Chimeric TßRII-SE/Fc overexpression by a lentiviral vector exerts strong antitumoral activity on colorectal cancer-derived cell lines in vitro and on xenografts.

The TGF signaling pathway is a key regulator of cancer progression. In this work, we report for the first time the antitumor activity of TßRII-SE/Fc, a novel peptibody whose targeting domain is comprised of the soluble endogenous isoform of the human TGF-ß type II receptor (TßRII-SE). Overexpression of TßRIISE/Fc reduces in vitro cell proliferation and migration while inducing cell cycle arrest and apoptosis in human colorectal cancer-derived cell lines. Moreover, TßRII-SE/Fc overexpression reduces tumorigenicity in BALB/c nude athymic mice. Our results revealed that TRII-SE/Fc-expressing tumors were significantly reduced in size or were even incapable of developing. We also demonstrated that the novel peptibody has the ability to inhibit the canonical TGF-ß and BMP signaling pathways while identifying SMAD-dependent and independent proteins involved in tumor progression that are modulated by TßRII-SE/Fc. These findings provide insights into the underlying mechanism responsible for the antitumor activity of TßRII-SE/Fc. Although more studies are required to demonstrate the effectiveness and safety of the novel peptibody as a new therapeutic for the treatment of cancer, our initial in vitro and in vivo results in human colorectal tumor-derived cell lines are highly encouraging. Our results may serve as the foundation for further research and development of a novel biopharmaceutical for oncology.

Compound A attenuates proinflammatory cytokine-induced endoplasmic reticulum stress in beta cells and displays beneficial therapeutic effects in a mouse model of autoimmune diabetes.

Type 1 diabetes (T1D) is characterized by an immune-mediated progressive destruction of the insulin-producing ß-cells. Proinflammatory cytokines trigger endoplasmic reticulum (ER) stress and subsequent insulin secretory deficiency in cultured ß-cells, mimicking the islet microenvironment in T1D. ß-cells undergo physiologic ER stress due to the high rate of insulin production and secretion under stimulated conditions. Severe and uncompensated ER stress in ß-cells is induced by several pathological mechanisms before onset and during T1D. We previously described that the small drug Compound A (CpdA), a selective glucocorticoid receptor (GR/NR3C1, nuclear receptor subfamily 3, group C, member 1) ligand with demonstrated inflammation-suppressive activity in vivo, is an effective modulator of effector T and dendritic cells and of macrophages, yet, in a GR-independent manner. Here, we focus on CpdA's therapeutic potential in T1D cellular and animal models. We demonstrate that CpdA improves the unfolded protein response (UPR) by attenuating ER stress and favoring the survival and function of ß-cells exposed to an environment of proinflammatory cytokines. CpdA administration to NODscid mice adoptively transferred with diabetogenic splenocytes (from diabetic NOD mice) led to a delay of disease onset and reduction of diabetes incidence. Histological analysis of the pancreas showed a reduction in islet leukocyte infiltration (insulitis) and preservation of insulin expression in CpdA-treated normoglycemic mice in comparison with control group. These new findings together with our previous reports justify further studies on the administration of this small molecule as a novel therapeutic strategy with dual targets (effector immune and ß-cells) during autoimmune diabetes.

A Novel Splice Variant of Human TGF-ß Type II Receptor Encodes a Soluble Protein and Its Fc-Tagged Version Prevents Liver Fibrosis in vivo.

We describe, for the first time, a new splice variant of the human TGF-ß type II receptor (TßRII). The new transcript lacks 149 nucleotides, resulting in a frameshift and the emergence of an early stop codon, rendering a truncated mature protein of 57 amino acids. The predicted protein, lacking the transmembrane domain and with a distinctive 13-amino-acid stretch at its C-terminus, was named TßRII-Soluble Endogenous (TßRII-SE). Binding predictions indicate that the novel 13-amino-acid stretch interacts with all three TGF-ß cognate ligands and generates a more extensive protein-protein interface than TßRII. TßRII-SE and human IgG1 Fc domain were fused in frame in a lentiviral vector (Lv) for further characterization. With this vector, we transduced 293T cells and purified TßRII-SE/Fc by A/G protein chromatography from conditioned medium. Immunoblotting revealed homogeneous bands of approximately 37 kDa (reduced) and 75 kDa (non-reduced), indicating that TßRII-SE/Fc is secreted as a disulfide-linked homodimer. Moreover, high-affinity binding of TßRII-SE to the three TGF-ß isoforms was confirmed by surface plasmon resonance (SPR) analysis. Also, intrahepatic delivery of Lv.TßRII-SE/Fc in a carbon tetrachloride-induced liver fibrosis model revealed amelioration of liver injury and fibrosis. Our results indicate that TßRII-SE is a novel member of the TGF-ß signaling pathway with distinctive characteristics. This novel protein offers an alternative for the prevention and treatment of pathologies caused by the overproduction of TGF-ß ligands.

The mRNA levels of TGF-ß Type II receptor splice variants in monocytes are associated with disease activity in patients with rheumatoid arthritis.

OBJECTIVES: In rheumatoid arthritis (RA) patients, TGF-ß exerts a singular effect on lymphocytes, macrophages, and polymorphonuclear leukocytes. Moreover, evidences indicate that TGF-ß1 stimulation affects the expression levels of TGF-ß receptors. Therefore, we analysed in different leukocyte subpopulations, whether the mRNA abundance of TGFBR2 splice variants might be related to RA. METHODS: We isolated different leukocyte subpopulations from peripheral blood from 9 healthy control volunteers and 9 RA patients, matched by gender and age (cohort 1), and 8 additional RA patients (cohort 2). Then we quantified, by RT-qPCR, the mRNA relative abundance of TGFBR2 splice variants (namely TGFBR2A and TGFBR2B) in PMNs, and PBMCs (monocytes and non-monocytes). We first checked whether the TGFBR2-splice variant mRNA profile could be associated with any particular blood cell type both, in healthy control volunteers and in RA patients. In addition, PBMC and PMN mRNA levels were correlated, using Spearman's rank-order correlation test, with clinical and biochemical determinations of RA patients. RESULTS: We have shown that TGFBR2 exhibits an alternative splicing pattern in different subpopulations of human leucocytes from healthy controls, and the lack of it in the same cell type from RA samples. Furthermore, our study yields initial evidence that TGFBR2 mRNA expression levels in monocytes might mirror RA disease activity. CONCLUSIONS: mRNA abundance of TGFBR2 splice variants in monocytes shows changes linked to RA disease activity.

Effect of poly (l-lactic acid) scaffolds seeded with aligned diaphragmatic myoblasts overexpressing connexin-43 on infarct size and ventricular function in sheep with acute coronary occlusion.

Diaphragmatic myoblasts (DM) are stem cells of the diaphragm, a muscle displaying high resistance to stress and exhaustion. We hypothesized that DM modified to overexpress connexin-43 (cx43), seeded on aligned poly (l-lactic acid) (PLLA) sheets would decrease infarct size and improve ventricular function in sheep with acute myocardial infarction (AMI). Sheep with AMI received PLLA sheets without DM (PLLA group), sheets with DM (PLLA-DM group), sheets with DM overexpressing cx43 (PLLA-DMcx43) or no treatment (control group, n = 6 per group). Infarct size (cardiac magnetic resonance) decreased ∼25% in PLLA-DMcx43 [from 8.2 ± 0.6 ml (day 2) to 6.5 ± 0.7 ml (day 45), p < .01, ANOVA-Bonferroni] but not in the other groups. Ejection fraction (EF%) (echocardiography) at 3 days post-AMI fell significantly in all groups. At 45 days, PLLA-DM y PLLA-DMcx43 recovered their EF% to pre-AMI values (PLLA-DM: 61.1 ± 0.5% vs. 58.9 ± 3.3%, p = NS; PLLA-DMcx43: 64.6 ± 2.9% vs. 56.9 ± 2.4%, p = NS), but not in control (56.8 ± 2.0% vs. 43.8 ± 1.1%, p < .01) and PLLA (65.7 ± 2.1% vs. 56.6 ± 4.8%, p < .01). Capillary density was higher (p < .05) in PLLA-DMcx43 group than in the remaining groups. In conclusion, PLLA-DMcx43 reduces infarct size in sheep with AMI. PLLA-DMcx43 and PLLA-DM improve ventricular function similarly. Given its safety and feasibility, this novel approach may prove beneficial in the clinic.

Human Adipose-Derived Mesenchymal Stem/Stromal Cells Handling Protocols. Lipid Droplets and Proteins Double-Staining.

Human Adipose-derived mesenchymal stem/stromal cells (hASCs) are of great interest because of their potential for therapeutic approaches. The method described here covers every single step necessary for hASCs isolation from subcutaneous abdominal adipose tissue, multicolor phenotyping by flow cytometry, and quantitative determination of adipogenic differentiation status by means of lipid droplets (LDs) accumulation, and Western blot analysis. Moreover, to simultaneously analyze both LDs accumulation and cellular proteins localization by fluorescence microscopy, we combined Oil Red O (ORO) staining with immunofluorescence detection. For LDs quantification we wrote a program for automatic ORO-stained digital image processing implemented in Octave, a freely available software package. Our method is based on the use of the traditional low cost neutral lipids dye ORO, which can be imaged both by bright-field and fluorescence microscopy. The utilization of ORO instead of other more expensive lipid-specific dyes, together with the fact that the whole method has been designed employing cost-effective culture reagents (standard culture medium and serum), makes it affordable for tight-budget research laboratories. These may be replaced, if necessary or desired, by defined xeno-free reagents for clinical research and applications.

Neumodisección con CO2 para lipotransferencia en reconstrucción mamaria / Pneumodissection with CO2 for transfer fat in breast reconstruction

Introducción La incorporación de la transferencia grasa como método accesorio a la reconstrucción con implantes permite hoy mayor seguridad y estabilidad de resultado en el tiempo. Es por ello que la optimización en el tratamiento de la lodge de la mastectomía con CO2 y campana de succión externa cobra un valor agregado en el mejoramiento tegumentario para aumentar la distribución y volumen de cobertura. Objetivos El objetivo es la preparación con succión externa y disección con CO2 del lecho receptor para la realización de reconstrucción mamaria diferida en pacientes mastectomizadas con radioterapia postmastectomía mediante la utilización de lipotransferencia y material protésico. Material y método Abordamos un grupo de 12 pacientes consecutivos con radioterapia postmastectomía que presentaban falta de cobertura, retracción cutánea, irregularidad de superficie, con una media de 43 años de edad ­entre un rango de 28 a 59 años­. Todas habían finalizado su tratamiento radioterapéutico hacía 1 año. La serie tuvo un seguimiento de 7 meses desde el primer procedimiento de lipotransferencia. Resultados En la comparación con nuestra serie general de 80 lipotransferencias en pacientes con radioterapia (sin neumodisector), se evidenció un mejoramiento clínico significativo de la lodge, por el aumento en la distribución a diferentes profundidades de un mismo plano del autoinjerto graso, impactando en una mayor superficie de cobertura y cambios estructurales de la piel. El espesor de los colgajos de la mastectomía fue evaluado con ecografías seriadas y rnm en la misma zona pre y postinjerto. Conclusiones Es evidente el mejoramiento de los tejidos mediante la utilización de la succión externa y aumento del grosor del lecho receptor con insuflación de CO2, con el consiguiente aumento de espesor en la distribución de grasa, que predispone a un mejor grado de prendimiento del autoinjerto y cobertura, pudiendo estos ser expandibles e indicar una reconstrucción con menor grado de morbilidad y mejor aceptación por la paciente.

Introduction The incorporation of transfer fat as accessory method to the reconstruction with implants today enables greater security and stability of result in time, so that the optimization in the treatment of the lodge of mastectomy with CO2 and external suction bell takes on an added value in the integumentary improvement to increase the distribution and volume of coverage. Objectives The aim is the preparation with external suction and dissection with CO2 of the receiving bed to perform breast reconstruction deferred in patients mastectomized and post-mastectomy radiotherapy using lipotransference and prosthetic material. Materials and method We approach a group of 12 consecutive post-mastectomy radiotherapy patients presenting with lack of coverage, cutaneous retraction, irregularity of surface, with an average of 43 years old, ­among a range of 28 to 59 years­. All had finished her radiotherapy treatment for 1 year; series was followed for 7 months from the first procedur eof lipotransference. Results Compared with our overall series 80 lipotransferences in patients with radiotherapy (without neumodissector), there is a significant clinical improvement of the lodge, by the increase in the distribution at different depths of the same level of fatty autograft, impacting on a larger surface coverage and structural changes of the skin. The thickness of the flaps of the mastectomy were assessed with serial ultrasounds and mri in the same area pre and post graft. Conclusions It is obvious the improvement of tissues, using external suction and increase of the thickness of the receptor bed with CO2 insufflation, with the consequent increase in thickness in the distribution of fat, which predisposes to a better degree of catch of autograft and coverage, which can be expandaded and indicate a reconstruction with lower degree of morbidity and better acceptance by the patient.

Aligned ovine diaphragmatic myoblasts overexpressing human connexin-43 seeded on poly (L-lactic acid) scaffolds for potential use in cardiac regeneration.

Diaphragmatic myoblasts (DMs) are precursors of type-1 muscle cells displaying high exhaustion threshold on account that they contract and relax 20 times/min over a lifespan, making them potentially useful in cardiac regeneration strategies. Besides, it has been shown that biomaterials for stem cell delivery improve cell retention and viability in the target organ. In the present study, we aimed at developing a novel approach based on the use of poly (L-lactic acid) (PLLA) scaffolds seeded with DMs overexpressing connexin-43 (cx43), a gap junction protein that promotes inter-cell connectivity. DMs isolated from ovine diaphragm biopsies were characterized by immunohistochemistry and ability to differentiate into myotubes (MTs) and transduced with a lentiviral vector encoding cx43. After confirming cx43 expression (RT-qPCR and Western blot) and its effect on inter-cell connectivity (fluorescence recovery after photobleaching), DMs were grown on fiber-aligned or random PLLA scaffolds. DMs were successfully isolated and characterized. Cx43 mRNA and protein were overexpressed and favored inter-cell connectivity. Alignment of the scaffold fibers not only aligned but also elongated the cells, increasing the contact surface between them. This novel approach is feasible and combines the advantages of bioresorbable scaffolds as delivery method and a cell type that on account of its features may be suitable for cardiac regeneration. Future studies on animal models of myocardial infarction are needed to establish its usefulness on scar reduction and cardiac function.

Pluripotent Nontumorigenic Adipose Tissue-Derived Muse Cells have Immunomodulatory Capacity Mediated by Transforming Growth Factor-ß1.

Adult mesenchymal stromal cell-based interventions have shown promising results in a broad range of diseases. However, their use has faced limited effectiveness owing to the low survival rates and susceptibility to environmental stress on transplantation. We describe the cellular and molecular characteristics of multilineage-differentiating stress-enduring (Muse) cells derived from adipose tissue (AT), a subpopulation of pluripotent stem cells isolated from human lipoaspirates. Muse-AT cells were efficiently obtained using a simple, fast, and affordable procedure, avoiding cell sorting and genetic manipulation methods. Muse-AT cells isolated under severe cellular stress, expressed pluripotency stem cell markers and spontaneously differentiated into the three germ lineages. Muse-AT cells grown as spheroids have a limited proliferation rate, a diameter of ∼15 µm, and ultrastructural organization similar to that of embryonic stem cells. Muse-AT cells evidenced high stage-specific embryonic antigen-3 (SSEA-3) expression (∼60% of cells) after 7-10 days growing in suspension and did not form teratomas when injected into immunodeficient mice. SSEA-3+ -Muse-AT cells expressed CD105, CD29, CD73, human leukocyte antigen (HLA) class I, CD44, and CD90 and low levels of HLA class II, CD45, and CD34. Using lipopolysaccharide-stimulated macrophages and antigen-challenged T-cell assays, we have shown that Muse-AT cells have anti-inflammatory activities downregulating the secretion of proinflammatory cytokines, such as interferon-γ and tumor necrosis factor-α. Muse-AT cells spontaneously gained transforming growth factor-ß1 expression that, in a phosphorylated SMAD2-dependent manner, might prove pivotal in their observed immunoregulatory activity through decreased expression of T-box transcription factor in T cells. Collectively, the present study has demonstrated the feasibility and efficiency of obtaining Muse-AT cells that can potentially be harnessed as immunoregulators to treat immune-related disorders. Stem Cells Translational Medicine 2017;6:161-173.

Percepción de la AR en una población fuera del ámbito sanitario / Perception of RA in a population outside health services

Objetivos: El acceso temprano a la población de pacientes con artritis reumatoidea (AR) a la asistencia especializada es un predictor de buen pronóstico. Sin embargo, la demora en la consulta es una constante; fueron identificados factores tales como los vinculados al paciente, el médico generalista o al sistema de salud. El objeto del estudio fue el de explorar la percepción de la población fuera del ámbito asistencial en un escenario potencial de sintomatología compatible con AR. Material y métodos: Se realizó un muestreo aleatorio de 1073 individuos mayores de 18 años, se realizó en la vía pública de los partidos de Quilmes y Berazategui. La encuesta fue efectuada por voluntarias entrenadas a tal fin las que indagaron acerca de a qué profesional consultaría si tuviera dolor e inflamación de los nudillos y muñecas de ambas manos, si cree padecer estos síntomas y si conoce la diferencia entre artritis y artrosis. Resultados: Frente a la posibilidad de padecer síntomas que remedan AR, la población encuestada eligió a los siguientes especialistas: traumatólogo (61%), clínico (17%), reumatólogo (15%), otros (7%). Un tercio de los encuestados refirió padecer esos síntomas y la mayoría (61%) referían no conocer la diferencia entre artritis y artrosis. Quienes más eligieron al reumatólogo como opción (56%) fueron del sexo femenino de entre 51 y 80 años y que conocían la diferencia entre AR y OA. Conclusiones: La ausencia relativa del reumatólogo en la preferencia de la población encuestada, así como la confusión respecto de la presencia de sintomatología compatible con la enfermedad o de la diferencia entre artritis y artrosis señalan la necesidad de ejecutar campañas de concientización pública dirigidas a la reafirmación de la especialidad frente a la patología articular inflamatoria.

Objectives: Early access to specialized care is a predictor of goodprognosis in patients recently diagnosed rheumatoid arthritis (RA).However, the delay in the consultation is a constant; they wereidentified factors such as those related to the patient, the generalpractitioner or the health system. The purpose of the study wasto explore the perception of the population outside the healthcaresetting in a potential scenario of symptoms compatible with RA.Material and methods: A random sample of 1073 individualsaged 18 and over was conducted. It was held in public matches ofQuilmes and Berazategui. The survey was conducted by volunteerstrained for this purpose which were asked which professional (interms of specialty) would consult if they had pain and swellingof the knuckles and wrists of both hands, and "if you know thedifference between arthritis and osteoarthritis".Results: Facing the possibility of having symptoms that mimic RA,the population surveyed chose the following specialists: orthopedicsurgeons (61%), clinicians (17%), rheumatologists (15%), others(7%). A third of respondents have referred these symptoms and most(61%) reported not knowing the difference between arthritis andosteoarthritis. People who chose rheumatologist an option (56%)were female between 51 and 80 years and knew the differencebetween RA and OA.Conclusions: The relative absence of rheumatologist in thepreference of the surveyed population, as well as confusion aboutthe presence of symptoms compatible with the disease or thedifference between arthritis and osteoarthritis indicate the needto implement public awareness campaigns aimed at reaffirmingspecialty against inflammatory joint disease.

Effect of TGF-ß1 Stimulation on the Secretome of Human Adipose-Derived Mesenchymal Stromal Cells.

Adipose tissue is an attractive source of mesenchymal stromal cells (MSCs) owing to the relative ease of obtaining large volumes with more MSC abundance compared with other sources. Increasing evidence supports the fact that trophic factors secreted by MSCs play a pivotal therapeutic role. Several strategies in regenerative medicine use MSCs, mainly exploiting their immunosuppressive effect and homing capacity to sites of damage. Transforming growth factor-ß1 (TGF-ß1) is a pleiotropic cytokine that, depending on the cell niche, can display either anti-inflammatory or proinflammatory effects. TGF-ß1 expression increases in various tissues with damage, especially when accompanied by inflammation. Thus, we analyzed the effect of TGF-ß1 on the secretion by adipose-derived mesenchymal stromal cells (ASCs) of a panel of 80 cytokines/chemokines using an antibody array. To avoid a possible effect of fetal bovine serum (FBS) on ASCs secretion, we performed our analysis by culturing cells in FBS-free conditions, only supplemented with 0.1% of bovine serum albumin. We report the cytokine profile secreted by ASCs. We also found that TGF-ß1 exposure modulates 8 chemokines and 18 cytokines, including TGF-ß1 and -ß2, and other important cytokines involved in immunosuppression, allergic responses, and bone resorption.

Development of novel efficient SIN vectors with improved safety features for Wiskott-Aldrich syndrome stem cell based gene therapy.

Gene therapy is a promising therapeutic approach to treat primary immunodeficiencies. Indeed, the clinical trial for the Wiskott-Aldrich Syndrome (WAS) that is currently ongoing at the Hannover Medical School (Germany) has recently reported the correction of all affected cell lineages of the hematopoietic system in the first treated patients. However, an extensive study of the clonal inventory of those patients reveals that LMO2, CCND2 and MDS1/EVI1 were preferentially prevalent. Moreover, a first leukemia case was observed in this study, thus reinforcing the need of developing safer vectors for gene transfer into HSC in general. Here we present a novel self-inactivating (SIN) vector for the gene therapy of WAS that combines improved safety features. We used the elongation factor 1 alpha (EFS) promoter, which has been extensively evaluated in terms of safety profile, to drive a codon-optimized human WASP cDNA. To test vector performance in a more clinically relevant setting, we transduced murine HSPC as well as human CD34+ cells and also analyzed vector efficacy in their differentiated myeloid progeny. Our results show that our novel vector generates comparable WAS protein levels and is as effective as the clinically used LTR-driven vector. Therefore, the described SIN vectors appear to be good candidates for potential use in a safer new gene therapy protocol for WAS, with decreased risk of insertional mutagenesis.

Stem-cell gene therapy for the Wiskott-Aldrich syndrome.

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive primary immunodeficiency disorder associated with thrombocytopenia, eczema, and autoimmunity. We treated two patients who had this disorder with a transfusion of autologous, genetically modified hematopoietic stem cells (HSC). We found sustained expression of WAS protein expression in HSC, lymphoid and myeloid cells, and platelets after gene therapy. T and B cells, natural killer (NK) cells, and monocytes were functionally corrected. After treatment, the patients' clinical condition markedly improved, with resolution of hemorrhagic diathesis, eczema, autoimmunity, and predisposition to severe infection. Comprehensive insertion-site analysis showed vector integration that targeted multiple genes controlling growth and immunologic responses in a persistently polyclonal hematopoiesis. (Funded by Deutsche Forschungsgemeinschaft and others; German Clinical Trials Register number, DRKS00000330.).

Disease-modifying immunotherapy for the management of autoimmune diabetes.

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that destroys the insulin-secreting beta-cells of the pancreas. It is now possible to predict those candidates that will progress to T1D before the full onset of the disease. Prevention of uncontrollable autoimmunity against beta-cells in therapies for T1D is mandatory to preserve the beta-cell mass. Therefore, immunomodulatory strategies directed to inhibiting the activity of self-reactive T cell clones as well as induction of regulatory T cells would be beneficial for prevention of T1D or recurrence of beta-cell autoimmunity against islet cell allografts.

A novel human primary immunodeficiency syndrome caused by deficiency of the endosomal adaptor protein p14.

Lysosome-related organelles have versatile functions, including protein and lipid degradation, signal transduction and protein secretion. The molecular elucidation of rare congenital diseases affecting endosomal-lysosomal biogenesis has given insights into physiological functions of the innate and adaptive immune system. Here, we describe a previously unknown human primary immunodeficiency disorder and provide evidence that the endosomal adaptor protein p14, previously characterized as confining mitogen-activated protein kinase (MAPK) signaling to late endosomes, is crucial for the function of neutrophils, B cells, cytotoxic T cells and melanocytes. Combining genetic linkage studies and transcriptional profiling analysis, we identified a homozygous point mutation in the 3' untranslated region (UTR) of p14 (also known as MAPBPIP), resulting in decreased protein expression. In p14-deficient cells, the distribution of late endosomes was severely perturbed, suggesting a previously unknown role for p14 in endosomal biogenesis. These findings have implications for understanding endosomal membrane dynamics, compartmentalization of cell signal cascades, and their role in immunity.

Development of hematopoietic stem cell gene therapy for Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is a complex primary immunodeficiency disorder associated with microthrombocytopenia, autoinnmunity and susceptibility to malignant lymphoma. At the molecular level, this rare disorder is caused by mutations in the gene encoding the Wiskott-Aldrich syndrome protein (WASP). WASP is a cytosolic adaptor protein mediating the rearrangement of the actin cytoskeleton upon surface receptor signaling. Allogenic hematopoietic stem cell (HSC) transplantation represents a curative approach but remains problematic in light of severe risks and side effects. Recently, HSC gene therapy has emerged as an alternative treatment option. Cumulative preclinical data obtained from WASP-deficient murine models and human cells indicate a marked improvement of the impaired cellular and immunological phenotypes associated with WASP deficiency. The first clinical trial is currently being conducted to assess the feasibility, toxicity, and potential therapeutic benefit of transplanting autologous WASP-reconstituted hematopoietic stem cells.

Retroviral WASP gene transfer into human hematopoietic stem cells reconstitutes the actin cytoskeleton in myeloid progeny cells differentiated in vitro.

OBJECTIVE: Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency disorder characterized by recurrent infections, autoimmunity, microthrombocytopenia, and susceptibility to malignant tumors. Compared with the conventional treatment using allogeneic bone marrow transplantation, hematopoietic stem cell gene therapy might offer more specific and less toxic therapeutic options. METHODS: We investigated retroviral WAS protein (WASP) gene transfer to assess functional correction and potential toxicities in human CD34(+) cells from WAS patients and healthy individuals, respectively. RESULTS: WASP mRNA and protein levels were restored in CD14(+) cells derived from WASP-transduced hematopoietic stem cells. Functional reconstitution in WASP-transduced myeloid cells was documented by podosome formation and Fc gamma R-mediated phagocytosis. Importantly, overexpression of WASP in CD34(+) cells from healthy donors did not cause any discernible toxic effects. CONCLUSIONS: Our studies document the feasibility of WASP gene transfer into human CD34(+) cells and suggest that the phenotype of WASP-deficient myeloid cells can be restored upon retroviral gene transfer.

Selective suppression of cathepsin L by antisense cDNA impairs human brain tumor cell invasion in vitro and promotes apoptosis.

Invasion and metastasis of certain tumors are accompanied by increased mRNA protein levels and enzymatic activity of cathepsin L. Cathepsin L has also been suggested to play a role in the proteolytic cascades associated with apoptosis. To investigate the role of cathepsin L in brain tumor invasion and apoptosis, the human glioma cell line, IPTP, was stably transfected with full-length antisense and sense cDNA of cathepsin L. Down-regulation of cathepsin L by antisense cDNA significantly impaired (up to 70%) glioma cell invasion in vitro and markedly increased glioma cell apoptosis induced by staurosporine. Compared to control and parental cell lines, antisense down-regulation of cathepsin L was associated with an earlier induction of caspase-3 activity. Up-regulation of cathepsin L activity by sense cDNA was associated with reduced apoptosis and later induction of caspase-3 activity. Moreover, down-regulation of cathepsin L lowered the expression of antiapoptotic protein Bcl-2, whereas up-regulation increased the expression of Bcl-2, indicating that cathepsin L acts upstream of caspase-3. These data show that cathepsin L is an important protein mediating the malignancy of gliomas and its inhibition may diminish their invasion and lead to increased tumor cell apoptosis by reducing apoptotic threshold.

Lysosomal enzymes, cathepsins in brain tumour invasion.

The expression patterns of different classes of peptidases in central nervous system (CNS) tumours have been most extensively studied in astrocytomas and meningiomas. Although the two types of tumours are very different in most respects, both may invade locally into normal brain. This process of invasion includes increased synthesis and secretion of lysosomal proteolytic enzymes - cathepsins. Aspartic endopeptidase cathepsin (Cat) D levels were found to be elevated in high-grade astrocytoma and partial inhibition of glioblastoma cell invasion by anti-Cat D antibody suggests that the enzyme activity is involved in the invasion process. Several studies on cysteine endopeptidase (CP) Cat B in gliomas agreed that transcript abundance, protein level and activity of Cat B increased in high-grade astrocytoma cultures compared with low-grade astrocytoma cultures and normal brain. Moreover, in glioma biopsies Cat B levels correlated with evidence of clinical invasion and it has been demonstrated that Cat B both in tumour cells and in endothelial cells can serve as a new biological marker for prognosis in glioblastoma patients. A high level of Cat B protein was also a diagnostic marker for invasive types of meningioma, distinguishing between histomorphologically benign, but invasive meningiomas and noninvasive, so-called clear-benign meningiomas. Cat L was also significantly increased in high-grade astrocytoma compared with low-grade astrocytoma and normal brain. Specific Cat L antibodies and antisense Cat L RNA transfection significantly lowered glioblastoma cell invasion. In meningioma, Cat L was a less-significant marker of invasion than Cat B. In contrast to cathepsins, the activities of endogenous cysteine peptidase inhibitors (CPIs), including stefins, cystatins and kininogens, were significantly higher in benign and atypical meningioma cell extracts than in malignant meningioma, and low-grade compared to high-grade astrocytoma. However, very low levels of stefins A and B were found in meningioma and glioblastoma tissues. Further studies on the expression levels and balance between cysteine endopeptidases (CPs) and CPIs would improve the clinical application of cathepsins in prognosis, which would lead to more-informed therapeutic strategies.

Differential methylation of the human apolipoprotein AI (APO AI) gene in liver and leucocyte cell DNA

O padräo de metilaçäo do DNA dentro da regiäo transcrita do gene humano Apolipoproteína AI (ApoAI) foi analizado em uma preparaçäo de DNA de células de fígado näo cultivadas e de células de leucócito de cinco pacientes caucasianos sem parentesco. Enquanto os sítios CCGG foram totalmente metilados em DNA de leucócitos, DNA da célula de fígado mostrou demetilaçäo em três sítios específicos dentro do gene. Estes resultados confirmam dados prévios de células cultivadas mostrando que demetilaçäo dentro do gene Apo AI correlaciona com a expressäo do gene