammatory response syndrome and healthy controls. To evaluate whether these biomolecules may discriminate sepsis from systemic inflammatory response syndrome in adult and pediatric patients. DESIGN: Prospective cohort study. SETTING: University ICU and PICU. PATIENTS: Seventy-eight adults (sepsis/23; systemic inflammatory response syndrome/23; healthy controls/33), 67 children (sepsis/18; systemic inflammatory response syndrome/23; controls/27), mechanically ventilated. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Flow cytometry determined mean fluorescence intensity for monocyte or neutrophil heat shock protein expression. Resistin, adiponectin, and extracellular heat shock proteins were measured using enzyme-linked immunosorbent assay; energy expenditure by E-COVX (GE Healthcare). Genomic DNA was extracted with PureLink Genomic DNA kit (Invitrogen, Carlsbad, CA) to detect heat shock protein 72 single nucleotide polymorphisms. Similarly, in adult and pediatric patients, Acute Physiology and Chronic Evaluation-II/Acute Physiology and Pediatric Risk of Mortality-III, Simplified Acute Physiology Score-III, C-reactive protein, lactate, and resistin were higher and myocardial contractility, monocyte heat shock protein 72, oxygen consumption, CO2 production, energy expenditure, metabolic pattern, glucose, and albumin lower in sepsis compared with systemic inflammatory response syndrome or controls (p < 0.05). For discriminating sepsis from systemic inflammatory response syndrome, resistin, extracellular heat shock protein 90α, and lactate achieved a receiver operating characteristic curve greater than 0.80 in children and greater than 0.75 in adults (p < 0.05). In both, adults and children, genotype heat shock protein 72 analysis did not disclose any diagnosis or mortality group differences regarding either rs6457452 or rs1061581 haplotypes. CONCLUSIONS: Sepsis presents with similar profiles in adult and pediatric patients, characterized by enhanced inflammatory hormonal response and by repressed innate immunity, metabolism, and myocardial contractility. These features early distinguish sepsis from systemic inflammatory response syndrome across all age groups.

Enhanced activity of NLRP3 inflammasome in peripheral blood cells of patients with active rheumatoid arthritis.

INTRODUCTION: Interleukin-1ß (IL-1ß) is a major inflammatory cytokine, produced predominantly by innate immune cells through NLRP3-inflammasome activation. Both intrinsic and extrinsic danger signals may activate NLRP3. Genetic variations in NLRP3-inflammasome components have been reported to influence rheumatoid arthritis (RA) susceptibility and severity. We sought to assess the activity of NLRP3-inflammasome in patients with active RA compared to healthy individuals. METHOD: Intracellular protein expression of NLRP3, ASC, pro- and active caspase-1, pro- and active IL-1ß was assessed by immunoblotting both at baseline and upon inflammasome activation. NLRP3 function (IL-1ß secretion) was assessed upon priming of TLR2 (Pam(3)CysSK(4), TLR3 (poly(I:C)) or TLR4 (LPS) and ATP sequential treatment. We used caspase inhibitors (casp-1, 3/7 and 8) to assess their contribution to IL-1ß maturation. All experiments were performed in whole blood cells. RESULTS: Active RA patients (n = 11) expressed higher basal intracellular levels of NLRP3 (p < 0.008), ASC (p < 0.003), active caspase-1 (p < 0.02) and pro-IL-1ß (p < 0.001). Upon priming with TLR4 (LPS) and ATP, RA-derived cell extracts (n = 7) displayed increased expression of NLRP3 (p < 0.01) and active caspase-1 (p < 0.001). Secreted IL-1ß in culture supernatants from whole blood cells activated with TLR4 (LPS) or TLR3 agonist (poly(I:C)) plus ATP was higher in RA patients (n = 20) versus controls (n = 18) (p < 0.02 for both). Caspase-1 inhibition significantly reduced IL-1ß secretion induced by all stimuli, whereas caspase-8 inhibition affected only TLR4 and TLR3 cell priming. CONCLUSION: Patients with active RA have increased expression of NLRP3 and NLRP3-mediated IL-1ß secretion in whole blood cells upon stimulation via TLR3 and TLR4 but not TLR2. In these patients, IL-1ß secretion seems to be predominately driven by caspase-1 and caspase-8. Targeting NLRP3 or downstream caspases may be of benefit in suppressing IL-1ß production in RA.

Dysregulated production of interleukin-1ß upon activation of the NLRP3 inflammasome in patients with familial Mediterranean fever.

Familial Mediterranean fever (FMF) is caused by mutations in pyrin, a protein expressed in innate immune cells that interacts with caspase-1 and other inflammasome components to regulate interleukin (IL)-1ß maturation. Since NLRP3 inflammasome represents major source of IL-1ß, we studied its protein expression and function in FMF. We isolated peripheral white blood cells (WBCs) from 20 symptoms-free FMF patients and 21 healthy individuals. Intracellular protein expression of NLRP3, caspase-1, IL-1ß at baseline and after LPS/ATP sequential treatment for NLRP3 activation was assessed by immunoblotting. Secreted IL-1ß was quantified by ELISA. THP-1 cells were transfected with wild-type or mutant pyrin and IL-1ß secretion was measured. FMF WBCs exhibited lower NLRP3 and active caspase-1 protein expression compared to healthy individuals, and LPS/ATP treatment resulted in significantly lower intracellular IL-1ß levels in FMF patients. Likewise, LPS/ATP induced caspase-1-dependent IL-1ß release at significantly lower amounts in the FMF group (1182±192 versus 2134±245pg/mL in controls, p=0.004). Consistently, THP-1 cells transfected with FMF-associated M694V mutant pyrin displayed lower LPS/ATP-induced IL-1ß compared with wild-type pyrin-transfected cells. FMF WBCs demonstrate reduced NLRP3-mediated IL-1ß production. Additional studies are needed to define whether this finding represents a compensatory mechanism to control inflammation or is directly linked to disease pathogenesis.

The role of children's bone marrow mesenchymal stromal cells in the ex vivo expansion of autologous and allogeneic hematopoietic stem cells.

The recognition of the role of Mesenchymal Stromal Cells (MSC) in hematopoiesis, as part of the bone marrow microenvironment, renewed the interest for cord blood (CB) ex vivo expansion as a source of HSC for transplantation. MSC from children are recognized to have different biological properties compared to the ones from adults. The current study focuses on the evaluation of the effects of children's bone marrow MSC on the ex vivo expansion capacity of both allogeneic cord blood and autologous bone marrow (BM) CD34(+) hematopoietic stem cells (HSCs) when used as a cell feeder layer with or without recombinant cytokines. Our results showed that children's bone marrow-derived MSC expand more primitive populations in co culture with CD34 and that the expansion is further enhanced when the culture is supplemented with growth factors. No additive effect was seen either with the early- or late-acting growth factors' cocktails used. Biological features of CB hematopoietic progenitors seem to make them more suitable than their BM counterparts for ex vivo expansion. Clinical implementation will be facilitated by methodological standardization and guidelines' establishment.

Expression levels of ASNS in mesenchymal stromal cells in childhood acute lymphoblastic leukemia.

Increased levels of asparagine synthetase (ASNS), an enzyme producing intracellular asparagine, have been implicated in the development of asparaginase resistance. The aim of this study was to assess ASNS mRNA and protein expression in bone marrow cell populations of children with acute lymphoblastic leukemia (ALL). Bone marrow mononuclear cells at diagnosis, day 33 of treatment, and after completion of chemotherapy were isolated and studied. ASNS mRNA expression was assessed by real-time PCR, and protein levels by Western blot. Our results indicate that MSC ASNS mRNA expression is upregulated in ALL samples compared to controls. ASNS expression of mesenchymal stromal cells (MSC) was found to be 2.3 times higher than that of blasts at diagnosis of ALL. We also observed that the values of the ASNS mRNA of MSC seem to reach a peak at diagnosis, and tend to decline with treatment. No correlation was found between the ASNS mRNA and protein levels. Chemotherapy does not exert any effect on the protein expression. Variability of asparaginase-induced effect may be attributable to factors involved in the interaction of hematopoietic cells with their microenvironment.

Adult Onset Still's Disease: A Case Report with a Rare Clinical Manifestation and Pathophysiological Correlations.

Adult-onset Still's disease is an inflammatory multisystemic disease of unknown etiology. Pleuritis is the most common pulmonary manifestation and pleural effusions are usually exudates with a predominance of neutrophils. We report a case of an eosinophilic pleural effusion as a novel and hitherto unrecognized manifestation of active adult-onset Still's disease. We also observed a marked NLRP3 inflammasome activation with increased production of IL-1ß which coincided with the development and resolved upon remission of the pleural effusion suggesting a possible novel pathogenetic pathway for the development of pleural effusions in the context of the auto-inflammatory disorders.

Interleukin-21 is increased in active systemic lupus erythematosus patients and contributes to the generation of plasma B cells.

OBJECTIVES: Excessive interleukin- (IL-) 21 production by T cells has been implicated in the pathogenesis of systemic lupus erythematosus (SLE). We explored the expression and function of IL-21 in human SLE. METHODS: IL-21 and IL-21 receptor (IL-21R) expression was assessed by real-time PCR and flow cytometry in peripheral blood mononuclear cells (PBMCs) of SLE patients and healthy controls. PBMCs, purified CD19+CD27- naïve and CD19+CD27+ memory B cells were stimulated with IL-21 and CpG-ODN2006 (TLR-9 agonist) to examine generation of memory and plasma (CD19+CD38highIgD-) B cells. Apoptosis was assessed by 7AAD staining. RESULTS: Active SLE patients had 4-fold higher IL-21 mRNA and increased levels of intracellular IL-21 in peripheral blood CD4+ T cells (mean±SD fluorescence intensity, 1.7±0.1 in active versus 0.9±0.3 in inactive SLE and controls, p=0.035). IL-21R mRNA was comparable between SLE and healthy controls. Stimulation of PBMCs with IL-21 increased the proportion of memory and plasma cells; addition of CpG-ODN2006 enhanced these effects. Both naïve and memory B cells responded to IL-21/TLR-9 by increased generation of memory and plasma B cells, respectively; an anti-apoptotic effect was observed. In active SLE, PBMCs stimulation with IL-21 and/or CpG-ODN increased memory and plasma B cells, comparable to healthy controls. Addition of IL-21 to lupus autologous mixed lymphocyte cultures induced significant IgG production, and treatment with IL-21R.Fc to block IL-21/IL-21R interaction reduced the proportion of plasma cells. CONCLUSIONS: Increased IL-21 may synergise with TLR-9 signalling and contributes to generation of plasma cells in active SLE patients.

A common SNP in the CD40 region is associated with systemic lupus erythematosus and correlates with altered CD40 expression: implications for the pathogenesis.

BACKGROUND: In systemic lupus erythematosus (SLE) sustained CD40L expression by T cells and platelets activates a variety of cells via its receptor CD40 contributing to disease pathogenesis. Although CD40 has recently been identified in genome-wide association study as a novel rheumatoid arthritis susceptibility gene such an association has not been documented for SLE. OBJECTIVE: To investigate whether the rs4810485 CD40 single nucleotide polymorphism (SNP) is associated with increased risk for SLE and its impact on CD40 expression. MATERIALS AND METHODS: The primary sample set consisted of 351 patients with SLE and 670 matched healthy controls of Greek origin. 158 patients with SLE and 155 controls from Turkey were used as a replication sample. Genotyping of rs4810485 was performed by restriction fragment length polymorphism and the Sequenom MassArray technology. The expression of CD40 mRNA and protein was assessed in unstimulated and lipopolysaccharide-stimulated peripheral blood mononuclear cells by quantitative real time PCR and flow cytometry, respectively. RESULTS: The minor allele T of CD40 rs4810485 SNP was significantly under-represented in Greek patients with SLE compared with healthy controls (OR=0.65, 95% CI 0.54 to 0.79). The association was replicated in the Turkish cohort (OR=0.57, 95% CI 0.41 to 0.80; meta-analysis of 509 patients with SLE and 825 healthy controls: OR=0.63, 95% CI 0.53 to 0.74, p = 2×10(-8)). In both cases and controls, the rs4810485 G/T and T/T genotypes were associated with significantly reduced CD40 mRNA and protein expression in peripheral blood CD14+ monocytes and CD19+ B cells compared with G/G genotype, both under basal conditions and following stimulation. CONCLUSIONS: CD40 has been identified as a new susceptibility locus in Greek and Turkish patients with SLE. The rs4810485 minor allele T is under-represented in SLE and correlates with reduced CD40 expression in peripheral blood monocytes and B cells, with potential implications for the regulation of aberrant immune responses in the disease.

Genetic, immunologic, and immunohistochemical analysis of the programmed death 1/programmed death ligand 1 pathway in human systemic lupus erythematosus.

OBJECTIVE: A putative regulatory intronic polymorphism (PD1.3) in the programmed death 1 (PD-1) gene, a negative regulator of T cells involved in peripheral tolerance, is associated with increased risk for systemic lupus erythematosus (SLE). We undertook this study to determine the expression and function of PD-1 in SLE patients. METHODS: We genotyped 289 SLE patients and 256 matched healthy controls for PD1.3 by polymerase chain reaction-restriction fragment length polymorphism analysis. Expression of PD-1 and its ligand, PDL-1, was determined in peripheral blood lymphocytes and in renal biopsy samples by flow cytometry and immunohistochemistry. A crosslinker of PD-1 was used to assess its effects on anti-CD3/anti-CD28-induced T cell proliferation and cytokine production. RESULTS: SLE patients had an increased frequency of the PD1.3 polymorphism (30.1%, versus 18.4% in controls; P=0.006), with the risk A allele conferring decreased transcriptional activity in transfected Jurkat cells. Patients homozygous for PD1.3-but not patients heterozygous for PD1.3-had reduced basal and induced PD-1 expression on activated CD4+ T cells. In autologous mixed lymphocyte reactions (AMLRs), SLE patients had defective PD-1 induction on activated CD4+ cells; abnormalities were more pronounced among homozygotes. PD-1 was detected within the glomeruli and renal tubules of lupus nephritis patients, while PDL-1 was expressed by the renal tubules of both patients and controls. PD-1 crosslinking suppressed proliferation and cytokine production in both normal and lupus T cells; addition of serum from patients with active SLE significantly ameliorated this effect on proliferation. CONCLUSION: SLE patients display aberrant expression and function of PD-1 attributed to both direct and indirect effects. The expression of PD-1/PDL-1 in renal tissue and during AMLRs suggests an important role in regulating peripheral T cell tolerance.

Mannose-binding lectin MBL2 gene polymorphisms and outcome of hepatitis C virus-infected patients.

INTRODUCTION: Mannose-binding lectin (MBL) is involved in host's response to several infections including hepatitis B but little is known about MBL and hepatitis C virus (HCV) infection. The present study attempts to investigate whether MBL2 genotype and serum MBL levels affect the course of HCV infection. RESULTS AND DISCUSSIONS: We investigated the variant alleles in MBL2 gene promoter and exon-1 regions in 80 Caucasian HCV-infected patients. Mutations in MBL2 were determined by polymerase chain reaction and restriction fragment length polymorphisms analysis. Serum MBL levels were measured by ELISA. Polymorphism homozygosity in exon-1 region was significantly related to lower serum MBL levels (p < 0.001), to liver inflammation (p = 0.034, OR = 11.7) and, in a lesser degree, to fibrosis. Polymorphisms in promoter sites -221nt and -550nt were not shown to be related with serum MBL levels or progress to liver inflammation and fibrosis. Serum MBL levels were adversely associated with progression to fibrosis (p = 0.037). Response to antiviral treatment was related to hepatitis C virus genotype (p < 0.001, OR = 10.9), but not to MBL2 genotype or serum MBL levels. CONCLUSION: These findings suggest that polymorphisms in MBL2 gene exon-1 region are related to low serum MBL levels and progression of HCV infection to liver inflammation and fibrosis.

Expansion of toll-like receptor 9-expressing B cells in active systemic lupus erythematosus: implications for the induction and maintenance of the autoimmune process.

OBJECTIVE: Toll-like receptors (TLRs) are pattern-associated receptors in innate immunity that may be involved in the recognition of self antigens and the production of pathogenic autoantibodies. This study was undertaken to examine the expression and function of various TLRs in subpopulations of peripheral blood mononuclear cells (PBMCs) of patients with systemic lupus erythematosus (SLE). METHODS: The expression of TLRs in PBMCs from 50 SLE patients with active disease (SLE Disease Activity Index [SLEDAI] score >or=8; n = 26) or inactive disease (SLEDAI score <8; n = 24) and 20 healthy controls was studied by flow cytometry. TLR expression was assessed on various subpopulations of PBMCs (TLR-2 and TLR-4 by membrane staining; TLR-3 and TLR-9 by intracellular staining). TLR function was accessed by stimulating PBMCs with specific ligands. RESULTS: The proportion of B cells and monocytes expressing TLR-9 was higher among patients with active SLE (mean +/- SD 49.5 +/- 24.4% and 30.7 +/- 24.1%, respectively) than among patients with inactive disease (22.8 +/- 19.6% and 14.3 +/- 8.4%, respectively; P = 0.02 and P = 0.03). Among B cells, the proportion of plasma cells and memory B cells expressing TLR-9 was increased in patients with active SLE. Increased percentages of TLR-9-expressing B cells correlated with the presence of anti-double-stranded DNA antibodies (P = 0.007). Treatment with serum from patients with active disease increased the percentage of TLR-9-expressing plasma cells in serum from healthy controls. Enhanced induction of HLA-DR after TLR-9 stimulation was documented in B cells from patients with active disease. CONCLUSION: In patients with active SLE, the proportion of peripheral blood memory B cells and plasma cells expressing TLR-9 is increased. Endogenous nucleic acids released during apoptotic cell death may stimulate B cells via TLR-9 and contribute to SLE pathogenesis.