Local, systemic and immunologic safety comparison between xenogeneic equine umbilical cord mesenchymal stem cells, allogeneic canine adipose mesenchymal stem cells and placebo: a randomized controlled trial.

Mesenchymal stem cells are multipotent cells with a wide range of therapeutic applications, including, among others, tissue regeneration. This work aims to test the safety (EUC-MSC) of intra-articular administration of equine umbilical cord mesenchymal stem cells in young healthy dogs under field conditions following single and repeated administration. This was compared with the safety profile of allogenic canine adipose derived mesenchymal stem cells (CAD-MSC) and placebo in order to define the safety of xenogeneic use of mesenchymal stem cells when administered intra-articular. Twenty-four police working dogs were randomized in three groups in a proportion 1:1:1. EUC-MSCs and CAD-MSCs were obtained from healthy donors and were manufactured following company SOPs and under GMP and GMP-like conditions, respectively, and compliant all necessary controls to ensure the quality of the treatment. The safety of the treatment was evaluated locally, systemically and immunologically. For this purpose, an orthopedic examination and Glasgow test for the assessment of pain in the infiltrated joint, blood tests, clinical examination and analysis of the humoral and cellular response to treatment were performed. No adverse events were detected following single and repeated MSC administration despite both equine and canine MSC generate antibody titres in the dogs. The intra-articular administration of equine umbilical cord mesenchymal stem cells in dogs has demonstrated to be safe.

In search of cytotoxic selectivity on cancer cells with biogenically synthesized Ag/AgCl nanoparticles.

Green synthesis may be a useful approach to achieve selective cytotoxicity of silver nanoparticles on cancer cells and healthy cells. In this study, the concomitant biosynthesis of silver (Ag)/silver chloride (AgCl) nanoparticles from pineapple peel extracts and their behavior on the breast cancer cell line MCF-7 is shown. Bioreactions were monitored at different temperatures. Fourier-transform infrared spectroscopy (FTIR), ultraviolet-visible spectroscopy (UV-vis), thermogravimetric analysis (TGA), X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy (EDX), and transmission electron microscopy (TEM) techniques were used to characterize nanoparticle development. The breast cancer cell line MCF-7 was used as a test model to study the cytotoxic behavior of Ag/AgCl nanoparticles and, as a counterpart, the nanoparticles were also tested on mononuclear cells. Ag/AgCl nanoparticles with spherical and triangular morphology were obtained. The size of the nanoparticles (10-70 nm) and the size distribution depended on the reaction temperature. A dose close to 20 µg/mL of Ag/AgCl nanoparticles considerably decreased the cell viability of the MCF-7 line. The best cytotoxicity effects on cancer cells were obtained with nanoparticles at 60 and 80 °C where cell viability was reduced up to 80% at a concentration of 50 µg/mL. A significant preference was observed in the cytotoxic effect of Ag/AgCl nanoparticles against cancer cells in comparison to monocytes.

Addition of proteinase K during the culture alter the physiology of Bacillus thuringiensis culture and the cry1Ac, nprX, nprA, and spo0A gene transcription.

Bacillus thuringiensis is the major bioinsecticide worldwide produced due to the Cry protein activity. Several studies have been done to improve the cost-productivity relation. The neutral protease A (NprA) is the major extracellular protein massively produced during the stationary phase by this bacterium, contributing to the Cry proteins' degradation. Also, the deletion of aprA and nprA genes enhanced the yield of Cry protein, stabilizing it. Therefore, to increase Cry production, one possibility is to degrade the NprA protease in the culture media. In the present study, proteinase K was used to hydrolyze the NprA to increase Cry production. Proteinase K was added during the exponential growth of B. thuringiensis culture. The bacilli and endospores were measured along all culture, while the Cry protein was measured at the end of the culture. The addition of PK affects the bacilli and spore kinetics positively but negatively to the Cry protein (there is no Cry protein detection). Therefore, the gene expression of the cry1Ac, nprX, nprA, and spo0A was measured. The expression of each gene was followed along all culture. Results demonstrated that PK alters both the transcriptional levels and the expression order of the genes.

Intrathecal administration of autologous mesenchymal stromal cells for spinal cord injury: Safety and efficacy of the 100/3 guideline.

BACKGROUND AIMS: Cell therapy with autologous mesenchymal stromal cells (MSCs) in patients with spinal cord injury (SCI) is beginning, and the search for its better clinical application is an urgent need. METHODS: We present a phase 2 clinical trial in patients with chronic SCI who received three intrathecal administrations of 100 x 106 MSCs and were followed for 10 months from the first administration. Efficacy analysis was performed on nine patients, and safety analysis was performed on 11 patients. Clinical scales, urodynamic, neurophysiological and neuroimaging studies were performed previous to treatment and at the end of the follow-up. RESULTS: The treatment was well-tolerated, without any adverse event related to MSC administration. Patients showed variable clinical improvement in sensitivity, motor power, spasms, spasticity, neuropathic pain, sexual function or sphincter dysfunction, regardless of the level or degree of injury, age or time elapsed from the SCI. In the course of follow-up three patients, initially classified as ASIA A, B and C, changed to ASIA B, C and D, respectively. In urodynamic studies, at the end of follow-up, 66.6% of the patients showed decrease in postmicturition residue and improvement in bladder compliance. At this time, neurophysiological studies showed that 55.5% of patients improved in somatosensory or motor-evoked potentials, and that 44.4% of patients improved in voluntary muscle contraction together with infralesional active muscle reinnervation. CONCLUSIONS: The present guideline for cell therapy is safe and shows efficacy in patients with SCI, mainly in recovery of sphincter dysfunction, neuropathic pain and sensitivity.

Cell therapy with autologous mesenchymal stromal cells in post-traumatic syringomyelia.

BACKGROUND AIMS: Recently, clinical studies show that cell therapy with mesenchymal stromal cells (MSCs) improves the sequelae chronically established in paraplegic patients, being necessary to know which of them can obtain better benefit. METHODS: We present here a phase 2 clinical trial that includes six paraplegic patients with post-traumatic syringomyelia who received 300 million MSCs inside the syrinx and who were followed up for 6 months. Clinical scales, urodynamic, neurophysiological, magnetic resonance (MR) and studies of ano-rectal manometry were performed to assess possible improvements. RESULTS: In all the cases, MR at the end of the study showed a clear reduction of the syrinx, and, at this time, signs of improvement in the urodynamic studies were found. Moreover, four patients improved in ano-rectal manometry. Four patients improved in neurophysiological studies, with signs of improvement in evoked potentials in three patients. In the American Spinal Injury Association (ASIA) assessment, only two patients improved in sensitivity, but clinical improvement in neurogenic bowel dysfunction was observed in four patients and three patients described improvement in bladder dysfunction. Spasms reduced in two of the five patients who had them previous to cell therapy, and spasticity was improved in the other two patients. Three patients had neuropathic pain before treatment, and it was reduced or disappeared completely during the study. Only two adverse events ocurred, without relation to the cell therapy. CONCLUSIONS: Cell therapy can be considered as a new alternative to the treatment of post-traumatic syringomyelia, achieving reduction of syrinx and clinical improvements in individual patients.

Repeated subarachnoid administrations of autologous mesenchymal stromal cells supported in autologous plasma improve quality of life in patients suffering incomplete spinal cord injury.

BACKGROUND AIMS: Cell therapy with mesenchymal stromal cells (MSCs) offers new hope for patients suffering from spinal cord injury (SCI). METHODS: Ten patients with established incomplete SCI received four subarachnoid administrations of 30 × 106 autologous bone marrow MSCs, supported in autologous plasma, at months 1, 4, 7 and 10 of the study, and were followed until the month 12. Urodynamic, neurophysiological and neuroimaging studies were performed at months 6 and 12, and compared with basal studies. RESULTS: Variable improvement was found in the patients of the series. All of them showed some degree of improvement in sensitivity and motor function. Sexual function improved in two of the eight male patients. Neuropathic pain was present in four patients before treatment; it disappeared in two of them and decreased in another. Clear improvement in bladder and bowel control were found in all patients suffering previous dysfunction. Before treatment, seven patients suffered spasms, and two improved. Before cell therapy, nine patients suffered variable degree of spasticity, and 3 of them showed clear decrease at the end of follow-up. At this time, nine patients showed infra-lesional electromyographic recordings suggesting active muscle reinnervation, and eight patients showed improvement in bladder compliance. After three administrations of MSCs, mean values of brain-derived neurotrophic factor, glial-derived neurotrophic factor, ciliary neurotrophic factor, and neurotrophin 3 and 4 showed slight increases compared with basal levels, but without statistically significant difference. CONCLUSIONS: Administration of repeated doses of MSCs by subarachnoid route is a well-tolerated procedure that is able to achieve progressive and significant improvement in the quality of life of patients suffering incomplete SCI.

An approach to personalized cell therapy in chronic complete paraplegia: The Puerta de Hierro phase I/II clinical trial.

BACKGROUND AIMS: Cell transplantation in patients suffering spinal cord injury (SCI) is in its initial stages, but currently there is confusion about the results because of the disparity in the techniques used, the route of administration, and the criteria for selecting patients. METHODS: We conducted a clinical trial involving 12 patients with complete and chronic paraplegia (average time of chronicity, 13.86 years; SD, 9.36). The characteristics of SCI in magnetic resonance imaging (MRI) were evaluated for a personalized local administration of expanded autologous bone marrow mesenchymal stromal cells (MSCs) supported in autologous plasma, with the number of MSCs ranging from 100 × 10(6) to 230 × 10(6). An additional 30 × 10(6) MSCs were administered 3 months later by lumbar puncture into the subarachnoid space. Outcomes were evaluated at 3, 6, 9 and 12 months after surgery through clinical, urodynamic, neurophysiological and neuroimaging studies. RESULTS: Cell transplantation is a safe procedure. All patients experienced improvement, primarily in sensitivity and sphincter control. Infralesional motor activity, according to clinical and neurophysiological studies, was obtained by more than 50% of the patients. Decreases in spasms and spasticity, and improved sexual function were also common findings. Clinical improvement seems to be dose-dependent but was not influenced by the chronicity of the SCI. CONCLUSION: Personalized cell therapy with MSCs is safe and leads to clear improvements in clinical aspects and quality of life for patients with complete and chronically established paraplegia.

Cell therapy with bone marrow stromal cells after intracerebral hemorrhage: impact of platelet-rich plasma scaffolds.

BACKGROUND AIMS: Cell therapy using bone marrow stromal cells (BMSCs) has been considered a promising strategy for neurologic sequelae after intracerebral hemorrhage (ICH). However, after intracerebral administration of BMSCs, most of the cells die, partly because of the absence of extracellular matrix. Intracerebral transplantation of BMSCs, supported in a platelet-rich plasma (PRP) scaffold, optimizes this type of cell therapy. METHODS: ICH was induced by stereotactic injection of 0.5 IU of collagenase type IV in the striatum of adult Wistar rats (n = 40). Two months later, the rats were subjected to intracerebral administration of 5 × 10(6) allogeneic BMSCs embedded in a PRP scaffold (n = 10), 5 × 10(6) allogeneic BMSCs in saline (n = 10), PRP-derived scaffold only (n = 10) or saline only (n = 10). Functional improvements in each group over the next 6 months were assessed using Rotarod and Video-Tracking-Box tests. Endogenous neurogenesis and survival of transplanted BMSCs were examined at the end of follow-up. RESULTS: Our study demonstrated neurologic improvement after BMSC transplantation and significantly better functional improvement for the group of animals that received BMSCs in the PRP-derived scaffold compared with the group that received BMSCs in saline. Histologic results showed that better functional outcome was associated with strong activation of endogenous neurogenesis. After intracerebral administration of BMSCs, donor cells were integrated in the injured tissue and showed phenotypic expression of glial fibrillary acidic protein and neuronal nucleus. CONCLUSIONS: PRP-derived scaffolds increase the viability and biologic activity of BMSCs and optimize functional recovery when this type of cell therapy is applied after ICH.

The severity of brain damage determines bone marrow stromal cell therapy efficacy in a traumatic brain injury model.

BACKGROUND: Patients who survive traumatic brain injury (TBI) can undergo serious sensorial and motor function deficits. Once damage occurs, there is no effective treatment to bring patients to full recovery. Recent studies, however, show bone marrow stromal cells (BMSC) as a potential therapy for TBI. METHODS: This study was designed to determine whether the degree of neurologic deficits influences the efficacy of cell therapy using intracerebral transplantation of BMSC in an experimental model of chronically established TBI. Adult Wistar rats were subjected to weight-drop impact causing TBI. Two months later, the animals were classified according to levels of neurologic deficits. To achieve this, we used two different functional tests: the modified Neurologic Severity Score test and internal zone Permanence Time in Video-Tracking-Box analysis. Saline only or saline containing BMSC was injected into injured brain tissue of the animals that were classified having moderate or severe neurologic damage depending on the level of established functional deficits. All experimental groups were evaluated in the course of the following 2 months to study the efficacy of BMSC administration. The animals were then killed and their brains were studied. RESULTS: Our results showed that significant functional improvement was seen when BMSC was injected into animals with moderate brain damage, but no significant improvement was found in animals with severe functional deficits when compared with controls. CONCLUSION: These findings suggest that the severity of neurologic damage may determine the potential effect of cell therapy when applied to chronically established TBI.

Failure of delayed intravenous administration of bone marrow stromal cells after traumatic brain injury.

Traumatic brain injury (TBI) is a leading cause of mortality and morbidity worldwide. Currently, there is no effective strategy to treat the functional sequelae associated with TBI. Experimental evidence shows that the intravenous administration of bone marrow stromal cells (BMSC) during the first week after TBI prevents neurological deficits, but no experimental studies have shown evidence of the effect of intravenous BMSC on chronic brain injury sequelae. Here we studied the effect of intravenous administration of BMSC on functional outcomes 2 months after experimental TBI in rats. Adult Wistar rats were subjected to weight-drop impact causing severe brain injury, and 2 months later BMSC in saline, or saline alone, was intravenously injected. All experimental groups were evaluated by means of the modified Neurological Severity Score (mNSS), and internal zone Permanence Time (izPT) after video-tracking box (VTB) analysis, over the following 2 months to test the efficacy of BMSC therapy. At the end of the study period the animals were sacrificed and their brains were studied to evaluate possible differences between groups. Two months after BMSC administration no significant differences were detected in the motor and sensory evaluation between animals treated with BMSC and controls, and no differences were detected after histological study of the brains. Our present results suggest that intravenous administration of BMSC after TBI, when the neurological deficits are well established, has no beneficial effect in neurological outcomes or on brain tissue.

Allogeneic bone marrow stromal cell transplantation after cerebral hemorrhage achieves cell transdifferentiation and modulates endogenous neurogenesis.

BACKGROUND AIMS: When a severe neurologic lesion occurs as a consequence of intracerebral hemorrhage (ICH), there is no effective treatment available for improving the outcome. However, cell therapy has opened new perspectives on reducing neurologic sequels subsequent to this disease. METHODS: In this study, ICH was induced by stereotactic injection of 0.5 U collagenase type IV in the striatum of adult Wistar rats, and 2 h later a group of animals (n = 48) was subjected to intracerebral injection of 2 × 10(6) allogeneic bone marrow stromal cells (BMSC), while a control group (n = 48) received saline only. Eight animals from each group were killed at 48 h, 72 h, 7 days, 14 days, 21 days and 28 days. At these time-points, endogenous neurogenesis and survival of transplanted BMSC were studied. RESULTS: Our findings show that after allogeneic BMSC transplantation, donor cells can survive in the brain tissue expressing neuronal and astroglial markers. Furthermore, BMSC transplantation enhances endogenous neurogenesis and inhibits apoptosis of newborn neural cells. CONCLUSIONS: Although these results should be extrapolated to human disease with caution, it is obvious that cell therapy using allogeneic BMSC transplantation offers great promise for developing novel and efficacious strategies in patients suffering ICH.

TP53 induced glycolysis and apoptosis regulator (TIGAR) knockdown results in radiosensitization of glioma cells.

BACKGROUND AND PURPOSE: The TP53 induced glycolysis and apoptosis regulator (TIGAR) functions to lower fructose-2,6-bisphosphate (Fru-2,6-P(2)) levels in cells, consequently decreasing glycolysis and leading to the scavenging of reactive oxygen species (ROS), which correlate with a higher resistance to cell death. The decrease in intracellular ROS levels in response to TIGAR may also play a role in the ability of p53 to protect from the accumulation of genomic lesions. Given these good prospects of TIGAR for metabolic regulation and p53-response modulation, we analyzed the effects of TIGAR knockdown in U87MG and T98G glioblastoma-derived cell lines. METHODS/RESULTS: After TIGAR-knockdown in glioblastoma cell lines, different metabolic parameters were assayed, showing an increase in Fru-2,6-P(2), lactate and ROS levels, with a concomitant decrease in reduced glutathione (GSH) levels. In addition, cell growth was inhibited without evidence of apoptotic or autophagic cell death. In contrast, a clear senescent phenotype was observed. We also found that TIGAR protein levels were increased shortly after irradiation. In addition, avoiding radiotherapy-triggered TIGAR induction by gene silencing resulted in the loss of capacity of glioblastoma cells to form colonies in culture and the delay of DNA repair mechanisms, based in γ-H2AX foci, leading cells to undergo morphological changes compatible with a senescent phenotype. Thus, the results obtained raised the possibility to consider TIGAR as a therapeutic target to increase radiotherapy effects. CONCLUSION: TIGAR abrogation provides a novel adjunctive therapeutic strategy against glial tumors by increasing radiation-induced cell impairment, thus allowing the use of lower radiotherapeutic doses.

Late transplantation of allogeneic bone marrow stromal cells improves neurologic deficits subsequent to intracerebral hemorrhage.

BACKGROUND AIMS: Stem cell therapy seems to be a promising therapeutic tool for treating central nervous system (CNS) injuries. Bone marrow stromal cell (BMSC) transplantation influences functional outcome subsequent to intracerebral hemorrhage (ICH), and enhances endogenous neurogenesis in acute condition studies. We investigated whether late administration of BMSC improves functional deficits subsequent to ICH. METHODS: Experimental ICH was induced by stereotactic injection of 0.5 IU collagenase type IV in the striatum of adult female Wistar rats, and 2 months later intralesional administration of 5 × 10(6) allogeneic BMSC from male donors rats in saline (n = 10), or saline only (n = 10), was performed. In the following 6 months, functional outcome was evaluated in each animal by rotarod, modified neurologic severity score (mNSS) and video-tracking box (VTB) tests. To study the behavior of BMSC after transplantation, in situ hybridization studies were performed, with double labeling of the chromosome Y-linked SrY-gene, and neuronal nuclei (NeuN) protein or gliofibrillary acidic protein (GFAP). RESULTS: The assessment test revealed significant improvements in functional outcome for the BMSC-treated animals after 2 months of follow-up. Histologic results showed that functional outcome was associated with strong reactivation of endogenous neurogenesis. Furthermore, intralesional BMSC not only integrated in the injured tissue but also showed phenotypic expression of GFAP and NeuN. CONCLUSIONS: Late intracerebral transplantation of allogeneic BMSC induces functional recovery after ICH. The possibility of using this type of cell therapy to reverse the consequences of hemorrhagic stroke in humans should be considered.

Relevance of viral context and diversity of antigen-processing routes for respiratory syncytial virus cytotoxic T-lymphocyte epitopes.

Antigen processing of respiratory syncytial virus (RSV) fusion (F) protein epitopes F85-93 and F249-258 presented to cytotoxic T-lymphocytes (CTLs) by the murine major histocompatibility complex (MHC) class I molecule Kd was studied in different viral contexts. Epitope F85-93 was presented through a classical endogenous pathway dependent on the transporters associated with antigen processing (TAP) when the F protein was expressed from either RSV or recombinant vaccinia virus (rVACV). At least in cells infected with rVACV encoding either natural or cytosolic F protein, the proteasome was required for epitope processing. In cells infected with rVACV encoding the natural F protein, an additional endogenous TAP-independent presentation pathway was found for F85-93. In contrast, epitope F249-258 was presented only through TAP-independent pathways, but presentation was brefeldin A sensitive when the F protein was expressed from RSV, or mostly resistant when expressed from rVACV. Therefore, antigen-processing pathways with different mechanisms and subcellular localizations are accessible to individual epitopes presented by the same MHC class I molecule and processed from the same protein but in different viral contexts. This underscores both the diversity of pathways available and the influence of virus infection on presentation of epitopes to CTLs.

Virus-specific effector CD4+ T-cell responses in hemodialysis patients with hepatitis C virus infection.

Patients with chronic renal failure undergoing hemodialysis who are infected with hepatitis C virus (HCV) may test consistently anti-HCV negative. Because CD4(+) T-cells provide help for antibody production virus-specific effector CD4(+) T-cell responses were investigated in relation to anti-HCV positivity in 15 hemodialysis patients grouped according to HCV antibody and viremia. CD4(+) T-cell reactivity was studied in peripheral blood mononuclear cells by standard lymphocyte proliferation assay and phenotypic/functional characterization (cell-surface staining/cytokine secretion) by flow cytometry. HCV-specific CD4(+) T-cell proliferation in viremic hemodialysis patients was weak or absent independently of their anti-HCV status. Virus-specific CD4(+) T-cells displayed a memory phenotype and showed low to undetectable capacity to secrete effector interferon (IFN)-gamma. Impaired activation-induced cytokine secretion appeared to be Th1 (IFN-gamma) but not Th2 (interleukin-4)-directed and was virus-specific as cytomegalovirus responses were preserved. The frequency ex vivo of CD3(+)CD4(+)IFN-gamma(+) T-cells was independent of the HCV antibody status and comparable between viremic (range: 0.08-1.54%) or non-viremic (0.11-3.2%) hemodialysis patients and healthy donors (0.13-1.10%; P = 0.58). The numbers of CD3(+)CD4(+)IFN-gamma(+) T-cells augmented slightly (P = 0.047) in HCV-infected hemodialysis patients but markedly in only one (greater than ninefold) after HCV stimulation. In conclusion, hemodialysis patients show limited HCV-specific effector CD4(+) Th1-cell responses which nonetheless seem unrelated to the anti-HCV status and are not more impaired due to the ongoing hemodialysis.

Features of the CD4+ T-cell response in liver and peripheral blood of hepatitis C virus-infected patients with persistently normal and abnormal alanine aminotransferase levels.

BACKGROUND/AIMS: The liver is the primary site of hepatitis C virus (HCV) replication; intrahepatic T-cell responses may influence liver disease severity. METHODS: HCV-specific CD4(+) T-cell reactivity was investigated ex vivo in paired liver tissue and peripheral blood from 42 chronic HCV patients. RESULTS: The frequencies with which HCV-specific HLA class-II-restricted CD4(+) T-cell proliferation were observed were 29% in liver and 36% in peripheral blood. Among responses, non-structural-3 protein (NS3)-specific T-cell proliferation was dominant but non-exclusive and did rarely occur concurrently in liver infiltrate and peripheral blood suggesting liver compartmentalization of a CD4(+) T-cells population. Compared with 24 patients with abnormal ALT levels, 18 HCV carriers with persistently normal ALT levels had similar serum and liver viral loads but showed: (i) a low-activity grade and stage chronic hepatitis (P<0.001); (ii) less intrahepatic CD4(+) T-lymphocytes (P<0.01); (iii) less frequent intrahepatic (17 vs. 33%) and peripheral (17 vs. 38%) NS3-specific CD4(+) T-cell proliferation; (iv) less often in vitro T-helper (Th)1 (interferon-gamma) cytokine production (2 vs. 18%; P<0.001). CONCLUSIONS: Our data show a low frequency of intrahepatic HCV-specific HLA class-II-restricted CD4(+) Th1 responses in patients with chronic HCV. However, these Th1 responses are detected more often in those patients with overt clinical and histological disease.