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Within the paradigm of smart mobility, the development of innovative materials aimed at improving resilience against structural failure in lightweight vehicles and electromagnetic interferences (EMI) due to wireless communications in guidance systems is of crucial relevance to improve safety, sustainability, and reliability in both aeronautical and automotive applications. In particular, the integration of intelligent structural health monitoring and electromagnetic (EM) shielding systems with radio frequency absorbing properties into a polymer composite laminate is still a challenge. In this paper, we present an innovative system consisting of a multi-layered thin panel which integrates nanostructured coatings to combine EM disturbance suppression and low-energy impact monitoring ability. Specifically, it is composed of a stack of dielectric and conductive layers constituting the sensing and EM-absorbing laminate (SEAL). The conductive layers are made of a polyurethane paint filled with graphene nanoplatelets (GNPs) at different concentrations to tailor the effective electrical conductivity and the functionality of the material. Basically, the panel includes a piezoresistive grid, obtained by selectively spraying onto mylar a low-conductive paint with 4.5 wt.% of GNPs and an EM-absorbing lossy sheet made of the same polyurethane paint but properly modified with a higher weight fraction (8 wt.%) of graphene. The responses of the grid’s strain sensors were analyzed through quasi-static mechanical bending tests, whereas the absorbing properties were evaluated through free-space and waveguide-based measurement techniques in the X, Ku, K, and Ka bands. The experimental results were also validated by numerical simulations.

Hypervirulent Klebsiella pneumoniae (Hv-Kp) strains have emerged as pathogens causing life-threatening, invasive disease even in immunocompetent hosts. Systemic dissemination usually occurs following perturbations of the gut microbiota and is facilitated by Hv-Kp resistance to phagocytosis and complement activity. Hv-Kp are usually associated with K1 or K2 capsular types, produce several iron uptake systems (e.g., aerobactin and salmochelin) and are often but not invariably, capsular material hyper-producers (hypermucoviscous phenotype: HMV). Whether Hv-Kp escape the immune response at mucosal site is unknown. In this work, we studied the effects of Hv-Kp on human dendritic cells (DCs), central players of the IL-23/IL-17 and IL-12/IFN-γ axis at mucosal sites, essential for pathogen clearance. Four Hv-Kp and HMV strains were selected and their activity on DC maturation and cytokine production was compared to that of non-virulent Kp strains with classic or HMV phenotypes. While the maturation process was equally induced by all Kp strains, significant differences between virulent and non-virulent strains were found in the expression of genes for cytokines involved in T-cell activation and differentiation. The non-virulent KP04C62 and the classic Kp, KPC157 induced high expression of TH1 (IL-12p70 and TNFα) and TH17 cytokines (IL-23, IL-1β and IL-6), while Hv-Kp poorly activated these cytokine genes. Moreover, conditioned media from DCs cultured with non-virulent Kp, either classical or hypercapsulated, induced the activation of IL-17 and IFN-γ genes in preactivated CD4+-cells suggesting their TH17/TH1 differentiation. Conditioned media from Hv-Kp poorly activated IL-17 and IFN-γ genes. In summary, our data indicate that Hv-Kp interfere with DC functions and T-cell differentiation and suggest that the escape from the IL-23/IL-17 and IL-12/IFN-γ axes may contribute to pathogen dissemination in immunocompetent hosts.

Hypervirulent

MCR-1 is a plasmid-encoded phosphoethanolamine transferase able to modify the lipid A structure. It confers resistance to colistin and was isolated from human, animal, and environmental strains of Enterobacteriaceae, raising serious global health concerns. In this paper, we used recombinant mcr-1-expressing Escherichia coli to study the impact of MCR-1 products on E. coli-induced activation of inflammatory pathways in activated THP-1 cells, which was used as a model of human macrophages. We found that infection with recombinant mcr-1-expressing E. coli significantly modulated p38-MAPK and Jun N-terminal protein kinase (JNK) activation and pNF-κB nuclear translocation as well as the expression of genes for the relevant proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-12 (IL-12), and IL-1β compared with mcr-1-negative strains. Caspase-1 activity and IL-1β secretion were significantly less activated by mcr-1-positive E. coli strains than the mcr-1-negative parental strain. Similar results were obtained with clinical isolates of mcr-1-positive E. coli, suggesting that, in addition to colistin resistance, the expression of mcr-1 allows the escape of early host innate defenses and may promote bacterial survival.

Interference with transforming growth factor-β-mediated pathways helps several parasites to survive for long periods in immunocompetent hosts. Macrophages and dendritic cells infected by Toxoplasma, Leishmania and Plasmodium spp. produce large amounts of transforming growth factor-β and induce the differentiation of antigen-specific T-regulatory cells. Mechanisms not mediated by antigen-presentation could also account for the expansion of T-regulatory cells in parasitic diseases and they also might be mediated through transforming growth factor-β-receptor activated pathways. We explored the properties of soluble extracts from Leishmania infantum promastigotes, Toxoplasma gondii tachyzoites, Trichinella spiralis muscle larvae to expand the pool of T-regulatory cells in a population of polyclonally activated T cells in the absence of accessory cells, and compared their effects to those induced by Plasmodium falciparum extracts. Similarly to P. falciparum, L. infantum extracts activate the latent soluble form of transforming growth factor-β and that bound to the membrane of activated T lymphocytes. The interaction of the active cytokine with transforming growth factor-β receptor induces Foxp3 expression by activated lymphocytes, favoring their conversion through the T-regulatory phenotype. Both Toxoplasma gondii and L. infantum extracts are able to induce transforming growth factor-β production by activated T cells in the absence of accessory cells.

Increased numbers of T regulatory cells (Tregs), key mediators of immune homeostasis, were reported in human and murine malaria and it is current opinion that these cells play a role in balancing protective immunity and pathogenesis during infection. However, the mechanisms governing their expansion during malaria infection are not completely defined. In this article we show that soluble extracts of Plasmodium falciparum (PfSEs), but not equivalent preparation of uninfected erythrocytes, induce the differentiation of polyclonally activated CD4(+) cells in Tregs endowed with strong suppressive activity. PfSEs activate latent TGFβ bound on the membrane of Treg cells, thus allowing the cytokine interaction with TGFβ receptor, and inducing Foxp3 gene expression and TGFβ production. The activation of membrane-bound latent TGFβ by PfSEs is significantly reduced by a broad-spectrum metalloproteinases inhibitor with Zn(++) -chelating activity, and completely inhibited by the combined action of such inhibitor and antibodies to a P. falciparum thrombospondin-related adhesive protein (PfTRAP). We conclude that Pf-Zn(++) -dependent proteinases and, to a lesser extent, PfTRAP molecules are involved in the activation of latent TGFβ bound on the membrane of activated Treg cells and suggest that, in malaria infection, this mechanism could contribute to the expansion of Tregs with different antigen specificity.

Cytokines are a group of polypeptide hormones endowed with pleiotropic biological properties. Normal B lymphocytes produce a number of these factors that subserve important regulatory functions in the combined processes of proliferation and differentiation. Also neoplastic B cells can release cytokines and, simultaneously, respond to the same factors in an autocrine circuit that supports their malignant growth. In addition, tumor cells can make use of the factors released by normal cells, either spontaneously or under the influence of inductive signals from the neoplastic cells. Inappropriate or excessive release of cytokines may have an important role in the pathophysiology of some clinical features. Thus, neutralization of cytokine biologic activity in vivo could be a therapeutic strategy for treatment of human B-cell neoplasias.

The detection of a γ-heavy chain disease protein in the serum and urine of a patient with a history of Hodgkin's disease in complete remission is reported. The protein has been characterized as a γ1 dimer, MW 66000, beginning at Asp 221 residue in the hinge region. No clinical or laboratory findings of malignancy have been observed in the patient since the abnormal protein was detected. The pathogenesis of this association, unreported to date, is discussed on the basis of previously administered high-dose anti-neoplastic treatments and of immunological impairment.

Candida spp. usually colonize ulcerative lesions of atrophic mucosa in patients with chemotherapy-induced oral mucositis inducing severe inflammation. The spread of antifungal-resistant strains strongly encouraged the search of complementary or alternative therapeutic strategies to cure inflamed mucosa. In this paper, we studied the effects of a near-infrared (NIR) laser system with dual-wavelength emission (808 nm + 904 nm) on the survival and inflammatory potential of C. albicans, C. glabrata, and C. parapsilosis. Laser treatment was performed with a Multiwave Locked System laser. Survival and apoptosis of fungal strains were evaluated by colony-forming units (CFU) counting and annexin V staining. Cytokine production was evaluated by ImmunoPlex array. Laser treatment significantly affected the survival of Candida spp. by inducing apoptosis and induced a lower production of inflammatory cytokines by dendritic cells compared to untreated fungi. No differences in the survival and inflammatory potential were recorded in treated or untreated Saccharomyces cerevisiae cells, used as the control non-pathogenic microorganism. Laser treatment altered the survival and inflammatory potential of pathogenic Candida spp. These data provide experimental support to the use of NIR laser radiation as a co-adjuvant of antifungal therapy in patients with oral mucositis (OM) complicated by Candida infections.

The antiapoptotic role of Bcl-2 can be regulated by its phosphorylation in serine and threonine residues located in a nonstructured loop that links BH3 and BH4 domains. p38 MAPK has been identified as one of the kinases able to mediate such phosphorylation, through direct interaction with Bcl-2 protein in the mitochondrial compartment. In this study, we identify, by using mass spectrometry techniques and specific anti-phosphopeptide antibodies, Ser87 and Thr56 as the Bcl-2 residues phosphorylated by p38 MAPK and show that phosphorylation of these residues is always associated with a decrease in the antiapoptotic potential of Bcl-2 protein. Furthermore, we obtained evidence that p38 MAPK-induced Bcl-2 phosphorylation plays a key role in the early events following serum deprivation in embryonic fibroblasts. Both cytochrome c release and caspase activation triggered by p38 MAPK activation and Bcl-2 phosphorylation are absent in embryonic fibroblasts from p38α knock-out mice (p38α-/- MEF), whereas they occur within 12 h of serum withdrawal in p38α+/+ MEF; moreover, they can be prevented by p38 MAPK inhibitors and are not associated with the synthesis of the proapoptotic proteins Bax and Fas. Thus, Bcl-2 phosphorylation by activated p38 MAPK is a key event in the early induction of apoptosis under conditions of cellular stress.

BACKGROUND Alterations in fibroblast growth factors (FGFs) production and/or FGF receptors expression have been described to play key roles in prostate tumor progression, particularly in androgen-independent tumors. However, the role of androgen receptor (AR) in altering FGF-mediated growth and survival of prostatic neoplastic cells has not been completely defined. In this study, we investigated the alterations in FGF2 production and utilization by the PC3 cell line, after transfection with a full-length AR. METHODS FGF1,2,7, FGF-binding protein (FGF-BP) production and FGF receptor (FGFR) 1–4 expression were investigated by polymerase chain reaction (PCR) and Western blot analysis. RESULTS De novo AR expression by PC3 cells restores FGFR2 IIIb isoform expression and sensitivity to FGF7 and FGF2. Androgen stimulation induces AR+ PC3 clones to secrete FGF-BP, likely responsible for activation and mobilization from the extracellular matrix of the high amounts of FGF2 produced by the same cells. In addition to the effects on cell proliferation, FGF2 maintains the survival of AR+ PC3 clones through a positive modulation of the Bcl-2 protein and down-modulates AR protein expression, allowing the escape of selected clones from androgen regulation. CONCLUSION In the presence of an active AR, the combined production of FGF2 and FGF-BP may play an important role in the progression of prostate cancer through the selection of AR− clones expressing high levels of Bcl-2. Prostate 53: 310–321, 2002. © 2002 Wiley-Liss, Inc.

The spread of KPC-type carbapenemases is mainly attributed to the global dissemination of Klebsiella pneumoniae (KP) strains belonging to the clonal group (CG) 258, including sequence type (ST) 258 and other related STs. Two distinct clades of CG258-KP have evolved, which differ mainly for the composition of their capsular polysaccharides, and recent studies indicate that clade 1 evolved from an ancestor of clade 2 by recombination of a genomic fragment carrying the capsular polysaccharide (cps) locus. In this paper, we investigated the ability of two ST258-KP strains, KKBO-1 and KK207-1, selected as representatives of ST258-KP clade 2 and clade 1, respectively, to activate an adaptive immune response using ex vivo-stimulation of PBMC from normal donors as an experimental model. Our data showed that KKBO-1 (clade 2) induces a Th17 response more efficiently than KK207-1 (clade 1): the percentage of CD4+IL17+ cells and the production of IL-17A were significantly higher in cultures with KKBO-1 compared to cultures with KK207-1. While no differences in the rate of bacterial internalization or in the bacteria-induced expression of CD86 and HLA-DR by monocytes and myeloid dendritic cells were revealed, we found that the two strains significantly differ in inducing the production of cytokines involved in the adaptive immune response, as IL-1β, IL-23 and TNF-α, by antigen-presenting cells, with KKBO-1 being a more efficient inducer than KK207-1. The immune responses elicited by KK207-1 were comparable to those elicited by CIP 52.145, a highly virulent K. pneumoniae reference strain known to escape immune-inflammatory responses. Altogether, present results suggest that CG258-KP of the two clades are capable of inducing a different response of adaptive immunity in the human host.

Survival of memory B lymphocytes is tightly linked to the integrity of the Bcl-2 protein and is regulated by a nerve growth factor (NGF) autocrine circuit. In factor-starved memory B cells, the addition of exogenous NGF promptly induced p38 mitogen-activated protein kinase (MAPK), but not c-Jun N-terminal kinase (JNK), dephosphorylation. Conversely, withdrawal of endogenous NGF was followed by p38 MAPK activation and translocation onto mitochondria, whereby it combined with and phosphorylated Bcl-2, as assessed by co-immunoprecipitation and kinase assays in vivo and in vitro. Mitochondria isolated from human memory B cells, then exposed to recombinant p38 MAPK, released cytochrome c, as did mitochondria from Bcl-2-negative MDCK cells loaded with recombinant Bcl-2. Apoptosis induced by NGF neutralization could be blocked by the specific p38 MAPK inhibitor SB203580 or by Bcl-2 mutations in Ser-87 or Thr-56. These data demonstrate that the molecular mechanisms underlying the survival factor function of NGF critically rely upon the continuous inactivation of p38 MAPK, a Bcl-2-modifying enzyme.

The optimal immune response to malaria infection comprises rapid induction of inflammatory responses promptly counteracted by regulatory mechanisms to prevent immunopathology. To evaluate the role of dendritic cells (DC) in the balance of parasite-induced inflammatory/anti-inflammatory mechanisms, we studied the activity of monocyte-derived dendritic cells (MDDC), previously exposed to soluble extracts of Plasmodium falciparum -infected red blood cells ( Pf SE), in the differentiation of CD4 cells isolated from donors never exposed to malaria infection. We show that MDDC exposed to Pf SE are extremely efficient to induce a contemporary differentiation of TH1 effector cells and T regulatory (Treg) cells in CD4 T cells even when exposed to low concentrations of parasitic extracts. Treg cells induced by MDDC infected with Pf SE (MDDC- Pf SE) produce transforming growth factor beta (TGF-β) and interleukin 10 (IL-10) and are endowed with strong suppressive properties. They also show phenotypical and functional peculiarities, such as the contemporary expression of markers of Treg and TH1 differentiation and higher sensitivity to TLR4 ligands both inducing an increasing production of suppressive cytokines. On the whole, our data indicate that MDDC exposed to Pf SE orchestrate a well-balanced immune response with timely differentiation of TH1 and Treg cells in CD4 cells from nonimmune donors and suggest that, during the infection, the role of MDCC could be particularly relevant in low-parasitemia conditions.

IL-1 receptor antagonist (IL-1ra) is a newly described member of the IL-1 family, isolated from supernatants of Ig stimulated monocytes, that binds competitively to IL-1 receptors without stimulating target cells (1-3). Also epithelial cells produce IL-1ra in a form which lacks a secretory signal sequence (4).Here we have compared the biosynthesis and secretion of IL-1ra in monocytes and keratinocytes. Our data show that monocytes produce two molecular forms of IL-1ra, of 18 Kd and 23 Kd respectively, which differ in the degree of glycosylation. Both forms are secreted via the "classical" endoplasmic reticulum (ER)-Golgi secretory pathway. By contrast keratinocytes produce IL-1ra in a molecular form of 20 Kd, which is not N-glycosylated: 20 Kd IL-1ra is detectable in supernatants of keratinocytes, although in small amounts. The presence of IL-1ra in keratinocytes cultures fluids is not inhibited by Brefeldin A (BFA), suggesting a possible secretion through the leaderless secretory pathway.

Exploitation of the biologic activity of neurotrophins is desirable for medical purposes, but their protein nature intrinsically bears adverse pharmacokinetic properties. Here, we report synthesis and biologic characterization of a novel class of low molecular weight, non-peptidic compounds with NGF (nerve growth factor)-mimetic properties. MT2, a representative compound, bound to Trk (tropomyosin kinase receptor)A chain on NGF-sensitive cells, as well as in cell-free assays, at nanomolar concentrations and induced TrkA autophosphorylation and receptor-mediated internalization. MT2 binding involved at least two amino-acid residues within TrkA molecule. Like NGF, MT2 increased phosphorylation of extracellular signal-regulated kinase1/2 and Akt proteins and production of MKP-1 phosphatase (dual specificity phosphatase 1), modulated p38 mitogen-activated protein kinase activation, sustained survival of serum-starved PC12 or RDG cells, and promoted their differentiation. However, the intensity of such responses was heterogenous, as the ability of maintaining survival was equally possessed by NGF and MT2, whereas the induction of differentiation was expressed at definitely lower levels by the mimetic. Analysis of TrkA autophosphorylation patterns induced by MT2 revealed a strong tyrosine (Tyr)490 and a limited Tyr785 and Tyr674/675 activation, findings coherent with the observed functional divarication. Consistently, in an NGF-deprived rat hippocampal neuronal model of Alzheimer Disease, MT2 could correct the biochemical abnormalities and sustain cell survival. Thus, NGF mimetics may reveal interesting investigational tools in neurobiology, as well as promising drug candidates.

BackgroundSusceptibility to viral infections as well as their severity are higher in men than in women. Heightened antiviral responses typical of women are effective for rapid virus clearance, but if excessively high or prolonged, can result in chronic/inflammatory pathologies. We investigated whether this variability could be in part attributable to differences in the response to the Toll-Like Receptors (TLR) more involved in the virus recognition.MethodsCytokine production by peripheral blood mononuclear cells (PBMCs) from male and female healthy donors after stimulation with Toll-like receptors (TLR) 3, 7, 8, 9 ligands or with viruses (influenza and Herpes-simplex-1) was evaluated.ResultsCompared to females, PBMCs from males produced not only lower amounts of IFN-α in response to TLR7 ligands but also higher amounts of the immunosuppressive cytokine IL10 after stimulation with TLR8 and TLR9 ligands or viruses. IL10 production after TLR9 ligands or HSV-1 stimulation was significantly related with plasma levels of sex hormones in both groups, whereas no correlation was found in cytokines produced following TLR7 and TLR8 stimulation.ConclusionsGiven the role of an early production of IL10 by cells of innate immunity in modulating innate and adaptive immune response to viruses, we suggest that sex-related difference in its production following viral nucleic acid stimulation of TLRs may be involved in the sex-related variability in response to viral infections.

Previous interethnic comparative studies on the susceptibility to malaria performed in West Africa showed that Fulani are more resistant to Plasmodium falciparum malaria than are sympatric ethnic groups. This lower susceptibility is not associated to classic malaria-resistance genes, and the analysis of the immune response to P. falciparum sporozoite and blood stage antigens, as well as non-malaria antigens, revealed higher immune reactivity in Fulani. In the present study we compared the expression profile of a panel of genes involved in immune response in peripheral blood mononuclear cells (PBMC) from Fulani and sympatric Mossi from Burkina Faso. An increased expression of T helper 1 (TH1)-related genes (IL-18, IFNγ, and TBX21) and TH2-related genes (IL-4 and GATA3) and a reduced expression of genes distinctive of T regulatory activity (CTLA4 and FOXP3) were observed in Fulani. Microarray analysis on RNA from CD4 + CD25 + (T regulatory) cells, performed with a panel of cDNA probes specific for 96 genes involved in immune modulation, indicated obvious differences between the two ethnic groups with 23% of genes, including TGFβ, TGFβRs, CTLA4, and FOXP3, less expressed in Fulani compared with Mossi and European donors not exposed to malaria. As further indications of a low T regulatory cell activity, Fulani showed lower serum levels of TGFβ and higher concentrations of the proinflammatory chemokines CXCL10 and CCL22 compared with Mossi; moreover, the proliferative response of Fulani to malaria antigens was not affected by the depletion of CD25 + regulatory cells whereas that of Mossi was significantly increased. The results suggest that the higher resistance to malaria of the Fulani could derive from a functional deficit of T regulatory cells.

In vitro proliferation of leukemic cells purified from 10 cases of acute myeloblastic leukemia (AML) was analyzed in basal conditions or in the presence of exogenous recombinant (r) Interleukin (IL) 1. In parallel, blasts from 5 of these patients were studied for granulocyte-macrophage colony-stimulating factor (GM-CSF) or granulocyte-CSF (G-CSF) mRNA. IL-1 augmented the spontaneous AML cell proliferation in all cases and induced de novo expression or increased amounts of GM-CSF and/or G-CSF transcripts in 4 of the 5 cases evaluated. IL-1-induced AML cell proliferation was modulated by neutralizing anti-GM-CSF or anti-G-CSF antibodies in those cases in which CSF mRNAs were induced or increased by exogenous cytokine. In the same cases, biosynthetic labelling and immunoprecipitation studies using monospecific anti-GM-CSF antibodies showed that IL-1 also increased the levels of GM-CSF protein synthesis. Addition of neutralizing anti-IL-1 antibodies to AML cell cultures completely abolished ongoing GM-CSF synthesis, suggesting that endogenous IL-1 is needed to maintain autocrine production of CSFs. The effects of rIL-2 were investigated in a larger series of 21 patients. The cytokine reduced spontaneous AML cell proliferation in 8 cases. It caused complete disappearance of GM-CSF mRNA in 1 case, and marked reduction of G-CSF mRNA in 2 cases. Increased AML cell proliferation was observed in 2 of 21 cases. These findings suggest that expression of CSF genes and cell proliferation in AML are under the control of different cytokines acting in autocrine or paracrine fashion.

The antiapoptotic role of Bcl-2 can be regulated by its phosphorylation in serine and threonine residues located in a nonstructured loop that links BH3 and BH4 domains. p38 MAPK has been identified as one of the kinases able to mediate such phosphorylation, through direct interaction with Bcl-2 protein in the mitochondrial compartment. In this study, we identify, by using mass spectrometry techniques and specific anti-phosphopeptide antibodies, Ser(87) and Thr(56) as the Bcl-2 residues phosphorylated by p38 MAPK and show that phosphorylation of these residues is always associated with a decrease in the antiapoptotic potential of Bcl-2 protein. Furthermore, we obtained evidence that p38 MAPK-induced Bcl-2 phosphorylation plays a key role in the early events following serum deprivation in embryonic fibroblasts. Both cytochrome c release and caspase activation triggered by p38 MAPK activation and Bcl-2 phosphorylation are absent in embryonic fibroblasts from p38alpha knock-out mice (p38alpha(-/-) MEF), whereas they occur within 12 h of serum withdrawal in p38alpha(+/+) MEF; moreover, they can be prevented by p38 MAPK inhibitors and are not associated with the synthesis of the proapoptotic proteins Bax and Fas. Thus, Bcl-2 phosphorylation by activated p38 MAPK is a key event in the early induction of apoptosis under conditions of cellular stress.

We have previously shown that the life cycles of several viruses are influenced by host-cell redox states. Reports of the antioxidant activities of the plant polyphenol resveratrol (RV) prompted us to investigate its effects on influenza virus replication in vitro and in vivo. We found that RV strongly inhibited the replication of influenza virus in MDCK cells but that this activity was not directly related to glutathione-mediated antioxidant activity. Rather, it involved the blockade of the nuclear-cytoplasmic translocation of viral ribonucleoproteins and reduced expression of late viral proteins seemingly related to the inhibition of protein kinase C activity and its dependent pathways. RV also significantly improved survival and decreased pulmonary viral titers in influenza virus-infected mice. No toxic effects were observed in vitro or in vivo. That RV acts by inhibiting a cellular, rather than a viral, function suggests that it could be a particularly valuable anti-influenza drug.

The sIgG(+) lymphoblastoid B cell line CESS spontaneously produces a high amount of nerve growth factor (NGF) and expresses both high affinity (p140(Trk-A)) and low affinity (p75(NTR)) NGF receptors. Autocrine production of NGF maintains the survival of CESS cells through the continuous deactivation of p38 MAPK, an enzyme able to induce Bcl-2 phosphorylation and subsequent cytochrome c release and caspase activation. In this paper, we show that NGF induces transcriptional activation and synthesis of MAPK phosphatase 1 (MKP-1), a dual specificity phosphatase that dephosphorylates p38 MAPK, thus preventing Bcl-2 phosphorylation. Furthermore, NGF increases MKP-1 protein stability by preventing its degradation through the proteasome pathway. Following NGF stimulation, MKP-1 protein mainly localizes on mitochondria, suggesting an interaction with p38 MAPK in this compartment. Incubation of CESS cells with MKP-1-specific antisense oligonucleotides induces cell death, which was not prevented by exogenous NGF. By contrast, overexpression of native MKP-1, but not of its catalytically impaired form, inhibits apoptosis induced by NGF neutralization in CESS cells. Thus, the molecular mechanisms underlying the survival function of NGF in CESS B cell line predominantly consist in maintaining elevated levels of MKP-1 protein, which controls p38 MAPK activation.

The sIgG(+) lymphoblastoid B cell line CESS spontaneously produces a high amount of NGF and expresses both high affinity (p140(Trk-A)) and low affinity (p75(NTR)) NGF receptors. Blocking NGF signals with neutralizing antibodies or specific Trk-A inhibitors induces a rapid phosphorylation of antiapoptotic Bcl-2 protein, followed by caspase activation, and apoptotic death of CESS cells. Bcl-2 phosphorylation in several sites within a approximately 60 aa "loop" domain of protein is known to regulate its antiapoptotic function. Accordingly, CESS cells expressing the loop deletional mutant cDNA constructs Bcl-2 Delta40-91 were completely resistant to apoptosis induced by NGF withdrawal, indicating that Bcl-2 phosphorylation is a critical event. NGF withdrawal induces p38 MAPK, but not JNK, activation in CESS cells, and SB203580, a specific inhibitor of p38 MAPK, is able to prevent both Bcl-2 phosphorylation and apoptosis, indicating that p38 MAPK is the enzyme responsible for these events.

The molecular mechanisms underlying the growth inhibition induced by interferon-alpha (IFN-alpha) in B16 murine melanoma cells were investigated. IFN-alpha did not induce cell apoptosis, but strongly interfered with the synthesis of basic fibroblast growth factor (bFGF), which acts as an autocrine growth factor in this system. Inhibition of bFGF synthesis was observed at the same concentrations (50-500 pM, 10-100 U/ml) of IFN-alpha able to induce growth arrest of B16 melanoma cells. Although the synthesis of acidic (a)FGF was only slightly affected by IFN-alpha, the cytokine induced release of an aFGF-related low-molecular-weight peptide, which was able to interfere with bFGF binding to surface receptors. Thus, the molecular mechanisms of IFN-alpha activity on melanoma cells include a specific modulation of the bFGF autocrine circuit.

Production of nerve growth factor (NGF) was assessed in cultures of human T and B lymphocytes and macrophages. NGF was constitutively produced by B cells only, which also expressed surface p140trk-A and p75NGFR molecules and hence efficiently bound and internalized the cytokine. Neutralization of endogenous NGF caused disappearance of Bcl-2 protein and apoptotic death of resting lymphocytes bearing surface IgG or IgA, a population comprising memory cells, while surface IgM/IgD “virgin” B lymphocytes were not affected. In vivo administration of neutralizing anti-NGF antibodies caused strong reduction in the titer of specific IgG in mice immunized with tetanus toxoid, nitrophenol, or arsonate and reduced numbers of surface IgG or IgA B lymphocytes. Thus, NGF is an autocrine survival factor for memory B lymphocytes.

In Acute Myelogenous Leukemia (AML), Interleukin 1 (IL-1) might sustain autocrine and paracrine loops of leukemic growth. An IL-1 inhibitor has been recently purified and cloned. This molecule binds to the IL-1 receptors but has no IL-1 like activity fulfilling the characteristics of a pure Interleukin-1 receptor antagonist (IL-1ra). We studied the in vitro effect of human recombinant IL-1ra on proliferation of AML blasts. Spontaneous as well as IL-1 stimulated AML proliferation was significantly inhibited by the addition of 50 ng/ml of recombinant IL-1ra in a dose dependent manner. The inhibitory effect of IL-1ra was measurable after 12 hours of culture and reached a plateau at 60 hrs. We found that IL-1ra could compete with IL-1 in binding to specific IL-1 receptors on AML cells. As expected, culture supernatants of unstimulated leukemic samples contained IL-1 beta and GM-CSF activity. The incubation of the same leukemic blasts with IL-1 ra was followed by reduction or disappearance of GM-CSF in culture supernatants whereas the IL-1 beta production was only partially modulated. By Northern blot experiments performed on freshly isolated, uncultured leukemic blasts, we found a constitutive expression of the IL-1 beta gene in 19 of 23 AML cases analyzed. On the contrary, only three of these patients express the IL-1 RA mRNA. All together these results suggest that imbalanced secretion of IL-1 and its natural receptor antagonist could contribute to the unrestricted growth of AML cells.

To elucidate mechanisms underlying neovascularization that accompanies certain chronic immune/inflammatory disorders, the effects of interferon-alpha (IFN-alpha) and interleukin 2 (IL-2) on endothelial cell (EC) growth in vitro and angiogenesis in vivo were studied. Preincubation of cultured human ECs with IFN-alpha, followed by exposure to IL-2, resulted in effective stimulation of cell growth, whereas either cytokine alone had only a slight effect. The combination of IFN-alpha/IL-2 induced an angiogenic response in the rabbit cornea. IL-2 receptor expression was enhanced on IFN-alpha-treated ECs: p55 was increased and p70 was induced. 125I-IL-2 binding to ECs treated with IFN-alpha was enhanced (Kd from approximately 7 nM to approximately 260 pM with IFN-alpha), and anti-p55 IgG blocked 125I-IL-2/EC interaction as well as IL-2-mediated EC proliferation. Consistent with these findings in cell culture, immunohistologic studies demonstrated p55 and p70 antigen in the vasculature of rheumatoid joints, but not in normal joint tissue. Exposure of cultured ECs to IFN-alpha increased levels of intracellular EC basic fibroblast growth factor (bFGF), and subsequent addition of IL-2 led to bFGF release into the medium. The observation that anti-bFGF IgG largely blocked EC proliferation in response to IFN-alpha/IL-2 suggested that bFGF was a critical agent in this setting. These data suggest a mechanism rendering ECs responsive to IL-2 which may be relevant in immune/inflammatory disorders: IFN-alpha-mediated induction of functional EC receptors for IL-2, which drives cell proliferation by a mechanism dependent on increased synthesis and release of bFGF.

In view of the key role played by interleukin 1 (IL-1) beta in inflammation, its production is likely to be precisely regulated. Previous studies have shown that IL-1 beta biosynthesis is controlled at the transcriptional and translational levels. We have investigated whether post-translational events also play a role in regulating the production of bioactive IL-1 beta. IL-1 beta, which lacks a signal sequence, is released by activated monocytes through a novel pathway of secretion, alternative to the classical endoplasmic reticulum-Golgi route. Secretion of mature 17 kDA IL-1 beta is increased when pulse-labelled activated monocytes are chased in the presence of heat-aggregated immunoglobulins or of various drugs. Febrile temperatures inhibit secretion of mature IL-1 beta, but only reduce its synthesis: treatment with cycloheximide restores secretion. Processing of the 33 kDa precursor to the 17 kDa mature molecule is inhibited when the external pH is 8 or higher: under these conditions, release of unprocessed, biologically inactive 33 kDa IL-1 beta is observed. Thus, secretion of IL-1 beta is regulated by post-translational mechanisms which operate at the level of both proteolytic processing and extracellular export.

Interleukin 1 (IL-1) is a major soluble mediator of inflammation. Two human IL-1 genes, alpha and beta, have been isolated, which encode polypeptides with only 20-30% amino acid sequence homology. Unlike most secreted proteins, the two cytokines do not have a signal sequence, an unexpected finding in view of their biological role. Here we show that IL-1 beta is actively secreted by activated human monocytes via a pathway of secretion different from the classical endoplasmic reticulum--Golgi route. Drugs which block the intracellular transport of IL-6, of tumour necrosis factor alpha and of other secretory proteins do not inhibit secretion of IL-1 beta. Secretion of IL-1 beta is blocked by methylamine, low temperature or serum free medium, and is increased by raising the culture temperature to 42 degrees C or by the presence of calcium ionophores, brefeldin A, monensin, dinitrophenol or carbonyl cyanide chlorophenylhydrazone. IL-1 beta is contained in part within intracellular vesicles which protect it from protease digestion. In U937 cells large amounts of IL-1 beta are made but none is secreted. In these cells IL-1 beta is not found in the vesicular fraction, and all the protein is accessible to protease digestion. This suggests that intracellular vesicles that contain IL-1 beta are part of the protein secretory pathway. We conclude that IL-1 beta is released by activated monocytes via a novel mechanism of secretion which may involve translocation of intracellular membranes and is increased by stress conditions.

Abnormalities of coagulation are common in patients with acute nonlymphoblastic leukemia, although the mechanisms involved are unclear, except in a few cases. To investigate the pathogenesis of this coagulopathy, suspensions of purified leukemic cells were prepared and tested for procoagulant activity. Neither the leukemic cells nor their supernatants directly accelerated the clotting of plasma. Since the leukemic cells did not possess direct procoagulant activity, their ability or inability to elaborate a mediator of cellular coagulant properties, interleukin-1, was studied. Leukemic cells from patients with coagulopathy elaborated interleukin-1, and addition of phytohemagglutinin increased interleukin-1 release. In contrast, no interleukin-1 was released, before or after stimulation with phytohemagglutinin, from leukemic cells from patients without coagulopathy. Leukemic cells from another group of patients with abnormalities of coagulation released interleukin-1 only after phytohemagglutinin treatment. In terms of the coagulation mechanism, interleukin-1 containing supematants from leukemic cell cultures induced the procoagulant receptor tissue factor, a co-factor in the initiation of coagulation, on the endothelial cell surface. There was coordinate suppression of the anticoagulant endothelial cell receptor thrombomodulin, a co-factor for the antithrombotic protein C pathway. Antibody to interleukin-1 prevented these changes in cellular coagulant properties. Taken together, these changes result in a shift in the balance of endothelial cell coagulant properties to an activated state in which mechanisms promoting procoagulant reactions on the vessel surface predominate. Synthesis and release of the mediator interleukin-1 by leukemic cells thus defines a new mechanism through which malignant cells can potentially activate the coagulation mechanism.

Interleukin-6 (IL-6) is a cytokine which is not only produced by a wide variety of different cells but one which also affects the function of diverse tissues. We have studied the expression of the IL-6 gene in freshly explanted human umbilical vein endothelial cells (HUVEC) and have also evaluated the effect of IL-6 on HUVEC proliferation. Cytokines like interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor (TNF) as well as bacterial products such as the lipopolysaccharide (LPS) rapidly enhance production of biologically active IL-6 by HUVEC (IL-6 bioassay: increase in alpha 1-antichymotrypsin secretion by Hep3B2 cells and its neutralization by antiserum to E. coli-derived human IL-6). The two inducible RNA start sites in the IL-6 gene that are used in cytokine-induced fibroblasts (at +1 and -21) are also used in the same relative proportion (+1 greater than -21) in cytokine or LPS-induced HUVEC as determined by S1-nuclease protection assays for IL-6 transcripts. Immunoaffinity chromatography followed by Western blotting shows that IL-6 species secreted by IL-1 alpha-induced HUVEC are of molecular mass 23-25, 27-30 and 45 kDa as judged by SDS-PAGE under reducing conditions. Finally, rIL-6 inhibits [3H]-thymidine incorporation by HUVEC in a dose-dependent manner. Thus IL-6 is not only produced by HUVEC but may also affect its proliferation. The ability of the vascular endothelium to rapidly secrete IL-6 in response to inflammation-associated cytokines is of strategic value since it generates a circulatory signal which helps mobilize the acute phase plasma protein response and enlists the immune system in host defence.

The lymphocyte surface phenotype of lymph nodes from patients with larynx or urinary bladder carcinoma was investigated by using a panel of monoclonal antibodies. The phenotype pattern of lymphocytes from lymph nodes invaded by malignant cells (as assessed by histopathology) was different from that of the cells from noninvaded or normal control nodes. Although the proportion of natural killer cells or macrophages was similar in the 3 groups of lymph nodes, invaded lymph nodes contained a higher proportion of T-cells and a lower B-cell percentage. Furthermore, cells from invaded nodes comprised 15-20% of T3+ T8+ cells that coexpressed the M1 marker and, to some extent, also the Leu 7 marker. A large proportion of cells with multiple markers were activated, as shown by the expression of Tac and HLA-DR antigens. In 2 patients activated T8+ cells expressing also M1 and Leu 7 markers infiltrated the tumor site. The presence of these activated cells both in involved nodes and tumor mass may indicate that they originate in response to cancer.

The initial tumors of 21 patients who earlier had presented with superficial noninvasive transitional cell carcinoma of the bladder and who subsequently underwent cystectomy for invasive disease were examined by the specific red cell adherence test. Eighteen of 21 initial tumors (85.7%) were antigen negative. The time from initial tumor resection ranged from twelve to 168 months (mean thirty-eight months). Loss of blood group antigens indicated enhanced biologic aggressiveness of tumors. However the interval to invasion is sufficiently variable as to preclude the use of this assay for timing of radical cystectomy.

Serum B2m concentrations were evaluated preoperatively in 40 patients with renal cell carcinoma and normal renal function, as assessed by serum creatinine less than 1.4 mg/dl, and compared with those of 23 age-matched controls. Mean value +/- SD was 3,088 +/- 966 ng/ml for renal cancer patients, while controls had a value of 1,800 +/- 240 ng/ml. Statistical analysis, performed by Student t test, revealed a very high degree of significance (p less than 0.0005). No statistically significant differences were found between groups of patients classified according to tumor stage and cell type. Seventy percent of renal cell carcinoma cases had preoperatively elevated serum levels of B2m.

Growing evidence indicates that viral replication is regulated by the redox state of the host cell. We demonstrate that cells of different origins display differential permissivity for influenza A virus replication, depending on their intracellular redox power as reflected by Bcl-2 expression and glutathione (GSH) content. Bcl-2 expressing cells were found to have higher intracellular levels of GSH and to produce lower amounts of virus than Bcl-2 negative cells. Two different steps in the virus life-cycle were involved in Bcl-2/GSH mediated viral inhibition: 1) expression of late viral proteins (in particular hemagglutinin and matrix); and 2) nuclear-cytoplasmic translocation of viral ribonucleoproteins (vRNPs). Buthionine-sulfoximine-induced inhibition of GSH synthesis in Bcl-2 expressing cells caused an increase in the expression of late viral proteins but did not restore vRNP export to the cytoplasm. Collectively, our findings show that both Bcl-2 expression and GSH content contribute to the host cell's ability to down-regulate influenza virus replication, although their effects are exerted at different stages of the viral life-cycle. In certain cell populations, this form of down-regulation might conceivably favor the establishment of persistent viral infection.

Proliferation of endothelial cells is regulated through the autocrine production of growth factors and the expression of cognate surface receptors. In this study, we demonstrate that interleukin 1 (IL-1) is an inhibitor of endothelial growth in vitro and in vivo. IL-1 arrested growing, cultured endothelial cells in G1 phase; inhibition of proliferation was dose dependent and occurred in parallel with occupancy of endothelial surface IL-1 receptors. In an angiogenesis model, IL-1 could inhibit fibroblast growth factor-induced vessel formation. The autocrine nature of the IL-1 effect on endothelial proliferation was demonstrated by the observation that occupancy of cell-surface receptors by endogenous IL-1 depressed cell growth. The potential significance of this finding was emphasized by the detection of IL-1 in the native endothelium of human umbilical veins. A mechanism by which IL-1 may exert its inhibitory effect on endothelial cell growth was suggested by studies showing that IL-1 decreased the expression of high-affinity fibroblast growth factor binding sites on endothelium. These results point to a potentially important role of IL-1 in regulating blood vessel growth and suggest that autocrine production of inhibitory factors may be a mechanism controlling proliferation of normal cells.

We have studied the effects of several interleukin-1 (IL-1) inhibitors--IL-1 receptor antagonist (IL-1ra), soluble IL-1 receptor (sIL-1R) types I and II, and neutralizing monoclonal antibody (mAb) specific for IL-1 receptor type I--on the osteoclast-activating factor (OAF) activity of recombinant IL-1beta and of culture supernatants of unfractionated bone marrow mononuclear cells from multiple myeloma (MM) patients. The latter activity sharply correlated with the IL-1 content of culture supernatants (r = 0.949; p0.001). IL-1ra and sIL-1R types I and II had a clear-cut modulating effect on the OAF activity of IL-1beta at saturating doses (2-10 ng/mL); their effect was evident at 2 ng/mL and was dose-dependent over a large range of concentrations. Similarly, the three reagents neutralized the OAF activities of all MM cell supernatants in a dose-dependent fashion and completely abolished them when tested at the fixed concentration of 5 nM. The bone-resorbing activity of tumor necrosis factor-alpha (TNF-alpha) or lymphotoxin (LT), tested alone or added to MM cell supernatants, was affected not at all by IL-1ra and only minimally by sIL-1R types I and II, suggesting that little or no endogenous IL-1 was produced by the rat cells in the assay under TNF-alpha or LT stimulation. Consistent with these findings, PGE2 production elicited by IL-1beta or IL-1-rich supernatants in the rat long-bone assay was abolished by each reagent. Also, mAbs to the IL-1R p80 (type I) chains could modulate the effects of IL-1--recombinant or plasma cell-derived--in the OAF assay, but their activity was markedly less pronounced when compared with the IL-1 inhibitors, since they could never completely abolish bone resorption. Taken together, these findings demonstrate that inhibition of IL-1 interaction with cognate surface receptors on bone cells effectively counteracts its biologic activity. The findings also strongly indicate that OAF activity in conditioned medium of unfractionated myeloma bone marrow cells is predominantly, if not solely, related to IL-1beta.