Thea Bensi

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.

The T cell activation molecule H4 and the CD28-like molecule ICOS are identical.

The recently cloned CD28‐like molecule ICOS displays striking similarities with H4, characterized some years ago in the mouse and recently in humans. Both molecules are selectively expressed by activated and germinal center T cells, display similar structure, and display co‐stimulatory activities. H4 displays lateral association with the CD3/TCR and is expressed by mature thymocytes. In the mouse, H4 is also expressed at high levels by thymic NKT cells that are resistant to negative selection. The aim of this work was to evaluate whether H4 and ICOS are the same molecule using the C398.4A (binding human and mouse H4) and F44 (binding human ICOS) monoclonal antibody (mAb) in parallel experiments on human T cells. ICOS and H4 displayed the same expression pattern in a panel of T cell lines and the same expression kinetics in phytohemagglutinin‐activated T cells. C398.4A completely blocked cell staining by F44, whereas F44 partially blocked C398.4A. H4 and ICOS immunoprecipitates displayed identical SDS‐PAGE patterns and H4 immunoprecipitation completely removed ICOS from cell lysates. Finally, the C398.4A mAb specifically stained cells transfected with the human or mouse ICOS. These data prove that H4 and ICOS are the same molecule and that F44 and C398.4A bind partially different epitopes.

Immunohistochemistry and molecular diagnostics of pleural malignant mesothelioma.

CONTEXT The pathologic approach to pleural-based lesions is stepwise and uses morphologic assessment, correlated with clinical and imaging data supplemented by immunohistochemistry (IHC), and more recently, molecular tests, as an aid for 2 main diagnostic problems: malignant mesothelioma (MM) versus other malignant tumors and malignant versus reactive mesothelial proliferations. OBJECTIVE To present the current knowledge regarding IHC and molecular tests with respect to MM diagnosis, and in particular, the differentiation of the epithelioid type of MM from carcinoma metastatic to the pleural cavity. DATA SOURCES A review of immunohistochemical features of 286 consecutive MMs from 459 cases of pleural pathology, diagnosed during routine practice from 2003 to 2009. A survey of biomedical journal literature from MedLine/PubMed (US National Library of Medicine) focused on MM and associated tissue-based diagnostic IHC markers and molecular tests. CONCLUSIONS The search for a single diagnostic marker of MM has so far been discouraging, given the biologic and phenotypic tumor heterogeneity of MM. The use of antibody panels has gained unanimous acceptance especially in the differential diagnosis between MM and metastatic carcinoma, whereas the usefulness of IHC is more limited when dealing with spindle cell malignancies or distinguishing malignant from reactive mesothelium. A great degree of interlaboratory variability in antibody combinations and clone selection within diagnostic panels still exists. Current investigations aim at selecting the most suitable and cost-effective combination of antibodies by using novel statistical approaches for assessing diagnostic performance beyond the traditional measures of sensitivity and specificity.

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.

Expression of the Novel T Cell Activation Molecule hpH4 in HIV-Infected Patients: Correlation with Disease Status

We have described hpH4, a surface glycoprotein selectively expressed by activated T cells and mature thymocytes and displaying weak lateral association with CD4. The hpH4 expression pattern and biochemical features, together with analysis of its tryptic digest by peptide mass searching using MALDI-MS, suggested that it is a novel molecule. The aim of this work was to evaluate the peripheral blood T cell expression of hpH4 in HIV-infected patients and the interplay between HIV gp120 and hpH4, since both molecules interact with CD4. hpH4 expression during HIV-1 infection was evaluated by assessing 55 patients at various disease stages and following up 3 patients with primary infection and 3 patients with AIDS. hpH4 expression displayed a peak in the early phase of primary infection, dropped to control levels in the asymptomatic phase, and was newly expressed, at low levels, as AIDS developed. The expression kinetics were different than those shown by HLA-DR, CD25, and CD38. The most striking findings were the transient hpH4 expression peak displayed in the earliest stage, which was unique for hpH4. Incubation of T cells from normal donors with HIV gp120 induced transient hpH4 expression in resting CD4+ T cells and potentiated the hpH4 lateral association with CD4 in activated T cells. Moreover, hpH4 triggering inhibited gp120-induced death of CD4+ cells. Therefore, H4 expression may be a response to avoid apoptosis induced by HIV products.

Effects of the human CD38 glycoprotein on the early stages of the HIV-1 replication cycle

CD38 displays lateral association with the HIV‐1 receptor CD4. This association is potentiated by the HIV‐1 envelope glycoprotein gp120. The aim of this work was to evaluate the CD38 role in T cell susceptibility to HIV‐1 infection. Using laboratory X4 HIV‐1 strains and X4 and X4/R5 primary isolates, we found that CD38 expression was negatively correlated to cell susceptibility to infection, evaluated as percentage of infected cells, release of HIV p24 in the supernatants, and cytopathogenicity. This correlation was at first suggested by results obtained in a panel of human CD4+ T cell lines expressing different CD38 levels (MT‐4, MT‐2, C8166, CEMx174, Supt‐1, and H9) and then demonstrated using CD38 transfectants of MT‐4 cells (the line with the lowest CD38 expression). To address whether CD38 affected viral binding, we used mouse T cells that are non‐permissive for productive infection. Gene transfection in mouse SR.D10.CD4~.F1 T cells produced four lines expressing human CD4 and/or CD38. Ability of CD4+CD38+ cells to bind HIV‐1 or purified recombinant gp120 was significantly lower than that of CD4+CD38~ cells. These data suggest that CD38 expression inhibits lymphocyte susceptibility to HIV infection, probably by inhibiting gp120/CD4‐dependent viral binding to target cells.—Savarino, A., Bottarel, F., Calosso, L., Feito, M. J., Bensi, T., Bragardo, M., Rojo, J. M., Pugliese, A., Abbate, I., Capobianchi, M. R., Dianzani, F., Malavasi, F., and Dianzani, U. Effects of the human CD38 glycoprotein on the early stages of the HIV‐1 replication cycle. FASEB J. 13, 2265–2276 (1999)

The cell death-inducing ability of glycoprotein 120 from different HIV strains correlates with their ability to induce CD4 lateral association with CD95 on CD4+ T cells.

CD4 cross-linking by HIV gp120 triggers CD4+ T cell death. Several authors have suggested that this effect is mediated by CD95, but this possibility is debated by other authors. In a previous work, we found by co-capping that gp120(451) and gp120MN, but not gp120(IIIB), induce lateral association of CD4 with CD95 on the T cell surface. In this work, we used fluorescence resonance energy transfer to confirm that CD4/CD95 lateral association is induced by gp120(451), but not gp120(IIIB). Moreover, we found that gp120 ability to induce the CD4/CD95 association correlates with ability to induce cell death, since gp120(451) and gp120MN induced higher levels of cell death than did gp120(IIIB) in PHA-derived CD4+ T cell lines. CD95 involvement in gp120-induced cell death was confirmed by showing that gp120(451) and gp120MN did not induce death in CD4+ T cells derived from patients with autoimmune/lymphoproliferative disease (ALD) and decreased CD95 function. Cell death induced by gp120MN was inhibited by a recombinant CD95/IgG.Fc molecule blocking the CD95/CD95L interaction. However, inhibition was late and only partial. These data suggest that the gp120-induced CD4/CD95 association exerts a dual effect: an early effect that is independent of CD95L and may be due to direct triggering of CD95 by gp120, and a late effect that may be due to sensitization of CD95 to triggering by CD95L. In line with the former effect, cell treatment with gp120MN activated caspase 3 in the presence of Fas/IgG.Fc, which shows that cell death induced by gp120MN independently of CD95L uses the same pathway as CD95.

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.

Evaluation of the antiretroviral effects of a PEG-conjugated peptide derived from human CD38.

Objective: Cell infection by HIV-1 is inhibited by both the expression of CD38 and a soluble peptide (sCD38p) corresponding to its extracellular membrane-proximal amino acid sequence (amino acids 51 – 74). We show here the effects of PEG conjugation to sCD38p and provide new insights into the mechanisms behind the anti-HIV-1 effects of CD38 and derived peptides. Research design/methods: In-vitro and in-silico study. Results: PEGylation of sCD38p increased its ability to inhibit replication of HIV-1 in MT-4 cells and syncytia formation in cocultures of MT-2 and persistently HIV-1IIIB-infected H9IIIB cells. In silico modeling suggests that sCD38p and CD4 form stable heterodimers involving, among others, an interaction between lysine 57 (K57) of CD38 and a groove in the CD4 receptor, which, in CD4/gp120 complexes, is partially occupied by a lysine residue of the HIV-1 envelope glycoprotein. K57 substitution with a glycine in sCD38p impaired its ability to inhibit syncytia formation in MT-2/H9IIIB cell cocultures and gp120 binding to CD4 in a mouse T cell line expressing human but not mouse CD4. Conclusions: PEGylation significantly improves the anti-HIV-1 activity of sCD38p, whose effect is probably due to competition with gp120 for a common binding site on CD4 although other mechanisms cannot be excluded so far. The inhibitory concentrations of the sCD38p-PEG as well as its poor toxicity, merit further consideration in anti-HIV-1 strategies.