Feasibility, safety, and preliminary proof of principles of autologous neural stem cell treatment combined with T-cell vaccination for ALS patients.

Uncontrolled activation of the innate immune system promotes the deterioration of neurons in different neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). T-cell vaccination (TCV) was developed by Irun Cohen and coworkers at the Weizmann Institute of Science (Israel) during the late 1970s and has been demonstrated to be an effective treatment for human autoimmune diseases and a regulator of macrophage activation in animal models. We treated seven ALS patients with this cell therapy and were able to slow or stop disease progression in the affected individuals. The median survival, which is 3.5 years, was extended to 6 years. They were also treated with autologous adult neural stem cells associated with effector T cells. The observed neurologic improvements after treatment lasted for at least 1 year. Clinical recovery in the treated ALS patients was confirmed by an independent, skilled neurologist using the ALS Functional Rating Scale-Revised (ALSFRS-R). TCV in conjunction with an autologous neural stem cell treatment might be a feasible, minimally invasive, safe, and effective approach to obtain enduring therapeutic effects in ALS patients.

Case report on the clinical results of a combined cellular therapy for chronic spinal cord injured patients.

OBJECTIVES: With the intention to ameliorate the clinical condition of patients with chronic spinal cord injury (SCI), a program that combines three cell therapies and an appropriate neurorehabilitation program were used to recreate and enhance the natural conditions of SCI repair. METHODS: Vascularization recovery is approached by selective artery infusion of BMMNCs (bone marrow mononuclear cells) to the disrupted area. Eighteen days later, with the aim to restore the specific inflammatory activity, an i.v. infusion of spinal cord specific ETCs (effector T cells) is carried out. With the intention of supplying cellular components for the process of repair, an infusion of autologous neural stem cells (NSCs) through selective feeding artery infusion is carried out, followed by an appropriate neurorehabilitation program. RESULTS: A total of eight ASIA (American Spinal Injury Association) A patients (five with jeopardized brachial plexus and three without) received the treatment. No severe adverse events was observed in any of the receptor patients: five patients evolved from ASIA A to ASIA D and regained the ability to stand up and, with varying effectiveness, to walk; two patients remained in the same condition, but exhibited motor and sensitive improvements; and one patient could not be evaluated. CONCLUSIONS: These reports suggest that the biological characteristics of acute SCI may be recreated in a comprehensive, safe and effective manner.

Autoreactive T cells induce in vitro BM mesenchymal stem cell transdifferentiation to neural stem cells.

BACKGROUND: The degree of post-injury inflammation of the damaged area of a spinal cord is the main difference between the natural successful repair in inferior vertebrates and failure in superior vertebrates. The treatment of rats with anti-myelin lymphocytes after experimental spinal cord injury induces their functional recovery. On the other hand, mesenchymal stem cells (MSC) from adult BM implanted in injured areas recover the morphology and function of spinal cord in mammals. The purpose of this study was to determine whether there is a direct relationship between anti-nervous tissue T cells and MSC reparatory properties. METHODS: Circulating autoreactive lymphocytes of patients with spinal cord injuries and amyotrophic lateral sclerosis were isolated and activated in vitro. These cells were cocultured with autologous MSC for 2-15 days. Cocultures of non-selected lymphocytes were used as controls. RESULTS: After 48 h of coculture, MSC adopted a spindle shape with polarization of the cytoplasm that resembled bipolar neurons. Their nuclei diminished the nucleolus number and the chromatin lost its granular appearance. After 15 days of culture the cells developed the typical structure of a neural network. No morphologic changes were observed in control cultures. The differentiated cells reacted positively to tubuline III, GFAP and nestin. No differences were observed between the different patient cell sources. DISCUSSION: We observed that autoreactive cells may induce the transdifferentiation of MSC to neural stem cells. This T-cell-MSC interaction may be a common phenomenon during physiologic nerve tissue repair.

Combined protocol of cell therapy for chronic spinal cord injury. Report on the electrical and functional recovery of two patients.

BACKGROUND: This is a preliminary report on successful results obtained during treatment of two patients with chronic spinal cord injury. The therapeutic approach was based on the generation of controlled inflammatory activity at the injury site that induced a microenvironment for the subsequent administration of autologous, BM-driven transdifferentiated neural stem cells (NSC). METHODS: BM mesenchymal stem cells (MSC) were cocultured with the patient's autoimmune T (AT) cells to be transdifferentiated into NSC. Forty-eight hours prior to NSC implant, patients received an i.v. infusion of 5 x 10(8) to 1 x 10(9) AT cells. NSC were infused via a feeding artery of the lesion site. Safety evaluations were performed everyday, from the day of the first infusion until 96 h after the second infusion. After treatment, patients started a Vojta and Bobath neurorehabilitation program. RESULTS: At present two patients have been treated. Patient 1 was a 19-year-old man who presented paraplegia at the eight thoracic vertebra (T8) with his sensitive level corresponding to his sixth thoracic metamere (T6). He received two AT-NSC treatments and neurorehabilitation for 6 months. At present his motor level corresponds to his first sacral metamere (S1) and his sensitive level to the fourth sacral metamere (S4). Patient 2 was a 21-year-old woman who had a lesion that extended from her third to her fifth cervical vertebrae (C3-C5). Prior to her first therapeutic cycle she had severe quadriplegia and her sensitive level corresponded to her second cervical metamere (C2). After 3 months of treatment her motor and sensitive levels reached her first and second thoracic metameres (T1-T2). No adverse events were detected in either patient. DISCUSSION: The preliminary results lead us to think that this minimally invasive approach, which has minor adverse events, is effective for the repair of chronic spinal cord lesions.

Development of tumor B-cell lymphocyte hybridoma (TBH) autovaccination. Results of a phase I-II clinical trial.

To improve patient immune recognition of autologous tumor cells, we have developed a tumor B-cell lymphocyte hybridoma (TBH) autovaccination protocol. This approach is based on immunization of a cancer patient with a hybridoma cell suspension derived from the fusion of autologous activated B-cells and autologous cancer cells. This hybrid allows the host immune system to recognize and destroy oncocytes with low toxicity and high specificity. Of 21 treated patients with very advanced diseases, six complete responses and four partial responses were achieved. Overall, survival was prolonged. Side-effects and combination therapies with IL2, IL6 and gamma I/FN are discussed in this paper. Breast and colon cancer seem to be sensitive to this therapy.

Antagonism of interferon beta on interferon gamma: inhibition of signal transduction in vitro and reduction of serum levels in multiple sclerosis patients.

Interferon gamma (IFN-gamma acts as a mediator of multiple sclerosis (MS) exacerbations through a number of biological effects, such as induction of major histocompatibility class II complexes (MHC-II), macrophage activation and potentiation of tumor necrosis factor (TNF-alpha). The clinical efficacy of interferon beta (IFN-beta) therapy in reducing exacerbations of relapsing-remitting MS has been related to antagonistic effects on various activities of IFN-gamma, including MHC-II gene induction. However, there is no model to explain such antagonistic effects of IFN-beta and IFN-gamma, and the two cytokines are also known to act synergistically against viruses and in the induction of MHC-I. We show that IFN-beta does inhibit an immediate molecular event of IFN-gamma, namely activation and DNA binding of the transcription factor Stat1. We propose a model of direct interference of the IFN-gamma and IFN-alpha,beta signal transduction pathways accounting for antagonistic effects on some genes, which in turn activate MHC-II transcription, as well as for synergistic effects on other genes. In addition, study of MS patients treated with natural IFN-beta shows that IFN-beta significantly reduces serum levels of IFN-gamma while increasing IL-4, strongly suggesting that IFN-beta also controls the relative activation of TH1- and TH2-type T lymphocytes.

Incorporation and intracellular hydrolysis of 131 I-albumin injected in rat testis.

A method of cell separation by enzymatic attack and filtration was applied after injecting the testes. The results indicated that in the case of the interstitial injections, the fraction rich in interstitial cells captures from 30 to 50 times more RISA than that rich in germinal cells, and from 10 to 20 times more than that rich in Sertoli cells. Following the intratubular injection, radioactivity incorporation was greatly reduced and there were no differences in RISA (radioactivity/mg of protein) among the cell fractions. It was concluded that Sertoli cells do not have in its apical side a phagocytic activity comparable to that of the interstitial macrophages and possibly they incorporate only a small amount of tracer by a process of moderate pinocytosis.

Hipertension portal en el trasplante insular de pancreas. / Portal hypertension in pancreatic islet transplantation

El autotrasplante de tejido insular pancreatico, obtenido por metodo mecanico-enzimatico y realizado mediante la embolizacion portal, ha provocado la hipertension portal aguda en la totalidad de los 43 animales transplantados. La utilizacion de heparina y aprotinina logro reducir parcialmente las cifras tensionales, pero solo una anastomosis portocava laterolateral no amplia, evito este fenomeno. La hipertension portal aguda, causada esencialmente por coagulacion intravascular es capaz de conducir a la muerte, alejando este procedimiento de la aparente inocuidad senalada inicialmente

Hipertension portal en el trasplante insular de pancreas. / Portal hypertension in pancreatic islet transplantation

El autotrasplante de tejido insular pancreatico, obtenido por metodo mecanico-enzimatico y realizado mediante la embolizacion portal, ha provocado la hipertension portal aguda en la totalidad de los 43 animales transplantados. La utilizacion de heparina y aprotinina logro reducir parcialmente las cifras tensionales, pero solo una anastomosis portocava laterolateral no amplia, evito este fenomeno. La hipertension portal aguda, causada esencialmente por coagulacion intravascular es capaz de conducir a la muerte, alejando este procedimiento de la aparente inocuidad senalada inicialmente

The blood-testis barrier in the toad (Bufo arenarum Hensel): a freeze-fracture and lanthanum tracer study.

Intercellular junctions between Sertoli cells in the toad testis were studied by freeze-fracture and electron-opaque intercellular markers. These junctional specializations are characterized in thin sections by a series of focal fusions on the outer leaflets of both adjacent cell plasmalemmas, associated with bundles of fine filaments in the subjacent Sertoli cell cytoplasms. However, the wide subsurface cisterna of the endoplasmic reticulum, a component constantly associated with Sertoli cell junctions in mammals, is absent in the toad. The intravascularly injected lanthanum hydroxide, used as a tracer compound, gains access to the seminiferous tubules and surrounds spermatogonia and leptotene spermatocytes, but is persistently excluded from germ cells in later stages of development. This indicates that, as is the case in the mammalian testis, a permeability barrier to lanthanum is established which isolates all germ cells beyond leptotene spermatocytes. Freeze-fracture reveals the characteristic occluding junctions between Sertoli cells, but a variation in their geometric patterns was clearly observed in different regions of the toad seminiferous epithelium. The membrane-fractured faces of Sertoli cells embracing differentiating spermatids exhibit a deep junctional complex: up to 50 rows of particles between adjacent Sertoli cells separate these late germ cells from the periphery of the seminiferous tubules. Sertoli cells surrounding early germ cells generally exhibit, instead, a discontinuous, poorly developed network of interconnected rows of particles with few widely spaced strands. This seems to permit the percolation of the intercellular marker in areas of the seminiferous epithelium containing spermatogonia and leptotene spermatocytes.

Lysosomal enzymes in cells separated from rat testis.

Fractions enriched with Sertoli cell (S), germ cells (G), and interstitial cells (I) were separated from rat testis after enzymic treatment and double filtration through nylon meshes. The fractions were analysed for protein content and for enzymic activity of 4 acid hydrolases known to be of lysosomal nature in other tissues. Acid phosphatase activity was preferentially recovered in Fraction G, the highest activity of beta-glucuronidase was found in Fraction I while the activity of aryl sulphatase and beta-N-acetyl-D-glucosaminidase was prominent in Fraction S. With the exception of acid phosphatase, the enzymes were mostly recovered in a subcellular fraction of whole testis homogenate separated between 600 and 27 000 g. The results may reflect the peculiar enzyme composition of the lysosomal apparatus of each cell type.

Fine structure of the testis in the toad (Bufo arenarum hensel): a freeze-fracture study.

The present work reports novel findings in the toad testis using freeze-fracture techniques. Tight and gap junctions are observed in the nonfenestrated endothelial cells. Numerous gap junctions are present between interstitial cells. Sertoli-Sertoli junctional specializations in the toad are similar to those described in mammals although they appear to be less elaborate. The appearance of nuclear pores, in the seminiferous epithelium, ranges from a uniformly and apparently random distribution in Sertoli cells to large aggregations of closely spaced pores separated by free areas in the nuclear envelope of spermatocytes. Inasmuch as these features have also been described in mammals, the fact that they are present in amphibia might indicate that they represent a widespread pattern. Concomitantly with increasing chromatin condensation, nuclear pores diminish in round spermatids and seem to disappear in the more mature germ cells. The en face freeze-fracture views of annulate lamellae in the postnuclear cytoplasm and membranous features of the acrosome formation, flagellum, and undulating membrane of elongating spermatids are also described.