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Increased expression of the urokinase-type plasminogen activator (uPA) system is associated with tumor invasion, neo-angiogenesis, and metastatic spread, and has been shown to positively correlate with a poor prognosis in several cancer types, including thyroid carcinomas. In recent years, several uPA inhibitors were found to have anticancer effects in preclinical studies and in some phase II clinical trials, which prompted us to evaluate uPA as a potential therapeutic target for the treatment of patients affected by the most aggressive form of thyroid cancer, the anaplastic thyroid carcinoma (ATC). In this study, we evaluated the in vitro and in vivo effects of WX-340, a highly specific and selective uPA inhibitor, on two ATC-derived cell lines, CAL-62 and BHT-101. The results obtained indicated that WX-340 was able to reduce cell adhesion and invasiveness in a dose-dependent manner in both cell lines. In addition, WX-340 increased uPA receptor (uPAR) protein levels without affecting its plasma membrane concentration. However, this compound was unable to significantly reduce ATC growth in a xenograft model, indicating that uPA inhibition alone may not have the expected therapeutic effects.

Parasitic diseases cause significant global morbidity and mortality particularly in the poorest regions of the world. Schistosomiasis, one of the most widespread neglected tropical diseases, affects more than 200 million people worldwide. Histone deacetylase (HDAC) inhibitors are prominent epigenetic drugs that are being investigated in the treatment of several diseases, including cancers and parasitic diseases. Schistosoma mansoni HDAC8 (SmHDAC8) is highly expressed in all life cycle stages of the parasite, and selective inhibition is required in order to avoid undesirable off-target effects in the host. Herein, by X-ray crystal structures of SmHDAC8-inhibitor complexes, biochemical and phenotypic studies, we found two schistosomicidal spiroindoline derivatives binding a novel site, next to Trp198, on the enzyme surface. We determined that by acting on this site, either by mutation of the Trp198 or by compound binding, a decrease in the activity of the enzyme is achieved. Remarkably, this allosteric site differs from the human counterpart; rather, it is conserved in all Schistosoma species, as well as Rhabidoptera and Trematoda classes, thus paving the way for the design of HDAC8-selective allosteric inhibitors with improved properties.

Schistosomiasis is the most significant neglected tropical parasitic disease caused by helminths in terms of morbidity and mortality caused by helminths. In this work, we present the antischistosomal activity against Schistosoma mansoni of a rationally selected small set of thiazinoquinone derivatives, some of which were previously found to be active against Plasmodium falciparum and others synthesized ad hoc. The effects on larvae, juvenile, and adult parasite viability as well as on egg production and development were investigated, resulting in the identification of new multistage antischistosomal hit compounds. The most promising compounds 6, 8, 13, and 14 with a LC50 value on schistosomula from ∼5 to ∼15 μM also induced complete death of juvenile (28 days old) and adult worm pairs (7 weeks old) and a detrimental effect on egg production and development in vitro. Structure-activity relationships (SARs) were analyzed by means of computational studies leading to the hypothesis of a redox-based mechanism of action with a one-electron reduction bioactivation step and the subsequent formation of a toxic semiquinone species, similarly to what was previously observed for the antiplasmodial activity. Our results also evidenced that the selective toxicity against mammalian cells or parasites as well as specific developmental stages of a parasite can be addressed by varying the nature of the introduced substituents.

[Image: see text] Histone deacetylase inhibitors (HDACi) have emerged as promising therapeutics for the treatment of neurodegeneration, cancer, and rare disorders. Herein, we report the development of a series of spiroindoline-based HDAC6 isoform-selective inhibitors based on the X-ray crystal studies of the hit 6a. We identified compound 6j as the most potent and selective hHDAC6 inhibitor of the series. Biological investigation of compounds 6b, 6h, and 6j demonstrated their antiproliferative activity against several cancer cell lines. Western blotting studies indicated that they were able to increase tubulin acetylation, without significant variation in histone acetylation state, and induced PARP cleavage indicating their apoptotic potential at the molecular level. 6j induced HDAC6-dependent pSTAT3 inhibition.

Schistosomiasis is one of the major parasitic diseases with more than 200 million people infected worldwide every year. Praziquantel is the drug of choice against the schistosomiasis although the use of a single drug to treat such a large amount of infected people appears particularly worrisome. For this reason, the search of new schistosomicidal compounds is viewed as an urgent goal and a number of screening campaigns have been carried out in the past years. The larval stage of Schistosoma (schistosomula) has been widely used in order to identify new compounds against the parasite. Here we describe detailed practical procedures for a luminescence-based assay proven to be highly effective for the selection of schistosomicidal compounds on small and medium-high scale. The assay is based on the quantitation of the parasite ATP, a good indicator of metabolically active cells, as measure of schistosomula viability. This assay is fast and reproducible, and it is suitable either for manual or for semiautomated screenings.

Schistosomiasis is a neglected tropical disease mainly affecting the poorest tropical and subtropical areas of the world with the impressive number of roughly 200 million infections per year. Schistosomes are blood trematode flukes of the genus Schistosoma causing symptoms in humans and animals. Organ morbidity is caused by the accumulation of parasite eggs and subsequent development of fibrosis. If left untreated, schistosomiasis can result in substantial morbidity and even mortality. Praziquantel (PZQ) is the most effective and widely used compound for the treatment of the disease, in prevention and control programs in the last 30 years. Unfortunately, it has no effect on juvenile immature schistosomes and cannot prevent reinfection or interfere with the schistosome life cycle; moreover drug-resistance represents a serious threat. The search for an alternative or complementary treatment is urgent and drug repurposing could accelerate a solution. The anti-anginal drug perhexiline maleate (PHX) has been previously shown to be effective on larval, juvenile, and adult stages of S. mansoni and to impact egg production in vitro. Since PHX is a racemic mixture of R-(+)- and S-(−)-enantiomers, we designed and realized a stereoselective synthesis of both PHX enantiomers and developed an analytical procedure for the direct quantification of the enantiomeric excess also suitable for semipreparative separation of PHX enantiomers. We next investigated the impact of each enantiomer on viability of newly transformed schistosomula (NTS) and worm pairs of S. mansoni as well as on egg production and vitellarium morphology by in vitro studies. Our results indicate that the R-(+)-PHX is mainly driving the anti-schistosomal activity but that also the S-(−)-PHX possesses a significant activity towards S. mansoni in vitro.

Schistosomiasis is one of the major parasitic diseases with more than 200 million people infected worldwide every year. Praziquantel is the drug of choice against the schistosomiasis although the use of a single drug to treat such a large amount of infected people appears particularly worrisome. For this reason, the search of new schistosomicidal compounds is viewed as an urgent goal and a number of screening campaigns have been carried out in the past years. The larval stage of Schistosoma (schistosomula) has been widely used in order to identify new compounds against the parasite. Here we describe detailed practical procedures for a luminescence-based assay proven to be highly effective for the selection of schistosomicidal compounds on small and medium-high scale. The assay is based on the quantitation of the parasite ATP, a good indicator of metabolically active cells, as measure of schistosomula viability. This assay is fast and reproducible, and it is suitable either for manual or for semiautomated screenings.

Neglected tropical diseases (NTDs), caused by several parasitic agents (Plasmodium spp., Schistosoma spp., Leishmania spp.), are still characterized by a high morbidity percentage worldwide. The therapeutic alternatives for neglected pathologies are very limited, first due to the drug-resistance phenomena but also due to unfavourable toxicity profiles and the difficult administration procedures of the few available chemical entities. A further drawback is represented by multiparasitism, namely the concurrent infestation of a single host by more than one parasite species, which poses additional diagnostic and therapeutic challenges [1]. In this view, a research aimed to discover a new chemical entity active against several parasites is crucial and the marine environment may be important resource [2]. As part of our own continuing search for new leads in the treatment of NTDs [3–5] we have explored the anti infective properties of the sponge derived metabolites avarone and avarol, as well as those of the semisynthetic thiazoavarone, obtained by converting the quinone avarone in the relevant thiazinoquinone. All compounds were shown to be active against the D10 and W2 strains of P. falciparum, the juvenile and adult forms of the worm S. mansoni, and both the promastigote and amastigote stages of L. infantum and L. tropica.

Schistosomiasis is the most significant neglected tropical parasitic disease caused by helminths in terms of morbidity and mortality caused by helminths. In this work, we present the antischistosomal activity against

Schistosomiasis (also known as bilharzia) is a neglected tropical disease caused by platyhelminths of the genus

In this work we describe the synthesis of potent and selective quinolone-based histone deacetylase 6 (HDAC6) inhibitors. The quinolone moiety has been exploited as an innovative bioactive cap-group for HDAC6 inhibition; its synthesis was achieved by applying a multicomponent reaction. The optimization of potency and selectivity of these products was performed by employing computational studies which led to the discovery of the diethylaminomethyl derivatives 7g and 7k as the most promising hit molecules. These compounds were investigated in cellular studies to evaluate their anticancer effect against colon (HCT-116) and histiocytic lymphoma (U9347) cancer cells, showing good to excellent potency, leading to tumor cell death by apoptosis induction. The small molecules 7a, 7g and 7k were able to strongly inhibit the cytoplasmic and slightly the nuclear HDAC enzymes, increasing the acetylation of tubulin and of the lysine 9 and 14 of histone 3, respectively. Compound 7g was also able to increase Hsp90 acetylation levels in HCT-116 cells, thus further supporting its HDAC6 inhibitory profile. Cytotoxicity and mutagenicity assays of these molecules showed a safe profile; moreover, the HPLC analysis of compound 7k revealed good solubility and stability profile.

Background Novel anti-schistosomal multi-stage drugs are needed because only a single drug, praziquantel, is available for the treatment of schistosomiasis and is poorly effective on larval and juvenile stages of the parasite. Schistosomes have a complex life-cycle and multiple developmental stages in the intermediate and definitive hosts. Acetylation and deacetylation of histones play pivotal roles in chromatin structure and in the regulation of transcription in eukaryotic cells. Histone deacetylase (HDAC) inhibitors modulate acetylation of several other proteins localized both in the nucleus and in the cytoplasm and therefore impact on many signaling networks and biological processes. Histone post-translational modifications may provide parasites with the ability to readily adapt to changes in gene expression required for their development and adaptation to the host environment. The aim of the present study was to screen a HDAC class I inhibitor library in order to identify and characterize novel multi-stage hit compounds. Methods We used a high-throughput assay based on the quantitation of ATP in the Schistosoma mansoni larval stage (schistosomula) and screened a library of 1500 class I HDAC inhibitors. Subsequently, a few hits were selected and further characterized by viability assays and phenotypic analyses on adult parasites by carmine red and confocal microscopy. Results Three compounds (SmI-124, SmI-148 and SmI-558) that had an effect on the viability of both the schistosomula larval stage and the adult worm were identified. Treatment with sub-lethal doses of SmI-148 and SmI-558 also decreased egg production. Moreover, treatment of adult parasites with SmI-148, and to a lesser extent Sm-124, was associated with histone hyperacetylation. Finally, SmI-148 and SmI-558 treatments of worm pairs caused a phenotype characterized by defects in the parasite reproductive system, with peculiar features in the ovary. In addition, SmI-558 induced oocyte- and vitelline cell-engulfment and signs of degeneration in the uterus and/or oviduct. Conclusions We report the screening of a small HDAC inhibitor library and the identification of three novel compounds which impair viability of the S. mansoni larval stage and adult pairs. These compounds are useful tools for studying deacetylase activity during parasite development and for interfering with egg production. Characterization of their specificity for selected S. mansoni versus human HDAC could provide insights that can be used in optimization and compound design.

Medulloblastoma (MB), the tumor of the cerebellum, is the most frequent brain cancer in childhood and a major cause of pediatric mortality. Based on gene profiling, four MB subgroups have been identified, i.e., Wnt or Sonic Hedgehog (Shh) types, and subgroup 3 or 4. The Shh-type MB has been shown to arise from the cerebellar precursors of granule neurons (GCPs), where a hyperactivation of the Shh pathway leads to their neoplastic transformation. We have previously shown that the gene Tis21 (PC3/Btg2) inhibits the proliferation and promotes the differentiation and migration of GCPs. Moreover, the overexpression or the deletion of Tis21 in Patched1 heterozygous mice, a model of spontaneous Shh-type MB, highly reduces or increases, respectively, the frequency of MB. Here we tested whether Tis21 can inhibit MB allografts. Athymic nude mice were subcutaneously grafted with MB cells explanted from Patched1 heterozygous mice. MB allografts were then injected with adeno-associated viruses either carrying Tis21 (AAV-Tis21) or empty (AAV-CBA). We observed that the treatment with AAV-Tis21 significantly inhibited the growth of tumor nodules, as judged by their volume, and reduced the number of proliferating tumor cells (labeled with Ki67 or BrdU), relative to AAV-CBA-treated control mice. In parallel, AAV-Tis21 increased significantly tumor cells labeled with early and late neural differentiation markers. Overall the results suggest that Tis21-gene therapy slows down MB tumor growth through inhibition of proliferation and enhancement of neural differentiation. These results validate Tis21 as a relevant target for MB therapy.

Inflammatory caspases, including human caspase-4 (CASP4), play key roles in innate immune responses to promote fusion of phagosomes harboring pathogenic bacteria with lysosomes, halt intracellular replication of pathogens, maturation and secretion of pro-inflammatory cytokines. The role of inflammatory caspases in cancer cells remains poorly investigated. Here, we explored the consequences of modulating CASP4 expression levels on the migratory behavior of epithelial cancer cell lines. By a gene silencing approach and in vitro and in vivo studies we show that down-regulation of CASP4 leads to impaired cell migration and cell-matrix adhesion. This phenotype is accompanied by an increased actin cytoskeleton polymerization, changes in the overall organization of adherens junctions (AJs) and number and size of focal adhesions. Interestingly, the cell migration deficit could be reversed by epithelial growth factor treatment, and depletion of calcium ions unveiled a role of CASP4 in the novo assembly of AJs, suggesting that the role of CASP4 is not cell-autonomous. Finally, CASP4-silenced A431 cells exhibited a severe reduction in their ability to invade lung tissue, when injected into nude mice. Overall, our data support the emerging evidence that inflammatory caspases can regulate cell migration through actin remodeling and uncover a novel role of CASP4 in cancer cell behavior.

Schistosomiasis, one of the most prevalent neglected parasitic diseases affecting humans and animals, is caused by the Platyhelminthes of the genus Schistosoma. Schistosomes are the only trematodes to have evolved sexual dimorphism and the constant pairing with a male is essential for the sexual maturation of the female. Pairing is required for the full development of the two major female organs, ovary and vitellarium that are involved in the production of different cell types such as oocytes and vitellocytes, which represent the core elements of the whole egg machinery. Sexually mature females can produce a large number of eggs each day. Due to the importance of egg production for both life cycle and pathogenesis, there is significant interest in the search for new strategies and compounds not only affecting parasite viability but also egg production. Here we use a recently developed high-throughput organism-based approach, based on ATP quantitation in the schistosomula larval stage of Schistosoma mansoni for the screening of a large compound library, and describe a pharmacophore-based drug selection approach and phenotypic analyses to identify novel multi-stage schistosomicidal compounds. Interestingly, worm pairs treated with seven of the eight compounds identified show a phenotype characterized by defects in eggshell assemblage within the ootype and egg formation with degenerated oocytes and vitelline cells engulfment in the uterus and/or oviduct. We describe promising new molecules that not only impair the schistosomula larval stage but also impact juvenile and adult worm viability and egg formation and production in vitro., Author summary Schistosomiasis is a neglected disease caused by parasitic flatworms called schistosomes. The disease affects hundreds of millions of people in developing countries in the poorest tropical and subtropical regions of the world and it represents a major public health and socio-economical problem in several countries. In humans, these blood flukes reside in the mesenteric and vesicle venules. They have a life span of many years and produce hundreds of eggs daily, which are able to pass through the gut lumen or the bladder to be finally excreted into the environment for maintaining the life cycle. Part of the eggs can be trapped in host tissues inducing immunologically mediated granulomatous inflammation and fibrosis leading eventually to severe sequelae such as hepatosplenomegaly and even death. Importantly, schistosome infections increase susceptibility to other parasitic, bacterial and viral diseases. To date, essentially a single drug, praziquantel, is available to treat this parasitic disease. Despite its high tolerability and efficacy against adult parasites it has an incomplete efficacy across all stages of the S. mansoni life cycle and it does not prevent reinfection. Moreover the potential risk of drug resistance is an increasing concern. In search of novel schistosomicidal molecules we screened a large compound collection using the schistosomula, larval stage of the parasite. We identified eight novel molecules able to impair viability of schistosomula, juvenile and adult worms and also egg formation and production, two important features required for both disease transmission and progression.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, and several molecular pathways that underlie the molecular tumorigenesis of HNSCC have been identified. Among them, amplification or overexpression of Delta Np63 isoforms is observed in the majority of HNSCCs. Here, we unveiled a Delta Np63-dependent transcriptional program able to regulate the metabolism and the signaling of hyaluronic acid (HA), the major component of the extracellular matrix (ECM). We found that Delta Np63 is capable of sustaining the production of HA levels in cell culture and in vivo by regulating the expression of the HA synthase HAS3 and two hyaluronidase genes, HYAL-1 and HYAL-3. In addition, Delta Np63 directly regulates the expression of CD44, the major HA cell membrane receptor. By controlling this transcriptional program, Delta Np63 sustains the epithelial growth factor receptor (EGF-R) activation and the expression of ABCC1 multidrug transporter gene, thus contributing to tumor cell proliferation and chemoresistance. Importantly, p63 expression is positively correlated with CD44, HAS3, and ABCC1 expression in squamous cell carcinoma datasets and p63-HA pathway is a negative prognostic factor of HNSCC patient survival. Altogether, our data shed light on a Delta Np63-dependent pathway functionally important to the regulation of HNSCC progression.

The Innovative Medicines Initiative is a unique and novel long term european public-private partnership designed by the European Commission and the European Federation of Pharmaceutical Industries and Associations (EFPIA). The initiative was promoted to accelerate the discovery and development of better medicines by removing bottlenecks in the drug development process. It focuses on creating better methods and tools that improve and enhance the drug development process, rather than on developing specific, new medicines.

— Peripheral blood lymphocyte subsets involved in cytotoxicity to autologous hepatocytes have been characterized by isolation on antibody-coated Petri dishes in autoimmune and HBsAg-positive chronic active hepatitis (CAH). In autoimmune CAH and in HBsAg-positive CAH without HBcAg in liver tissue, cytotoxicity is sustained by non-T lymphocytes and is confined to M1-positive cells bearing Fc receptors: M1 cytotoxicity inhibition by adding aggregated IgG suggests that these cells are responsible for an antibody-dependent cell-mediated mechanism (ADCC). Moreover, when T-enriched fractions were separated in T4, T8 and 5/9 positive subsets, only the first one showed a significant cytotoxicity: T4 positive cells might act as cytotoxic T cells or might be involved in delayed type hypersensitivity (DTH) reactions. Cytotoxic T lymphocytes in HBsAg-positive CAH with HBcAg in liver tissue are confined in T8 positive subset, while helper/inducer T cells (T4 positive or 5/9 positive) seem to play an important role only in the induction of cell-mediated injury against hepatocytes. The inhibition of T cell-cytotoxicity by preincubating liver cells with monoclonal antibody (Mab) anti-HLA AB and not with Mab anti-HLA DR or aggregated IgG supports the involvement of the class I major histocompatibility complex (MHC) expressed on the hepatocyte surface.

T cell activation results from the integration of signals generated through the T cell antigen receptor-CD3 complex with those from additional positive and negative regulatory pathways mainly mediated by the engagement of costimulatory receptors on T cells. Disruption of this balance leads to a defective immune response or alternative over-activation of the immune system. CTLA-4 plays a critical role in downregulating T cell responses. Autoimmune diseases have shown genetic linkage to the CTLA4 locus. In this report we demonstrate that the 3' UTR of CTLA4 regulates firefly luciferase reporter gene expression, can confer instability to CTLA4 mRNA and can influence its translation efficiency in vitro.

Natural polyreactive IgM autoantibodies, encoded by unmutated germline Ig V genes, represent a major fraction of the normal circulating IgM repertoire. We have previously shown that therapeutic preparation of pooled IgM exerts immunomodulatory effects as assessed by in vitro and in vivo studies. Here, we show that the IgM preparation induces cell death in lymphoblastoid cell lines and in human peripheral blood mononuclear cells. The IgM-induced cell death involved classical features of apoptosis such as nuclear fragmentation and activation of caspases. Treatment of leukemic cells with IgM resulted in the cleavage of poly-(A)DP ribose polymerase, a substrate of caspase, and in a reduction in mitochondrial transmembrane potential during the early period of apoptosis induction. Natural IgM-induced apoptosis was inhibited by soluble Fas molecules and affinity-purified Fas antibodies from pooled IgM preparation induced apoptosis in lymphoblastoid cells, suggesting the involvement of the Fas receptor. Our results suggest a role for normal IgM in controlling cell death and proliferation, and imply a possible therapeutic role for IgM in autoimmune and lymphoproliferative disorders.

Background Schistosomiasis, one of the world’s greatest neglected tropical diseases, is responsible for over 280,000 human deaths per annum. Praziquantel, developed in the 1970s, has high efficacy, excellent tolerability, few and transient side effects, simple administration procedures and competitive cost and it is currently the only recommended drug for treatment of human schistosomiasis. The use of a single drug to treat a population of over 200 million infected people appears particularly alarming when considering the threat of drug resistance. Quantitative, objective and validated methods for the screening of compound collections are needed for the discovery of novel anti-schistosomal drugs. Methodology/Principal Findings The present work describes the development and validation of a luminescence-based, medium-throughput assay for the detection of schistosomula viability through quantitation of ATP, a good indicator of metabolically active cells in culture. This validated method is demonstrated to be fast, highly reliable, sensitive and automation-friendly. The optimized assay was used for the screening of a small compound library on S. mansoni schistosomula, showing that the proposed method is suitable for a medium-throughput semi-automated screening. Interestingly, the pilot screening identified hits previously reported to have some anti-parasitic activity, further supporting the validity of this assay for anthelminthic drug discovery. Conclusions The developed and validated schistosomula viability luminescence-based assay was shown to be successful and suitable for the identification of novel compounds potentially exploitable in future schistosomiasis therapies., Author Summary Schistosomiasis, one of the world’s greatest human neglected tropical diseases, is caused by a parasitic flatworm trematode of the genus Schistosoma. Among human parasitic diseases, schistosomiasis ranks second behind malaria in terms of socio-economic and public health importance in tropical and subtropical areas. More than 200 million people are currently infected in 77 countries, 85% of whom live in sub-Saharian Africa. To date no vaccine is available against schistosomiasis. As chemotherapy relies on a single drug, praziquantel, many initiatives have been promoted aiming to search for novel anti-schistosomal drugs that can represent a valid alternative to the current treatment or could be used in case of emerging resistance. Quantitative, objective and validated methods for compound collections screening are needed for the discovery of novel anti-schistosomal drugs. Here, we report the development and validation of a medium-throughput, luminescence-based assay for assessing viability at the schistosomulum stage of the human parasite S. mansoni. Our methodology enables a simple, reproducible, highly sensitive and objective quantitation of parasite viability. It is also automation compatible and enables the screening of compound collections thus hopefully contributing to the discovery of novel therapeutic strategies against schistosomiasis.

Therapeutic preparations of normal human IgG for i.v. use (IVIg) exhibit a broad spectrum of immunoregulatory activities in vitro and in vivo. IVIg has been shown to inhibit the proliferation of activated B and T lymphocytes and of several autonomously growing cell lines. In this study, we demonstrate that IVIg induces apoptosis in leukemic cells of lymphocyte and monocyte lineage and in CD40-activated normal tonsillar B cells, involving, at least in part, Fas (CD95/APO-1) and activation of caspases. IVIg-induced apoptosis was higher in Fas-sensitive HuT78 cells than in Fas-resistant HuT78.B1 mutant cells, and soluble Fas inhibited IVIg-induced apoptosis. IVIg immunoprecipitated Fas from Fas-expressing transfectants and recognized purified Fas/glutathione-S-transferase fusion proteins upon immunoblotting. Affinity-purified anti-Fas Abs from IVIg induced apoptosis of CEM T cells at a 120-fold lower concentration than unfractionated IVIg. Inhibitors of cysteine proteases of the caspase family, caspase 1 (IL-1β-converting enzyme) and caspase 3 (Yama/CPP32b), partially inhibited IVIg-induced apoptosis of CEM cells. Furthermore, cleavage of poly(A)DP-ribose polymerase into an 85-kDa signature death fragment was observed in CEM cells following IVIg treatment. Thus, normal IgG induces apoptosis in lymphocytes and monocytes. Our results provide evidence for a role of Fas, bring new insights into the mechanisms of action of IVIg in autoimmune diseases, and suggest a role of normal Ig in controlling cell death and proliferation.

The non-obese diabetic (NOD) mouse is widely used to study the pathogenesis of insulin-dependent diabetes mellitus. However, the mechanisms responsible for beta-cell destruction, in this model, are still poorly defined. The CD95/CD95L system among other effector systems has been implicated in beta-cell death. In this study we investigated in NOD, non-obese resistant (NOR) and Balb/c mice the expression of CD95 and CD95L in alpha and beta pancreatic cells by immunohistochemistry and immunofluorescence. We demonstrate that alpha cells in the islets of Langherans constitutively express CD95L forming a natural shield around beta cells.

The poor prognosis associated with patients afflicted with the acquired immunodeficiency syndrome and primary central nervous system lymphoma (AIDS-PCNSL) is due in part to the intrinsic resistance of this Epstein-Barr virus (EBV)-associated tumor to conventional antineoplastic therapy. Fas (CD95) is a transmembrane protein receptor that transmits an intracellular signal leading to rapid programmed cell death following ligation with its natural ligand or anti-Fas antibodies. Fas expression and function were assessed in AIDS-PCNSL biopsy samples and in EBV+ human B-cell tumors that spontaneously developed in severe combined immune deficient (SCID) mice engrafted with human lymphocytes (hu-PBL-SCID mice). All tumors samples showed high-density surface expression of Fas by flow cytometry or immunohistochemical staining. Cells from two AIDS-PCNSL biopsy samples that did not express pan B-cell markers did not express Fas antigen. All tumors examined were susceptible to Fas-mediated apoptosis, as measured by standard assays for endonucleolytic cleavage of DNA. The response to Fas-mediated apoptosis was dependent on log-fold increases in the concentration of immobilized anti-Fas antibody, but could also be induced with a mobilized anti-Fas antibody. No evidence for intrinsic resistance to Fas-mediated apoptosis (ie, secreted or truncated forms of Fas) could be shown. Radiation-induced apoptosis of neoplastic EBV+ B cells was enhanced by activation of Fas, and prolonged exposure to interleukin-2 increased both Fas expression and Fas-induced apoptosis. As the normal brain parenchyma appears to have either low-density or absent expression of Fas, and antineoplastic therapy can be selectively delivered to the CNS with little systemic toxicity, local delivery of Fas-activating molecules could prove to be a useful component in the multimodal treatment of AIDS-PCNSL.

The early signals generated following cross-linking of Fas/APO-1, a transmembrane receptor whose engagement by ligand results in apoptosis induction, were investigated in human HuT78 lymphoma cells. Fas/APO-1 cross-linking by mAbs resulted in membrane sphingomyelin hydrolysis and ceramide generation by the action of both neutral and acidic sphingomyelinases. Activation of a phosphatidylcholine-specific phospholipase C (PC-PLC) was also detected which appeared to be a requirement for subsequent acidic sphingomyelinase (aSMase) activation, since PC-PLC inhibitor D609 blocked Fas/APO-1-induced aSMase activation, but not Fas/APO-1-induced neutral sphingomyelinase (nSMase) activation. Fas/APO-1 cross-linking resulted also in ERK-2 activation and in phospholipase A2 (PLA2) induction, independently of the PC-PLC/aSMase pathway. Evidence for the existence of a pathway directly involved in apoptosis was obtained by selecting HuT78 mutant clones spontaneously expressing a newly identified death domain-defective Fas/APO-1 splice isoform which blocks Fas/APO-1 apoptotic signalling in a dominant negative fashion. Fas/APO-1 cross-linking in these clones fails to activate PC-PLC and aSMase, while nSMase, ERK-2 and PLA2 activates are induced. These results strongly suggest that a PC-PLC/aSMase pathway contributes directly to the propagation of Fas/APO-1-generated apoptotic signal in lymphoid cells.

T cell receptor (TCR) vaccination in rats prevents the development of experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis. The mechanism of this potential immunotherapy was examined by vaccinating mice with an immunogenic peptide fragment of the variable region of the TCR V beta 8.2 gene. Another immunogen that usually induces an immune response mediated by V beta 8.2+ T cells was subsequently inhibited because specific clonal unresponsiveness (anergy) had been induced. Depletion of CD8+ cells before TCR peptide vaccination blocked such inhibition. Thus, the clonal anergy was dependent on CD8+ T cells, and such immunoregulatory T cells may participate in the normal course of EAE.

Analyses of knockout and mutant transgenic mice as well as in vitro studies demonstrated a complex role of FADD in the regulation of cell fate. FADD is involved in death receptor induced apoptosis, cell cycle progression and cell proliferation. In a search for mechanisms that might regulate FADD functions, we identified, upon the screening of a lambda-phage cDNA library, calmodulin (CaM) as a novel FADD interacting protein. CaM is a key mediator of signals by the secondary messenger calcium and it is an essential regulator of cell cycle progression and cell survival. Here, we describe the identification and characterization of two calcium dependent CaM binding sites in the alpha helices 8-9 and 10-11 of FADD. Phosphorylation of human FADD at the C-terminal serine 194, by casein kinase I alpha (CKIalpha), has been shown to regulate FADD-dependent non-apoptotic activities. Remarkably, we showed that both FADD and CaM are CKIalpha substrates and that in synchronized HeLa cells, FADD, CaM and CKIalpha co-localize at the mitotic spindle in metaphase and anaphase. Moreover, complementation experiments in Jurkat FADD-/- T cells indicated that: a) cells expressing FADD mutants in the CaM binding sites are protected from Taxol-induced G2/M cell cycle arrest; b) FADD/CaM interaction is not required for Fas receptor-mediated apoptosis although Fas and CaM might compete for binding to FADD. We suggest that the interplay of FADD, CaM and CKIalpha may have an important role in the regulation of cell fate.

T cell clones recognizing the sperm whale myoglobin (SpWMb) epitope 110-121 in association with H-2d major histocompatibility complex class II molecules display a very limited heterogeneity of T cell receptor (TCR) V beta usage in DBA/2 mice. All clones previously tested used the same V beta 8.2 gene segment and very restricted junctional regions. To investigate the significance of this observation in vivo, we immunized DBA/2 mice with the intact SpW Mb protein or peptide 110-121. Only the V beta 8+ T cells showed any significant response to the 110-121 epitope. The response to peptide 110-121 was then analyzed in mice which, either as a consequence of antibody depletion or through genetic deletion of TCR V beta genes, lacked V beta 8+ peripheral T cells. DBA/2 mice depleted of V beta 8+ T cells by antibody treatment responded poorly to the 110-121 peptide, and only at high antigen concentrations. In contrast, DBA/2V beta a mice (homozygous for a deletion of multiple V beta gene segments including the V beta 8 family) made a response at least as great as that made by DBA/2 mice, even though the DBA/2V beta a mice had a very restricted TCR V beta repertoire compared with DBA/2 mice. Mechanisms which might determine differences in the 110-121 specific response of DBA/2, DBA/2V beta a and F23.1-treated DBA/2 mice are discussed.

Using the polymerase chain reaction and sequence-specific oligonucleotide probes, we have determined the distribution of the alleles at the polymorphic class II loci, DRB1, DQA1, DQB1, and DPB1 in 25 patients with Lyme arthritis. Comparison of the frequencies of the DRB1 and DPB1 alleles in Lyme arthritis patients to the Centre d'Etude du Polymorphisme Humaine control panel revealed statistically significant differences between the two groups. An increase in DRB1 ∗ 1301 is noted along with a unique distribution of the DR4 subtypes within the 9 DR4+ patients. In addition, an increase in the rate DPB1 ∗ 1001 and the more common DPB1 ∗ 0201 alleles is reported.

Two prior studies with a small number of T cell lines have shown that the presentation of native protein antigens by epidermal Langerhans cells (LC) is regulated. When freshly isolated, LC are efficient antigen-presenting cells (APC), but after a period of culture LC are inefficient or even inactive. The deficit in culture seems to be a selective loss in antigen processing, since cultured LC are otherwise rich in major histocompatibility complex (MHC) class II products and are active APC for alloantigens and mitogens, which do not require processing. We have extended the analysis by studying presentation to bulk populations of primed lymph node and a T-T hybrid. Only freshly isolated LC can be pulsed with the protein antigens myoglobin and conalbumin, but once pulsed, antigen is retained in an immunogenic form for at least 2 d. The acquisition of antigen, presumably as MHC-peptide complexes, is inhibited if the fresh LC are exposed to foreign protein in the presence of chloroquine or cycloheximide. The latter, in contrast, improves the efficacy of antigen pulsing in anti-Ig-stimulated B blasts. In additional studies of mechanism, we noted that both fresh and cultured LC endocytose similar amounts of an antigen, rhodamineovalbumin, into perinuclear granules. However, freshly isolated LC synthesize high levels class II MHC molecules and express higher amounts of the class II-associated invariant chain. Fresh LC are at least 5-10 times more active than many other cells types in the level of biosynthesis of MHC class II products. These findings provide a physiologic model in which newly synthesized MHC class II molecules appear to be the principal vehicle for effective antigen processing by APC of the dendritic cell lineage. Another APC, the B lymphoblast, does not appear to require newly synthesized MHC class II molecules for presentation.

BACKGROUND: It has been recently demonstrated that apoptosis is involved in beta-cell destruction in the NOD mouse model of diabetes. The aim of the present study was to investigate whether IL-15, a cytokine involved in the modulation of the apoptotic process, is capable of modifying the natural history of diabetes and/or insulitis in pre-diabetic NOD mice. The rationale for the use of IL-15-IgG2b recombinant cytokine is related to its long half-life (28 +/- 4 h). METHODS: At 10 weeks of age, 2 groups of 24 female mice were treated with single or multiple i.p. doses of IL-15-IgG2b respectively. As control, 2 groups of 24 age- and litter-matched female mice were injected intra-peritoneally with single or multiple doses of IgG2b immunoglobulin. RESULTS: Diabetes incidence at 33 weeks of age was lower in the group of mice treated with multiple doses than in the control group (p = 0.03). The cumulative incidence of diabetes at 33 weeks of age between single-dose treated mice and the control group was similar. No significant differences in the calculated index of insulitis were observed in all treated and control mice. CONCLUSIONS: We conclude that IL-15-IgG2b reduces the cumulative incidence of diabetes, without affecting the extent and severity of the insulitis process. Considering this and the well-defined anti-apoptotic effects of IL-15, we suggest that the reduction of diabetes incidence could be due to a down-regulation of beta-cell apoptosis.

Many anticancer drugs are able to induce apoptosis in tumor cells but the mechanisms underlying this phenomenon are poorly understood. Some authors reported that the p53 tumor suppressor gene may be responsible for drug-induced apoptosis; however, chemotherapy-induced apoptosis can also be observed in p53 negative cells. Recently, doxorubicin (DXR) was reported to induce CD95L expression to mediate apoptosis through the CD95/CD95L system. Thus, an impairment of such a system may be involved in drug resistance. We evaluated the in vitro antitumor activity of several cytotoxic drugs on two human p53-negative T-cell lymphoma cell lines, the HUT78-B1 CD95L-resistant cell line and the HUT78 parental CD95L-sensitive cell line. We demostrated by Western blotting assay that DXR and etoposide (VP-16) were able to induce CD95L expression after 4 h of treatment. In contrast, they were unable to induce the expression of p53. DXR, at concentrations ranging from 0.001 - 1 microg/ml, and VP16, at concentrations ranging from 0.05 - 1 microg/ml, were equally cytotoxic and induced apoptosis in both cell lines as assessed by fluorescence microscopy and flow cytometry analyses. Although we observed a slightly reduced percentage of apoptotic cells in HUT78B1 when compared with the parental HUT78 cells after few hours of drug exposure, this difference was no longer evident at 48 or 72 h. Similarly, the exposure of HUT78 cells to a CD95-blocking antibody partially reduced early apoptosis (24 h) without affecting the long-term effects of the drugs including cytotoxicity. Furthermore, as observed with DXR and VP-16, both the CD95L-sensitive and the CD95L-resistant cell lines resulted equally sensitive to the cytotoxic effects of a number of different cytotoxic drugs (vincristine, camptothecin, 5-fluorouracil and methotrexate). The treatment with the Caspase-3 tetrapeptide aldehyde inhibitor, Ac-DEVD-CHO, did not affect the DXR-induced apoptosis whereas it only modestly inhibited apoptosis and cytotoxicity of VP-16, while Z-VAD.FMK, a Caspase inhibitor that prevents the processing of Caspase-3 to its active form, was able to block DXR-induced apoptosis at 24 h but not at 48 h. Thus, our results do not confirm a crucial role for the CD95/CD95L system in drug-induced apoptosis and suggest the involvement of alternative p53-independent pathways at least in this experimental model system.

The CD95 death receptor plays an important role in several physiological and pathological apoptotic processes involving in particular the immune system. CD95 ligation leads to clustering of the receptor cytoplasmic "death domains" and recruitment of the zymogen form of caspase-8 to the cell surface. Activation of this protease through self-cleavage, followed by activation of downstream effector caspases, culminates in cleavage of a set of cellular proteins resulting in apoptosis with disassembly of the cell. It is very well known that the extracellular region of the CD95 receptor is required for CD95L interaction and that the death domain is necessary for the induction of the apoptotic signaling. Here, we identified and characterized a novel CD95 ligand- and death domain-independent oligomerization domain mapping to the NH(2)-terminal extracellular region of the CD95 receptor. In vitro and in vivo studies indicated that this domain, conserved among all soluble CD95 variants, mediates homo-oligomerization of the CD95 receptor and of the soluble CD95 proteins, as well as hetero-oligomerization of the receptor with the soluble variants. These results offer new insight into the mechanism of apoptosis inhibition mediated by the soluble CD95 proteins and suggest a role of the extracellular oligomerization domain in the regulation of the non-signaling state of the CD95 receptor.

The relationship between Fas-mediated apoptosis and Type 1 diabetes is currently under investigation. Fas/Fas ligand interaction could be involved both in the insulitis process and in beta-cell death. Nevertheless, different mechanisms appear to be involved in human Type 1 diabetes and in NOD mice. In the present work, we review recent evidence of the role of the Fas/Fas ligand system in human and NOD mouse diabetes, describing possible hypotheses for its involvement in the pathogenesis of the disease, with possible implications for therapy and islet transplantation.

Apoptosis may be triggered, in a variety of tissues, by interaction of the cell surface molecule CD95 with its specific ligand, CD95L. CD95 plays a physiological role in the regulation of the immune response; furthermore, alterations in CD95/CD95L function may contribute to the pathogenesis of a number of human diseases, including cancer, autoimmune diseases and viral infections. Many cells that express CD95, however, are not susceptible to CD95-mediated apoptosis. It is therefore important to identify the mechanisms that counteract the CD95 apoptotic process that are still poorly understood. Growth factors and lymphokines such as interleukin (IL)-4 that counteract CD95-mediated apoptosis may activate phosphatidylinositide 3-kinase (PI 3-kinase). We therefore used two different approaches to investigate the role of PI 3-kinase on CD95-mediated apoptosis. First we tested the effect of two pharmacological PI 3-kinase inhibitors, wortmannin and LY294002, on CD95 agonistic antibody-induced apoptosis in three different cell lines. Second, we co-expressed in COS7 cells CD95 with constitutively active PI 3-kinase. Results of both approaches indicate that active PI 3-kinase effectively protects against CD95-mediated apoptosis. Furthermore we extended our studies on the CD95 downstream mediator, FADD, and on the PI 3-kinase downstream mediator, the serine/threonine protein kinase PKB, using the co-expression approach in COS7 cells. We provide evidence that apoptosis induced by triggering the CD95 cell death receptor is counteracted by PI 3-kinase activation; moreover, PKB but not p70S6K represents the relevant downstream target of PI 3-kinase signaling.

Higher vertebrates frequently contain multigene families of related ligands and their receptors, often with overlapping specificities. Presumably such an organization allows for greater flexibility in the timing and tissue distribution of these molecules. A further level of complexity is introduced by the fact that variants of the same growth factor or receptor can be encoded as alternative transcripts of the same gene. Such variants may remain membrane associated or may be efficiently secreted.