Hippocampal SPARC regulates depression-related behavior.

SPARC (secreted protein acidic and rich in cysteine) is a matricellular protein highly expressed during development, reorganization and tissue repair. In the central nervous system, glial cells express SPARC during development and in neurogenic regions of the adult brain. Astrocytes control the glutamate receptor levels in the developing hippocampus through SPARC secretion. To further characterize the role of SPARC in the brain, we analyzed the hippocampal-dependent adult behavior of SPARC KO mice. We found that SPARC KO mice show increased levels of anxiety-related behaviors and reduced levels of depression-related behaviors. The antidepressant-like phenotype could be rescued by adenoviral vector-mediated expression of SPARC in the adult hippocampus, but anxiety-related behavior persisted in these mice. To identify the cellular mechanisms underlying these behavioral alterations, we analyzed neuronal activity and neurogenesis in the dentate gyrus (DG). SPARC KO mice have increased levels of neuronal activity, evidenced as more neurons that express c-Fos after a footshock. SPARC also affects cell proliferation in the subgranular zone of the DG, although it does not affect maturation and survival of new neurons. SPARC expression in the adult DG does not revert the proliferation phenotype in KO mice, but our results suggest a role of SPARC in limiting the survival of new neurons in the DG. This work suggests that SPARC could affect anxiety-related behavior by modulating neuronal activity, and that depression-related behavior is dependent upon the adult expression of SPARC, which affects adult brain function by mechanisms that need to be elucidated.

Mesenchymal stem cells as therapeutic tools and gene carriers in liver fibrosis and hepatocellular carcinoma.

Mesenchymal stem (stromal) cells (MSCs) are a source of circulating progenitors that are able to generate cells of all mesenchymal lineages and to cover cellular demands of injured tissues. The extent of their transdifferentiation plasticity remains controversial. Cells with MSC properties have been obtained from diverse tissues after purification and expansion in vitro. These cellular populations are heterogeneous and under certain conditions show pluripotent-like properties. MSCs present immunosuppressive and anti-inflammatory features and high migratory capacity toward inflamed or remodeling tissues. In this study we review available data regarding factors and signaling axes involved in the chemoattraction and engraftment of MSCs to an injured tissue or to a tissue undergoing active remodeling. Moreover, experimental evidence in support of uses of MSCs as vehicles of therapeutic genes is discussed. Because of its regenerative capacity and its particular immune properties, the liver is a good model to analyze the potential of MSC-based therapies. Finally, the potential application of MSCs and genetically modified MSCs in liver fibrosis and hepatocellular carcinoma (HCC) is proposed in view of available evidence.

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.

Bias in estimations of DNA content by competitive polymerase chain reaction.

Competitive PCR is a highly sensitive method for specific DNA quantification. Despite the lack of studies related to the accuracy of the method it has been widely used. Here we present a simulation model for competitive PCR, which takes into account the efficiency decay as a linear relationship of the total product yield. The model helped us to study the kind and magnitude of errors that arise from quantitative and semiquantitative competitive PCR protocols and to find ways to minimize them. The simulation data suggest that differences in amplification efficiency between target and standard templates induce stronger biases in quantitative than in semiquantitative competitive PCR. Quantitative competitive PCR can only be used when both efficiencies are equal. In contrast, semiquantitative competitive PCR can be used even when the target is amplified with a higher efficiency than the standard, since under such conditions the method tends to underestimate the differences in initial DNA content. These predictions have been confirmed with experimental data and show that the estimation of the amplification efficiencies is a prerequisite for the use of quantitative and semiquantitative competitive PCR. A simple method for this estimation is also presented.

Immuno- and genetic therapy in autoimmune diseases.

Animal models of autoimmune disease have been developed that mimic some aspects of the pathophysiology of human disease. These models have increased our understanding of possible mechanisms of pathogenesis at the molecular and cellular level and have been important in the testing, development and validation of new immunotherapies. The susceptibility to develop disease in the majority of these models is polygenic as is the case in humans. The exceptions to this rule are gene knock outs and transgenic models of particular genes which, in particular genetic backgrounds, have also contributed to the understanding of single gene function and their possible contribution to pathogenesis. Gene therapy approaches that target immune functions are being developed with encouraging results, despite the polygenic nature of these diseases. Basically this novel immuno-genetic therapy harnesses the knowledge of immunology with the myriad of biotechnological breakthroughs in vector design and delivery. Autoimmune disease is the result of genetic dysregulation which could be controlled by gene therapy. Here we summarize the genetic basis of these human diseases as well as some of the best characterized murine models. We discuss the strategies for their treatment using immuno- and gene therapy.

A novel doxycycline inducible autoregulatory plasmid which displays "on"/"off" regulation suited to gene therapy applications.

The development of transcriptionally controlled systems which function in eukaryotic cells are important for achieving regulated gene expression in gene therapy. In this study we combined the components of the tetracycline-inducible system in self-contained retroviral and plasmid vectors. Regulated reporter gene expression from the autoregulatory plasmid pGTRTL in response to doxycycline (Dox) induction surpasses the expression observed from other self-contained retroviral and plasmid vectors. Induction kinetics and expression levels of luciferase and the therapeutic molecule, truncated soluble complement receptor 1 (sCR1) were characterised in a mouse fibroblast and a human neuroblastoma cell line. The regulatory characteristics of the plasmids were shown to be optimal for gene therapy applications, as there was a rapid reduction in expression levels following removal of Dox. Co-transfection of cells with an autoregulatory plasmid and a Dox inducible enhanced green fluorescent protein (EGFP) plasmid demonstrated the feasibility of using this plasmid combination to achieve parallel regulation of two genes of interest in a single cell under the control of Dox. These novel autoregulatory plasmids display the requirements for gene therapy applications in chronic conditions which are remitting/relapsing such as rheumatoid arthritis or multiple sclerosis, where novel protein therapeutics and combination therapies are needed. Gene Therapy (2000) 7, 2061-2070.

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.

[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.

Cytokine gene therapy in experimental allergic encephalomyelitis by injection of plasmid DNA-cationic liposome complex into the central nervous system.

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system with many similarities to multiple sclerosis. The main effector cells involved are CD4+ T cells, recognizing encephalitogenic epitopes within the central nervous system, and macrophages, both of which secrete proinflammatory cytokines, such as IFN-gamma and TNF. Studies have shown that immunomodulation of this inflammatory response by anti-inflammatory cytokines (IL-4, IL-10, IFN-beta, and TGF-beta) can reduce clinical severity in EAE. The importance of TNF in EAE has been demonstrated by using soluble TNF-receptor molecules to inhibit EAE. However, the limitation of this type of therapy is the necessity for frequent administration of cytokine proteins due to their short biologic half-life. This study demonstrates that EAE can be inhibited by a single injection of therapeutic cytokine (IL-4, IFN-beta, and TGF-beta) DNA-cationic liposome complex directly into the central nervous system. DNA coding for a novel, dimeric form of human p75 TNF receptor also ameliorated clinical EAE. Local administration of DNA-cationic liposome complex has identified gene targets that may be more efficiently exploited using vectors producing more stable expression for effective treatment of neuroimmunologic disease.

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.

Prevention of collagen-induced arthritis by gene delivery of soluble p75 tumour necrosis factor receptor.

Collagen type II-induced arthritis (CIA) in DBA/1 mice can be passively transferred to SCID mice with spleen B- and T-lymphocytes. In the present study, we show that infection ex vivo of splenocytes from arthritic DBA/1 mice with a retroviral vector, containing cDNA for the soluble form of human p75 receptor of tumour necrosis factor (TNF-R) before transfer, prevents the development of arthritis, bone erosion and joint inflammation in the SCID recipients. Assessment of IgG subclass levels and studies of synovial histology suggest that down-regulating the effector functions of T helper-type 1 (Th1) cells may, at least in part, explain the inhibition of arthritis in the SCID recipients. In contrast, the transfer of splenocytes infected with mouse TNF-alpha gene construct resulted in exacerbated arthritis and enhancement of IgG2a antibody levels. Intriguingly, infection of splenocytes from arthritic DBA/1 mice with a construct for mouse IL-10 had no modulating effect on the transfer of arthritis. The data suggest that manipulation of the immune system with cytokines, or cytokine inhibitors using gene transfer protocols can be an effective approach to ameliorate arthritis.

Pathogenic lymphoid cells engineered to express TGF beta 1 ameliorate disease in a collagen-induced arthritis model.

Collagen-induced arthritis in DBA/1 mice is a model of rheumatoid arthritis with marked synovitis and erosions. The disease can be adoptively transferred to SCID mice with arthritogenic splenocytes from DBA/1 mice injected with bovine collagen type II. However, infection of arthritogenic splenocytes with a retrovirus expressing TGF beta 1 inhibits development of arthritis in SCID mice. When DBA/1 mice, at onset of arthritis have additional arthritogenic splenocytes transferred, exacerbation occurs, reflected in a rapid increase in the number of arthritic joints, increased paw swelling and higher levels of anti-collagen antibody. By infecting arthritogenic splenocytes ex vivo with TGF beta 1 retrovirus, this exacerbation was inhibited. TGF beta 1 was effective in lowering inflammation of joints with already established arthritis and inhibiting the spreading of the disease to other joints. Transient reduction in anti-collagen antibody levels could also be obtained using purified T cells infected with TGF beta 1 retrovirus. In addition, expression of TGF beta 1 in lymphocytes reduced the levels of gelatinase (MMP2) activity in inflamed joints.

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.

The expression of the secreted protein acidic and rich in cysteine (SPARC) is associated with the neoplastic progression of human melanoma.

SPARC (secreted protein acidic and rich in cysteine) is an extracellular protein associated with tissues exhibiting high rates of cell proliferation and matrix remodeling. The current work shows that the human melanoma cell lines IIB-MEL-LES, IIB-MEL-IAN, and IIB-MEL-J and different human metastatic melanomas expressed high levels of SPARC mRNA and protein. By western blot analysis we detected a single secreted 42-kDa band in human diploid fibroblasts-conditioned medium and a 45- to 40-kDa doublet in the three melanoma cell lines and all the metastatic melanomas tested. Part of the melanoma samples and cell lines showed an additional doublet of 36-34 kDa. SPARC mRNA was expressed by the three established cell lines, 14 metastatic melanoma samples, and tumors raised in nude mice, and no spliced variants were found. The heterogeneous pattern of SPARC secreted by human melanoma cells is the result of post-translational glycosylation and a specific extracellular leupeptin-inhibitable cleavage. Unlike human fibroblasts, melanoma cells did not overexpress SPARC on addition of TGF-beta. Immunohistochemical analysis showed that SPARC was strongly expressed in 100% of primary melanomas (7 of 7) and metastatic melanomas (29 of 29), moderately expressed in most of the positive dysplastic nevi (13 of 14), and only weakly expressed in nevocellular nevi (4 of 25). Normal melanocytes did not express SPARC. The data suggest that the expression of SPARC is associated with the neoplastic progression of human melanoma.

Tumor cells engineered to express interleukin-6 exhibit a reduced tumorigenicity depending on the tumor cell model.

Cytokine gene transfer to tumor cells has been demonstrated to induce tumor rejection in different murine models. However, controversial results were presented for different cytokines. In order to study the antitumorigenic activity that has been proposed for IL-6, the poorly immunogenic melanoma B16 and the colon adenocarcinoma CT26-murine cell lines, were transduced with recombinant retrovirus expressing rat IL-6. In vivo studies showed that IL-6-producing-B 16 cells inoculated s.c. in syngeneic mice, exhibited reduced tumorigenicity compared to vector-transduced B 16 cells. The histology of growing IL-6-producing tumors showed a "pseudo-nodular" pattern which correlated with a strong inhibition of the in vitro invasive capacity of these cells. IL-6-producing-B 16 cells did not develop tumors in athymic nude mice suggesting that the antitumor effect is not mediated by a normal host-T- and B-cell response. In contrast, IL-6-producing CT26 cells grew as tumors in syngeneic mice with a faster growth rate than parental and vector-transduced cells, in accordance with an increased in vitro growth kinetics. These results indicate that IL-6 expression by tumor cells demonstrate different effects depending on the tumor cell model.

[The role of SPARC gene in tumorigenic capacity of human melanoma cells]. / Rol del gen SPARC en la capacidad tumorigénica de células de melanoma humano.

Previous studies from our laboratory have demonstrated that human melanoma cell lines and tumors expressed high levels of the extracellular protein SPARC. In order to demonstrate its role in human melanoma progression, IIB-MEL-LES human melanoma cells were transfected with SPARC full length c-DNA in the antisense orientation. In vivo studies demonstrated that all the control mice injected with parental cells developed tumors, while none of the mice injected with cells obtained from three different clones with diminished levels of SPARC expression, developed tumors. These studies suggest that SPARC may play a key role in human melanoma progression.

Inhibition of transfer of collagen-induced arthritis into SCID mice by ex vivo infection of spleen cells with retroviruses expressing soluble tumor necrosis factor receptor.

Collagen-induced arthritis can be transferred into severe combined immunodeficiency (SCID) mice by spleen cells from diseased DBA/1 mice. The development of arthritis in SCID animals can be prevented by infection ex vivo of DBA/1 spleen cells with retroviruses expressing the monomeric soluble human p75 tumor necrosis factor (TNF) receptor (TNF-R). In addition, a vector engineered to express a polycystronic mRNA with TNF-R and the herpes simplex virus thymidine kinase (HSVtk) gene, while producing low levels of TNF-R, had a limited effect which could be blocked by treating the animals with ganciclovir. A retroviral vector expressing the HSVtk gene alone had no effect on this arthritis transfer model with or without ganciclovir. Serum levels of TNF-R did not correlate with clinical signs, however, lower anti-collagen antibody levels corresponded with lack of clinical symptoms. These results indicate that local production of cytokine inhibitor is essential for therapeutic purposes while systemic levels may not be required.

Biologic, immunocytochemical, and cytogenetic characterization of two new human melanoma cell lines: IIB-MEL-LES and IIB-MEL-IAN.

Two human melanoma cell lines, derived from metastases of two patients with epithelioid malignant amelanotic melanomas, and designated IIB-MEL-LES and IIB-MEL-IAN, have been established. Both cell lines have been in continuous culture over 2 years and were propagated continuously for 85 and 75 serial passages, respectively. Morphologically, IIB-MEL-LES is composed predominantly of spindle shaped cells, whereas IIB-MEL-IAN grows as a monolayer of cuboid and stellate shaped cells with many rounded cells in suspension. Immunocytochemical studies revealed that both cell lines express S-100 protein, vimentin, and GD3 and GD2 gangliosides but are negative for keratin and collagen. Both cell lines express HLA class I and HLA-DR antigens in variable proportions. The MAGE-1 gene is expressed only by the IIB-MEL-IAN cell line, as revealed by PCR analysis. Cytogenetic analysis of both cell lines revealed abnormal karyotypes; the modal chromosome numbers of IIB-MEL-LES and IIB-MEL-IAN were 48 and 81, respectively. IIB-MEL-LES cells presented rearrangements in chromosomes 1, 14 and X, gains in chromosomes 10, 20, and 21 losses in chromosomes 15 and Y. The most frequent markers observed in IIB-MEL-IAN cells were 7q+, 10p+, 2p+, i(6p), 2q+, and 10q-. Clonal gains were observed in chromosomes 12 and 21, whereas losses were seen in chromosomes 1, 2, 3, 4, 6, 7, 11, and 17. Both cell lines were capable of forming colonies in soft agar and developed tumors when transplanted into nude mice, reproducing and maintaining the characteristics of the original tumors. These cell lines and their xenografts appear to provide useful systems for studying the biology, genetics and histogenesis of human malignant melanoma and could be utilized for the development of melanoma vaccines.