Riccardo Mesturini

Osteopontin gene haplotypes correlate with multiple sclerosis development and progression

Osteopontin (OPN) is an inflammatory cytokine highly expressed in multiple sclerosis (MS) plaques. In a previous work, we showed that four OPN polymorphisms form three haplotypes (A, B, and C) and that homozygotes for haplotype-A display lower OPN levels than non-AA subjects. In this work, we evaluated the distribution of these OPN haplotypes in 425 MS patients and 688 controls. Haplotype-A homozygotes had about 1.5 lower risk of developing MS than non-AA subjects. Clinical analysis of 288 patients showed that AA patients displayed slower switching from a relapsing remitting to a secondary progressive form and milder disease with slower evolution of disability. MS patients displayed increased OPN serum levels, which were partly due to the increased frequency of non-AA subjects. Moreover in AA patients, OPN levels were higher than in AA controls and similar to those found in both non-AA patients and controls, which suggests a role of the activated immune response. These data suggest that OPN genotypes may influence MS development and progression due to their influence on OPN levels.

Serum levels of osteopontin are increased in SIRS and sepsis

ObjectiveIn sepsis, dysregulation of the immune response leads to rapid multiorgan failure and death. Accurate and timely diagnosis is lifesaving and should discriminate sepsis from the systemic inflammatory response syndrome (SIRS) caused by non-infectious agents. Osteopontin acts as an extracellular matrix component or a soluble cytokine in inflamed tissues. Its exact role in immune response and sepsis remains to be elucidated. Therefore, we investigated the role of osteopontin in SIRS and sepsis.DesignProspective, observational study.SettingIntensive care unit of a university hospital.Patients and participantsFifty-six patients with SIRS or sepsis and 56 healthy subjects were enrolled.InterventionsWe analyzed the serum levels of osteopontin and TH1–TH2 cytokines and investigated the role of osteopontin on interleukin 6 secretion by monocytes.Measurements and main resultsSerum osteopontin levels were strikingly higher in patients than in controls and in sepsis than in SIRS, and decreased during the resolution of both the disorders. Receiver operating characteristic curves showed that osteopontin levels have discriminative power between SIRS and sepsis with an area under the curve of 0.796. Osteopontin levels directly correlated with those of interleukin 6 and in vitro, recombinant osteopontin increased interleukin 6 secretion by monocytes in both the absence and presence of high doses of lipopolysaccharide.ConclusionThese data suggest that osteopontin might be a mediator involved in the pathogenesis of SIRS and sepsis, possibly by supporting interleukin 6 secretion.Descriptor45. SIRS/Sepsis: clinical studies.

Group I mGlu receptor stimulation inhibits activation-induced cell death of human T lymphocytes.

1 The effects of L‐glutamate on activation‐induced cell death (AICD) of human activated (1 μg ml−1 phytohemagglutinin plus 2 U ml−1 interleukin‐2; 8 days) T lymphocytes were studied by measuring anti‐CD3 monoclonal antibody (10 μg ml−1; 18 h)‐induced cell apoptosis (Annexin V and propidium iodide staining). 2 L‐Glutamate (1 × 10−8–1 × 10−4 M) significantly (P0.01) inhibited AICD in a concentration‐dependent manner (EC50=6.3 × 10−8 M; maximum inhibition 54.8±6.3% at 1 × 10−6 M). 3 The L‐glutamate inhibitory effect was pharmacologically characterized as mediated by group I mGlu receptors, since mGlu receptor agonists reproduced this effect. The EC50 values were: 3.2 × 10−7 M for (1S,3R)‐ACPD; 4.5 × 10−8 M for quisqualate; 1.0 × 10−6 M for (S)‐3,5‐DHPG; 2.0 × 10−5 M for CHPG. 4 Group I mGlu receptor antagonists inhibited the effects of quisqualate 1.0 × 10−6 M. The IC50 values calculated were: 8.7 × 10−5, 4.3 × 10−6 and 6.3 × 10−7 M for AIDA, LY 367385 and MPEP, respectively. 5 L‐Glutamate (1 × 10−6 M; 18 h) significantly (P0.05) inhibited FasL expression (40.8±11.3%) (cytofluorimetric analysis), whereas it did not affect Fas signalling. 6 Expression of both mGlu1 and mGlu5 receptor mRNA by T lymphocytes and T‐cell lines, as demonstrated by reverse transcriptase–PCR analysis, suggests that L‐glutamate‐mediated inhibition of AICD was exerted on T cells. 7 These data depict a novel role for L‐glutamate in the regulation of the immune response through group I mGlu receptor‐mediated mechanisms.

ICOS cooperates with CD28, IL-2 and IFN-γ and modulates activation of human naïve CD4+ T cells

Several sets of data indicate that ICOS regulates cytokine production in activated T cells, but is less effective on naïve T cells. This work evaluates ICOS function in human naïve CD4+ T cells through an assessment of the effect of soluble forms of the ICOS and CD28 physiological ligands on activation driven by anti‐CD3 mAb. ICOS strikingly potentiated secretion of IL‐2, IFN‐γ, IL‐10, and TNF‐α, but not IL‐4, promoted by optimal stimulation of CD3+CD28, and it was the key switching‐factor of activation when cells received suboptimal stimulation of CD3+CD28 or stimulation of CD3 alone in the presence of exogenous IL‐2. In these conditions, blockade of IL‐2 and IFN‐γ showed that ICOS builds up a positive feedback loop with IFN‐γ, which required IL‐2 and was inhibited by IL‐4. By contrast, in the absence of CD28 triggering or exogenous IL‐2, ICOS‐induced costimulation mainly supported expression of TGF‐β1 and FoxP3 and differentiation of regulatory T cells capable to inhibit proliferation of naïve CD4+ T cells driven by allogeneic cells. These data suggest that ICOS favors differentiation of Th effector cells when cooperates with appropriate activation stimuli such as CD3+CD28 or CD3+IL‐2, whereas it supports differentiation of regulatory T cells when costimulatory signals are insufficient.

Human CD38 interferes with HIV-1 fusion through a sequence homologous to the V3 loop of the viral envelope glycoprotein gp120.

CD38 is a progression marker in HIV‐1 infection, it displays lateral association with CD4, and down‐modulates gp120/CD4 binding. The aim of this study was to elucidate the mechanism behind the interplay between CD4, CD38, and HIV‐1. We used mouse cell transfectants expressing human CD4 and either CD38 or other CD4‐associated molecules to show that CD38 specifically inhibits gp120/CD4 binding. Human cell transfectants expressing truncated forms of CD38 and bioinformatic analysis were used to map the anti‐HIV activity and show that it is concentrated in the membrane‐proximal region. This region displayed significant sequence‐similarity with the V3 loop of the HIV‐1 gp120 glycoprotein. In line with this similarity, synthetic soluble peptides derived from this region reproduced the anti‐HIV effects of full‐length CD38 and inhibited HIV‐1 and HIV‐2 primary isolates from different subtypes and with different coreceptor use. A multiple‐branched peptide construct presenting part of the sequence of the V3‐like region potently and selectively inhibited HIV‐1 replication in the nanomolar range. Conversely, a deletion in the V3‐like region abrogated the anti‐HIV‐1 activity of CD38 and its lateral association with CD4. These findings may provide new insights into the early events of HIV‐1 fusion and strategies to intervene.

SAP-Mediated Inhibition of Diacylglycerol Kinase α Regulates TCR-Induced Diacylglycerol Signaling

Diacylglycerol kinases (DGKs) metabolize diacylglycerol to phosphatidic acid. In T lymphocytes, DGKα acts as a negative regulator of TCR signaling by decreasing diacylglycerol levels and inducing anergy. In this study, we show that upon costimulation of the TCR with CD28 or signaling lymphocyte activation molecule (SLAM), DGKα, but not DGKζ, exits from the nucleus and undergoes rapid negative regulation of its enzymatic activity. Inhibition of DGKα is dependent on the expression of SAP, an adaptor protein mutated in X-linked lymphoproliferative disease, which is essential for SLAM-mediated signaling and contributes to TCR/CD28-induced signaling and T cell activation. Accordingly, overexpression of SAP is sufficient to inhibit DGKα, whereas SAP mutants unable to bind either phospho-tyrosine residues or SH3 domain are ineffective. Moreover, phospholipase C activity and calcium, but not Src-family tyrosine kinases, are also required for negative regulation of DGKα. Finally, inhibition of DGKα in SAP-deficient cells partially rescues defective TCR/CD28 signaling, including Ras and ERK1/2 activation, protein kinase Cθ membrane recruitment, induction of NF-AT transcriptional activity, and IL-2 production. Thus SAP-mediated inhibition of DGKα sustains diacylglycerol signaling, thereby regulating T cell activation, and it may represent a novel pharmacological strategy for X-linked lymphoproliferative disease treatment.

Cholesteryl butyrate solid lipid nanoparticles inhibit the adhesion and migration of colon cancer cells

BACKGROUND AND PURPOSE Cholesteryl butyrate solid lipid nanoparticles (cholbut SLN) provide a delivery system for the anti‐cancer drug butyrate. These SLN inhibit the adhesion of polymorphonuclear cells to the endothelium and may act as anti‐inflammatory agents. As cancer cell adhesion to endothelium is crucial for metastasis dissemination, here we have evaluated the effect of cholbut SLN on adhesion and migration of cancer cells.

Defective Fas‐mediated T‐cell apoptosis predicts acute onset CIDP

Guillain‐Barré syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP) are immune‐mediated neuropathies. GBS is characterized by acute onset and subsequent remission of symptoms, whereas CIDP displays slow progression over at least 2 months. However, a small proportion of CIDP patients display acute onset CIDP (a‐CIDP) resembling that of GBS. The Fas receptor is involved in shutting off the immune response and its defective function predisposes to auto‐immune diseases. In CIDP patients, Fas function is lower than in GBS patients and healthy controls. This study is aimed at assessing whether evaluation of T‐cell Fas function helps in early discrimination between GBS and a‐CIDP. Fas function was evaluated in patients with acute onset polyneuropathy: 55 retrospective patients analyzed after development of GBS or a‐CIDP before year 2005; 50 prospective patients analyzed at onset after year 2005 and followed up for development of GBS or a‐CIDP. In both groups, a‐CIDP patients displayed defective Fas function, whereas GBS patients displayed normal function. These findings suggest that the evaluation of Fas function in acute onset polyneuropathy helps in early prediction of long‐term outcome.

Fas-mediated T-cell apoptosis is impaired in patients with chronic inflammatory demyelinating polyneuropathy.

Abstract  The Fas death receptor is expressed by activated lymphocytes and is involved in switching‐off the immune response. Its inherited defects cause auto‐immune lymphoproliferative syndrome. Impaired Fas function may also play a role in other auto‐immune diseases, such as multiple sclerosis and type 1 diabetes mellitus. The aim of this work was to evaluate Fas function in T cells from patients with chronic inflammatory demyelinating polyneuropathy (CIDP). We evaluated Fas‐induced apoptosis in T‐cell lines from 27 patients with CIDP, 12 patients with acute inflammatory demyelinating polyneuropathy (AIDP), and 110 controls. CIDP patients displayed lower Fas function than both AIDP patients and controls, whereas no statistically significant difference was found between AIDP patients and controls. Moreover, Fas function was lower in CIDP patients with progressive course than in those with relapsing–remitting course and lower in CIDP patients with axonal damage than in those with pure demyelination. These data suggest that defective Fas function favours CIDP development and aggressive evolution.

The 423Q polymorphism of the X-linked inhibitor of apoptosis gene influences monocyte function and is associated with periodic fever.

OBJECTIVE Hereditary periodic fever syndromes (HPFs) develop as a result of uncontrolled activation of the inflammatory response, with a substantial contribution from interleukin-1beta or tumor necrosis factor alpha (TNFalpha). The HPFs include familial Mediterranean fever (FMF), hyperimmunoglobulinemia D with periodic fever syndrome (HIDS), TNF receptor-associated syndrome (TRAPS), and cryopyrinopathies, which are attributable to mutations of the MEFV, MVK, TNFRSF1A, and CIAS1 genes, respectively. However, in many patients, the mutated gene has not been determined; therefore, the condition in these patients with an HPF-like clinical picture is referred to as idiopathic periodic fever (IPF). The aim of this study was to assess involvement of X-linked inhibitor of apoptosis (XIAP), which plays a role in caspase inhibition and NF-kappaB signaling, both of which are processes that influence the development of inflammatory cells. METHODS The XIAP gene (X-linked) was sequenced in 87 patients with IPF, 46 patients with HPF (13 with HIDS, 17 with TRAPS, and 16 with FMF), and 182 healthy control subjects. The expression of different alleles was evaluated by sequencing XIAP-specific complementary DNA mini-libraries and by real-time polymerase chain reaction and Western blot analyses. The functional effect of XIAP on caspase 9 activity was assessed by a fluorimetric assay, and cytokine secretion was evaluated by enzyme-linked immunosorbent assay. RESULTS Sequencing disclosed a 1268A>C variation that caused a Q423P amino acid substitution. The frequency of 423Q-homozygous female patients and 423Q-hemizygous male patients was significantly higher in the IPF group than in the control group (69% versus 51%; odds ratio 2.17, 95% confidence interval 1.23-3.87, P = 0.007), whereas no significant difference was detected in the HPF group (59%) compared with controls. In primary lymphocytes and transfected cell lines, 423Q, as compared with 423P, was associated with higher XIAP protein and messenger RNA expression and lower caspase 9 activation. In lipopolysaccharide-activated monocytes, 423Q was associated with higher secretion of TNFalpha. CONCLUSION These results suggest that 423Q is a predisposing factor for IPF development, possibly through its influence on monocyte function.