Differences between disease-associated endoplasmic reticulum aminopeptidase 1 (ERAP1) isoforms in cellular expression, interactions with tumour necrosis factor receptor 1 (TNF-R1) and regulation by cytokines.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) processes peptides for major histocompatibility complex (MHC) class I presentation and promotes cytokine receptor ectodomain shedding. These known functions of ERAP1 may explain its genetic association with several autoimmune inflammatory diseases. In this study, we identified four novel alternatively spliced variants of ERAP1 mRNA, designated as ΔExon-11, ΔExon-13, ΔExon-14 and ΔExon-15. We also observed a rapid and differential modulation of ERAP1 mRNA levels and spliced variants in different cell types pretreated with lipopolysaccharide (LPS). We have studied three full-length allelic forms of ERAP1 (R127-K528, P127-K528, P127-R528) and one spliced variant (ΔExon-11) and assessed their interactions with tumour necrosis factor receptor 1 (TNF-R1) in transfected cells. We observed variation in cellular expression of different ERAP1 isoforms, with R127-K528 being expressed at a much lower level. Furthermore, the cellular expression of full-length P127-K528 and ΔExon-11 spliced variant was enhanced significantly when co-transfected with TNF-R1. Isoforms P127-K528, P127-R528 and ΔExon-11 spliced variant associated with TNF-R1, and this interaction occurred in a region within the first 10 exons of ERAP1. Supernatant-derived vesicles from transfected cells contained the full-length and ectodomain form of soluble TNF-R1, as well as carrying the full-length ERAP1 isoforms. We observed marginal differences between TNF-R1 ectodomain levels when co-expressed with individual ERAP1 isoforms, and treatment of transfected cells with tumour necrosis factor (TNF), interleukin (IL)-1ß and IL-10 exerted variable effects on TNF-R1 ectodomain cleavage. Our data suggest that ERAP1 isoforms may exhibit differential biological properties and inflammatory mediators could play critical roles in modulating ERAP1 expression, leading to altered functional activities of this enzyme.

Suppression of SPARC expression by antisense RNA abrogates the tumorigenicity of human melanoma cells.

Acquisition of invasive/metastatic potential is a key event in tumor progression. Cell surface glycoproteins and their respective matrix ligands have been implicated in this process. Recent evidence reveals that the secreted glycoprotein SPARC (secreted protein, acidic and rich in cysteine) is highly expressed in different malignant tissues. The present study reports that the suppression of SPARC expression by human melanoma cells using a SPARC antisense expression vector results in a significant decrease in the in vitro adhesive and invasive capacities of tumor cells, completely abolishing their in vivo tumorigenicity. This is the first evidence that SPARC plays a key role in human melanoma invasive-metastatic phenotype development.

Recombinant galectin-1 and its genetic delivery suppress collagen-induced arthritis via T cell apoptosis.

Galectin-1 (GAL-1), a member of a family of conserved beta-galactoside-binding proteins, has been shown to induce in vitro apoptosis of activated T cells and immature thymocytes. We assessed the therapeutic effects and mechanisms of action of delivery of GAL-1 in a collagen-induced arthritis model. A single injection of syngeneic DBA/1 fibroblasts engineered to secrete GAL-1 at the day of disease onset was able to abrogate clinical and histopathological manifestations of arthritis. This effect was reproduced by daily administration of recombinant GAL-1. GAL-1 treatment resulted in reduction in anticollagen immunoglobulin (Ig)G levels. The cytokine profile in draining lymph node cells and the anticollagen IgG isotypes in mice sera at the end of the treatment clearly showed inhibition of the proinflammatory response and skewing towards a type 2-polarized immune reaction. Lymph node cells from mice engaged in the gene therapy protocol increased their susceptibility to antigen-induced apoptosis. Moreover, GAL-1-expressing fibroblasts and recombinant GAL-1 revealed a specific dose-dependent inhibitory effect in vitro in antigen-dependent interleukin 2 production to an A(q)-restricted, collagen type 2-specific T cell hybridoma clone. Thus, a correlation between the apoptotic properties of GAL-1 in vitro and its immunomodulatory properties in vivo supports its therapeutic potential in the treatment of T helper cell type 1-mediated autoimmune disorders.

Historical development of monoclonal antibody therapeutics.

Since the first publication by Kohler and Milstein on the production of mouse monoclonal antibodies (mAbs) by hybridoma technology, mAbs have had a profound impact on medicine by providing an almost limitless source of therapeutic and diagnostic reagents. Therapeutic use of mAbs has become a major part of treatments in various diseases including transplantation, oncology, autoimmune, cardiovascular, and infectious diseases. The limitation of murine mAbs due to immunogenicity was overcome by replacement of the murine sequences with their human counterpart leading to the development of chimeric, humanized, and human therapeutic antibodies. Remarkable progress has also been made following the development of the display technologies, enabling of engineering antibodies with modified properties such as molecular size, affinity, specificity, and valency. Moreover, antibody engineering technologies are constantly advancing to enable further tuning of the effector function and serum half life. Optimal delivery to the target tissue still remains to be addressed to avoid unwanted side effects as a result of systemic treatment while achieving meaningful therapeutic effect.

Identification of thyroid stimulating hormone receptor-specific T cells in Graves' disease thyroid using autoantigen-transfected Epstein-Barr virus-transformed B cell lines.

The importance of thyrotropin receptor (TSHR) agonist antibodies in the manifestations of Graves' disease (GD) is recognized. There are, however, no convincing reports of TSHR-specific T cells. We have previously cloned T cells specific for thyroglobulin and thyroid peroxidase (TPO) from GD lymphoid infiltrates and used autologous EBV-transformed B cell lines (EBVL) transfected with an expression vector encoding TPO to efficiently detect TPO-specific T cells. Here we used EBVL transfected with TSHR to seek TSHR-specific T cells in the GD infiltrates, after cloning the in vivo activated T cells without antigen. 3 out of 30 clones responded vigorously and reproducibly to EBVL-TSHR, with a mean stimulation index > 7. Their release of IL-2, IL-4, and IL-10 after stimulation with soluble anti-CD3 and phorbol ester was indistinguishable from the other clones from this thyroid. However, they produced relatively little IFN gamma (median IL-4/IFN gamma ratio of 0.80) compared with the other clones (median IL-4/IFN gamma ratio 0.06). Thus, this new potent method of antigen presentation, using autoantigen-transfected EBVL, has permitted the first unequivocal identification of TSHR T cells in GD thyroid, with distinct Th0/Th2 characteristics, unlike previously cloned TPO-responsive cells which have Th1 characteristics.

Family of human alpha-interferon-like sequences.

An interferon-alpha-like sequence was isolated from a human genomic library by hybridization with a 15-base oligonucleotide. The sequence also showed homology to alpha-interferon and was most closely related to the leukocyte interferon-M gene fragment. The original isolate cross-hybridized to a family of sequences, 10 of which were isolated as clones. Some of these sequences were located within a few kilobases of alpha-interferon genes, consistent with our assignment of several members of the family to human chromosome 9 which also has the beta 1- and alpha-interferon genes.

Predominance of the metastatic phenotype in somatic cell hybrids of the K-1735 murine melanoma.

The purpose of these studies was to determine whether the metastatic phenotype will dominate when metastatic and nonmetastatic clones of the K-1735 mouse melanoma are hybridized by somatic cell fusion. Three nonmetastatic and three metastatic clones were transfected with DNA from plasmids pSV2neo or pSV2hygro, which confer resistance to the drugs neomycin or hygromycin, respectively. The metastatic properties of the six clones were not altered by these transfections. The tumorigenicity and metastatic capacity of cell hybrids formed by somatic cell fusion of nonmetastatic and metastatic clones were examined. To do so, near-tetraploid hybrids containing a nearly complete chromosomal complement from both parental cells were injected i.v. into syngeneic mice, and the number of metastatic nodules in the lung was determined at 45 days or when the mice became moribund. Seven of nine hybrids produced from the fusion of metastatic and nonmetastatic clones exhibited a highly metastatic phenotype, although in most cases the metastatic potential of the hybrids was lower than that of the metastatic parent cells. Very similar results were obtained in athymic nude mice. The metastatic potential of the hybrids was directly correlated with their growth in the subcutis of nude mice. These results indicate that the metastatic capacity of K-1735 cells predominates in somatic cell hybrids between nonmetastatic and metastatic cells. When fusion of nonmetastatic and metastatic cells yields a hybrid with nonmetastatic properties, it may be due to suppression of growth.

Interferon-stimulated genes in interferon-sensitive and -resistant chronic myelogenous leukemia patients.

alpha-Interferon induces hematological and cytogenetic remissions in some individuals with newly diagnosed Philadelphia-positive chronic myelogenous leukemia. However, interferon-resistant disease occurs in a consistent patient subset (primary resistance) and develops during therapy in additional patients (secondary resistance). Several alpha-interferon-inducible genes have been characterized. In interferon-resistant cell line variants, defects in these genes have been implicated in the mechanisms mediating resistance. We have, therefore, evaluated mRNA expression of four interferon-stimulated genes (ISGs) following alpha-interferon therapy. Twenty-seven chronic myelogenous leukemia patients (ten interferon-sensitive patients, 17 interferon-resistant patients) were studied. Peripheral blood samples were collected prior to and 1 to 7 days after starting interferon therapy and analyzed for the expression of 2'-5' oligoadenylate synthetase, ISG-15, ISG-54, and 6-16 transcripts. Following therapy with alpha-interferon, 2'-5' oligoadenylate synthetase, ISG-54, and 6-16 transcripts were discerned in all patients regardless of their response to interferon. The ISG-15 message was detected in eight of nine interferon-sensitive and in 15 of 16 interferon-resistant patients, as well. Overall, no consistent defect in the ISG system could be identified. Therefore, lack of induction of these genes cannot explain resistance to alpha-interferon in chronic myelogenous leukemia patients. Other mechanisms such as posttranslational modification, leading to defects in the ISG corresponding proteins, may play a role in the development of resistance.

Mice vaccination with interleukin 12-transduced colon cancer cells potentiates rejection of syngeneic non-organ-related tumor cells.

Cell-based gene therapy after cytokine gene transfer is being investigated for autologous and allogeneic vaccination in cancer therapy. Here we show that mice vaccinated with 3-5 x 10(6) interleukin 12 (IL-12) gene-transduced CT26 colon cancer cells developed a long-lasting antitumor immune memory able to reject not only parental cells but also syngeneic, LM3 mammary, and MCE fibrosarcoma tumorigenic cells. In contrast, mice vaccinated with 0.5-1 x 10(6) CT26 cells transduced with pBabe neo IL-12 retrovirus cells (CT26-IL12) were only able to reject parental cells. An increase in the total circulating levels of IgG2a and a clear shift toward a systemic Th1 response developed, regardless of the amount of injected CT26-IL12 cells. On the contrary, a strong increase in anti-CT26-specific IgG2a levels was observed only when 3-5 x 10(6) CT26-IL12 cells were injected. Immunocompetent mice vaccinated with 3-5 x 10(6) CT26-IL12 cells developed local nodules for a few days, which then ceased growing. These nodules comprised mainly blood vessels, suggesting that an angiogenic process was taking place. CD8+ T cells were responsible for the anti-LM3 tumor cell memory, whereas CD4+ T cells were not involved. Splenocytes and lymphocytes obtained from mice immunized against CT26 cells were able to kill LM3 cells in vitro. Adoptive transfer of lymphocytes obtained from animals immunized against CT26 colon cancer cells suppressed LM3 mammary tumor growth in tumor-bearing mice. The present studies raised the possibility of isolating CTL clones and identifying CTL epitopes shared by different tumor cell types, which can be a target for cancer therapy.

Assembly PCR synthesis of optimally designed, compact, multi-responsive promoters suited to gene therapy application.

Gene therapy has the potential to provide innovative treatments for genetic and non-genetic diseases, with the ability to auto-regulate expression levels of therapeutic molecules so that they are produced locally and in direct response to disease activity. Generating disease responsive gene therapy vectors requires knowledge of the activation profile of transcription factors (TFs) during active disease, in order to assemble binding sites for these TFs into synthetic promoters, which can be appropriately activated by the disease process. In this study, we optimised a PCR random assembly approach to generate promoters with optimal spacing between TF binding sites (TFBSs) and their distance from the TATA box. In promoters with optimal spacing, it was possible to demonstrate activation by individual transcription pathways and either additive or synergistic promoter activation when transfected cells were treated with combined stimuli. The kinetics and sensitivity of promoter activation was further explored in transduced cells and when lentivirus was directly delivered to mouse paws a synthetic promoter demonstrated excellent activation by real-time imaging in response to local inflammation.

The murine p75 TNF receptor promoter region: DNA sequence and characterization of a cis-acting silencer.

The promoter region of the murine p75 TNF receptor (TNF-R) was isolated from a mouse genomic DNA cosmid library. The promoter region is devoid of TATA box and has the characteristics of a house keeping gene since it contains multiple SP1 binding sites. Its mRNA has different initiation sites in different lymphoid cell lines. This promoter confers transcriptional activity to heterologous reporter genes. Deletion analysis showed that there is a silencer element located upstream from position -841. This inhibitory sequence is active both in fibroblasts and T-cell lines transiently or permanently transfected. A fragment from -929 to -841 is capable of transferring this 'silencer' activity to the early SV40 promoter. This activity could be blocked in trans- when a plasmid containing the same sequence was co-transfected with the reporter plasmid indicating that a protein binds to this region of the promoter.

Constitutive in vitro binding of nuclear proteins to the 5'-flanking region of 6-16, a human gene inducible by alpha, beta-interferons.

Proteins in nuclear extracts of HeLa cells that constitutively bound in vitro to three regions upstream of the interferon-inducible gene 6-16 were separated partially by chromatography on DEAE-Sepharose. Region one, a CCAAT box in the non-coding strand at position --63 to --67, was protected from DNase digestion by the bound protein(s) and was required for transcription in vitro. Region two, a tandem duplication sequence at position --89 to --168 contains two copies of a sequence essential for strong induction of the 6-16 gene by interferon in vitro. Region three, a palindromic sequence at position --449 to --465, not necessary for induction of 6-16 by interferon, was also protected from DNase digestion by nuclear protein(s). Templates with or without regions of two and three were transcribed equally well in extracts from interferon-treated or untreated cells.

Different efficacy of in vivo herpes simplex virus thymidine kinase gene transduction and ganciclovir treatment on the inhibition of tumor growth of murine and human melanoma cells and rat glioblastoma cells.

Initial studies have demonstrated the therapeutic efficacy for cancer treatment of in vivo transfer of the herpes simplex virus thymidine kinase gene followed by ganciclovir (GCV) treatment. However, recent studies have questioned the validity of this approach. Using retroviral vector-producing cells (VPC) as a source for in vivo gene transfer, we evaluated the efficacy of in vivo transduction of malignant cells using three different tumor cell models: B16 murine and IIB-MEL-LES human melanomas and a C6 rat glioblastoma. In vitro studies showed a bystander effect only in C6 cells. In vivo studies showed an inhibition of tumor growth in the two melanoma models when tumor cells were coinjected with VPC-producing retroviral vectors carrying the herpes simplex virus thymidine kinase gene, followed by GCV treatment; however, 100% of mice developed tumors in both models. Under similar experimental conditions, 70% (7 of 10) of syngeneic rats completely rejected stereotactically transferred C6 tumor cells; most of them (5 of 10) showed a prolonged survival. Treating established C6 tumors with VPC-producing retroviral vectors carrying the herpes simplex virus thymidine kinase gene and GCV led to the cure of 33% (4 of 12) of the animals. Rats that rejected tumor growth developed an antitumor immune memory, leading to a rejection of a stereotactic contralateral challenge with parental cells. The immune infiltrate, which showed the presence of T lymphocytes, macrophages, and polymorphonuclear cells at the site of the first injection and mainly T lymphocytes and macrophages at the site of tumor challenge, strengthened the importance of the immune system in achieving complete tumor rejection.

The molecular and cellular basis of corticosteroid resistance.

Corticosteroids (CS) can modulate gene expression and are often used to treat a range of immunological and inflammatory diseases such as asthma, inflammatory bowel disease and rheumatoid arthritis. However, a proportion of patients fail to show an adequate response. On this basis patients have been subdivided into CS-sensitive (SS) and -resistant (SR) subgroups. The ability of CS to inhibit peripheral blood T cell proliferation in vitro has also been used similarly. In rheumatoid arthritis (RA), the in vitro-defined SS and SR subgroups correlate with the clinical responses to CS therapy. The mechanisms responsible for this observation are unknown but they appear to involve a number of known molecular events related to the described mechanisms of action of CS. These include alterations in the functional status of CS receptor-alpha, perturbations of the cytokine and hormonal milieu and intracellular signalling pathways. Peripheral blood mononuclear cells (MNCs) from SR significantly overexpress activated NF-kappaB. In vitro, CS fail to significantly inhibit concanavalin A (conA)-induced NF-kappaB activation in MNCs from SR RA patients. The alterations in the intracellular signalling pathways may explain in part our observations seen in SR RA subjects, CS fail to significantly inhibit conA-induced interleukin (IL)-2 and IL-4 secretion and lipopolysaccharide-induced IL-8 and IL-1beta secretion in vitro. CS therapy fails to reduce the circulating levels of IL-8 and IL-1beta in RA patients. In asthma, CS fail to induce L10 in SR asthma patients. Other molecular mechanisms such as enhanced AP-1 expression and alterations in the MAP kinase pathway are most likely to be involved too and we are currently investigating such possibilities. A full understanding of the molecular basis of SR will lead to the development of more rational therapeutic strategies.

Human kinesin light (beta) chain gene: DNA sequence and functional characterization of its promoter and first exon.

Kinesins are tubulin molecular motors whose function is to transport organelles within cells. Very little is known about the regulation of expression of these proteins. We have characterized the gene product of one differentially spliced mRNA of the human light chain kinesin and cloned its promoter region. A full-length kinesin cDNA was translated in vitro in a cell-free system, producing a 70-kDa protein. Using this cDNA as a probe, we isolated and sequenced the promoter, first exon, and part of the first intron of this gene from a genomic lambda EMBL3 human placental DNA library. The whole gene spans more than 90 kb. The beta kinesin promoter region confers only constitutive transcription to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene. In permanently transfected human HeLa and NB100 neuroblastoma cells, a reporter gene containing the promoter and part of the first exon of beta kinesin was 75-fold more active than the HSV-tk promoter. The first exon contains the 5'-untranslated sequence capable of forming a stable double-hairpin loop, which functions as a translational enhancer. Its deletion decreases the efficiency of in vitro translation of beta kinesin mRNA and confers increased translation to a CAT reporter gene.

Minimal tumor necrosis factor receptor binding protein: optimum biological activity of a truncated p55 soluble tumor necrosis factor receptor-IgG fusion protein.

We have previously demonstrated, using expressed deletion constructs, that the fourth membrane proximal cysteine-rich repeat of the p55 TNF receptor (TNF-R) is not required for binding of tumour necrosis factor-alpha (TNF) or lymphotoxin-alpha (LT; tumour necrosis factor-beta). We and others have also shown that the soluble p55 TNF-R, rendered dimeric by fusion to an IgG backbone is extremely effective at neutralizing the harmful effects of TNF overproduction, such as in toxic shock. Here we address the question of how the TNF binding properties of the truncated TNF-R comprising the three distal cysteine-rich repeats (delta4 TNF-R), when fused with an IgG backbone, compare with those of the full length soluble receptor. We constructed several versions of the soluble delta4 TNF-R, on a complete IgG heavy chain backbone and on an IgG lacking the CH1 (first constant region) domain. The constructs were expressed with an Ig or native TNF receptor leader sequence and altered or native N terminal sequence, to compare efficiency of expression. When compared with a full length, soluble receptor Ig fusion protein, the affinity of all for TNF was identical, as were their activities in in vitro binding and cytotoxicity assays. In vivo studies showed that the delta4 and wild type fusion proteins afforded equivalent protection against LPS-induced lethality. However, the delta4 proteins exhibited a significantly lower affinity for LT, and reduced activity in LT binding and cytotoxicity assays. We conclude that the truncated TNF receptor IgG fusion protein is as effective at neutralizing TNF activity as the full length soluble receptor fusion protein. Its lower affinity for LT may make it a more selective agent in blocking the action of TNF, while causing less interference with the action of LT. Also its smaller size may make it a more useful therapeutic agent as it may be less immunogenic than the full length receptor.

Gene therapy for rheumatoid arthritis. Theoretical considerations.

Current understanding of the pathogenesis of rheumatoid arthritis has provided evidence that therapeutic benefit can be achieved by using antagonists targeted to the inflammatory cytokines involved, mainly tumour necrosis factor-alpha and interleukin-1. Gene delivery of antagonists, which can inhibit the production or action of these cytokines and other mediators, has been achieved in experimental animal models. This new method of delivery can produce therapeutic effects at lower concentrations and in a local environment, overcoming the adverse effects that often accompany protein therapy. However, several technological and biological restraints preclude the immediate adaptation of this method to human treatment. Based on the experimental evidence, possible target therapeutic genes, cell types and vector systems that could be used are discussed in this article.

Inhibition of interleukin 2 production and alteration of interleukin 2 mRNA processing by human T-T cell hybridoma-derived suppressor factors.

We investigated the mechanisms by which two human T-T cell hybridoma-derived suppressor factors (SFs) (designated 160 and 169) (Platsoucas et al., Hybridoma 1987;6:589; Kunicka et al., Hybridoma 1989;8:127) inhibit the proliferative response to mitogens by human peripheral blood mononuclear cells (PBMCs). Interleukin 2 (IL-2) production by human PBMCs cultured with concanavalin A or OKT3 monoclonal antibody for 12 or 36 hr in the presence of 160 or 169 SF was found to be inhibited > 80% when compared to control PBMC cultures stimulated with mitogen in the absence of SFs. This suppression of IL-2 production was not due to the SFs interfering with IL-2-induced proliferation of the IL-2-dependent murine cell clone used to determine the levels of IL-2. The proliferative responses of SF-treated PBMCs could not be restored by addition of exogenous recombinant human IL-2 (rIL-2) (1-100 U/ml). Furthermore, inhibition of the proliferative responses by the SFs could not be reversed by addition of exogenous rIL-1, rIL-2, or rIL-4 alone or in paired combinations. The expression of IL-2 receptors (TAC Ag) on concanavalin A-activated cultures at 12- or 36-hr time points was not affected by treatment with the SFs. Both the 160 and 169 hybridoma-derived SFs were found to cause the accumulation of an mRNA of 2.8 kb that hybridized with an IL-2-specific oligonucleotide probe. This 2.8-kb transcript was in addition to the expected 1.0-kb, transiently expressed IL-2 message, and it could be superinduced in the presence of cycloheximide. These results suggest that these SFs may be influencing RNA splicing pathways. These SFs appear to be useful molecules for probing the regulatory controls of lymphocyte proliferation and may constitute important physiological regulators of the immune response. In addition, they may have clinical activity for the treatment of patients that received transplants, patients with autoimmune diseases, and others.

[Antitumor gene therapy using suicide genes]. / Terapia génica antitumoral mediante el uso de genes suicidas.

Tumor cells transduced with retrovirus carrying the herpes simplex-1 virus thymidine kinase (HSV-tk) are capable of transforming the antiviral drug ganciclovir (GVC) into a metabolic form only toxic to dividing cells. The efficiency of this suicide gene therapy is increased by a "bystander" effect resulting not only in the death of the recipient cell, but also in the death of non modified surrounding cells. Even though the mechanism of this "bystander" effect remains to be elucidated, strong evidence suggest that the immune system plays a main role to achieve complete tumor eradication. In the present study we evaluate the efficiency of this suicide system on three different tumor models: one human melanoma, one murine melanoma, and a rat glioblastoma. Tumors were established by injection of tumor cells s.c. in nude and C57Bl/6 mice, respectively, and stereotactically into the brain of Sprague Dawley rats. Animals in the treated group were co-injected with packaging cells producing recombinant retrovirus carrying the HSV-tk gene, and followed by i.p. administration of GVC. In short term studies, we observed inhibition of tumor growth for all the tumor models evaluated (p < 0.01). In long term studies, using the C6 rat glioma line, 50% of the animals survived longer than 75 days (p < 0.0001), and were able to reject a contralateral challenges with C6 parental cells. Histological and immunohistochemical analysis showed the presence at an inflammatory infiltrate composed by T lymphocytes, macrophages and polymorphonuclear cells. These data demonstrate that suicide genes might represent an attractive form of cancer gene therapy in the treatment of brain tumors and their intracerebral dissemination.