A simple method for identifying bone marrow mesenchymal stromal cells with a high immunosuppressive potential

Background aimsThe beneficial activity of mesenchymal stromal cells (MSC) in allogeneic hematopietic stem cell transplantation requires correct use in terms of cell dose and timing of infusion and the identification of biomarkers for selection. The immunosuppressive bone marrow (BM)-derived MSC (BM-MSC) functions have been associated with the production of soluble HLA-G molecules (sHLA-G) via interleukin (IL)-10. We have established a reliable method for evaluating BM-MSC HLA-G expression without the influence of peripheral blood mononuclear cells (PBMC).MethodsThirteen BM-MSC from donors were activated with recombinant IL-10 or co-cultured with 10 different phytohemagglutinin (PHA)-treated PBMC (PHA-PBMC). Membrane-bound and sHLA-G expression was evaluated by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively; lymphoproliferation was measured by (methyl-³H)thymidine.ResultsThe results demonstrated the ability of IL-10 to stimulate both membrane-bound and sHLA-G production by BM-MSC. The levels of HLA-G expression induced by IL-10 in BM-MSC were associated with the inhibition of PHA-PBMC proliferation (sHLA-G, P = 0.0008, r = 0.9308; membrane HLA-G, P = 0.0005, r = 0.9502).ConclusionsWe propose the evaluation of sHLA-G production in IL-10-treated BM-MSC cultures as a possible marker of immunoregulatory function.     

Lifespan of human amniotic fluid-derived multipotent mesenchymal stromal cells

Background aimsHuman multipotent mesenchymal stromal cells (hMSC) have become one of the main interests in regenerative medicine because of their ability to differentiate into different lineages. Human amniotic fluid is reported to contain MSC (hAMSC) and therefore may be a useful source of cells for clinical applications. However, our understanding of the behavior of these cells in indefinite in vitro culture conditions is very limited.MethodsWe systematically evaluated and characterized, throughout their whole lifespan, the expansion potential, chromosomal stability, surface and intracellular phenotype and differentiation potential of fibroblastoid hAMSC (F-type hAMSC).ResultsNine F-type hAMSC cultures could be expanded in in vitro culture conditions for 223.25 ± 24.44 days (mean ± SD), during which time 28.96 ± 1.5 passages were made giving rise to 54.95 ± 3.17 population doublings (PD) and an estimated number of accumulated cells of between 1.0 × 10²² and 9.7 × 10²³, with no visible alterations in the chromosome during their lifespan. All the cultures showed unchanged percentages of strongly positive expressions of the surface markers CD29, CD44, CD73, CD90, CD95, CD105 and HLA-ABC, as well as the embryonic intracellular markers Nanog and Sox2, during their lifespan, whereas the expression of the embryonic surface markers SSEA3, SSEA4, TRA-1-60 and TRA-1-81 fell until it disappeared with progression of the culture. These cells retained their differentiation capacities to adipogenic, chondrogenic and osteogenic lineages throughout their lifespan.ConclusionsF-type hAMSC exhibit reproducible biologic characteristics, confirming that these cells are ideal candidates for use in regenerative medicine.     

Cryopreservation of umbilical cord mesenchymal cells in xenofree conditions

Background aimsMesenchymal stromal cells (MSC) are being used to treat and prevent a variety of clinical conditions. To be readily available, MSC must be cryopreserved until infusion. However, the optimal cryopreservation methods, cryoprotector solutions and MSC sensitivity to dimethyl sulfoxide (DMSO) exposure are unknown. This study investigated these issues.MethodsMSC samples were obtained from human umbilical cord (n = 15), expanded with Minimal Essential Medium-alpha (α-MEM) 10% human serum (HS), resuspended in 25 mL solution (HS, 10% DMSO, 20% hydroxyethyl starch) and cryopreserved using the BioArchive^® system. After a mean of 18 ± 7 days, cell suspensions were thawed and diluted until a DMSO concentration of 2.5% was reached. Samples were tested for cell quantification and viability, immunophenotype and functional assays.ResultsPost-thaw cell recovery: 114 ± 2.90% (mean ± SEM). Recovery of viable cells: 93.46 ± 4.41%, 90.17 ± 4.55% and 81.03 ± 4.30% at 30 min, 120 min and 24 h post-thaw, respectively. Cell viability: 89.26 ± 1.56%, 72.71 ± 2.12%, 70.20 ± 2.39% and 63.02 ± 2.33% (P < 0.0001) pre-cryopreservation and 30 min, 120 min and 24 h post-thaw, respectively. All post-thaw samples had cells that adhered to culture bottles. Post-thaw cell expansion was 4.18 ± 0.17 ×, with a doubling time of 38 ± 1.69 h, and their capacity to inhibit peripheral blood mononuclear cells (PBMC) proliferation was similar to that observed before cryopreservation. Differentiation capacity, cell-surface marker profile and cytogenetics were not changed by the cryopreservation procedure.ConclusionsA method for cryopreservation of MSC in bags, in xenofree conditions, is described that facilitates their clinical use. The MSC functional and cytogenetic status and morphologic characteristics were not changed by cryopreservation. It was also demonstrated that MSC are relatively resistant to exposure to DMSO, but we recommend cell infusion as soon as possible.     

Intramyocardial and intracoronary autologous bone marrow-derived mesenchymal stromal cell treatment in chronic severe dilated cardiomyopathy

Background aimsMesenchymal stromal cells (MSC) may improve cardiac function following myocardial infarction. MSC can differentiate into cardiomyocytes and endothelial cells while exerting additional paracrine effects. There is limited information regarding the efficacy of route for MSC treatment of severe dilated cardiomyopathy (DCM). The aim of this study was to demonstrate the clinical safety, feasibility and efficacy of direct intramyocardial and intracoronary administration of autologous bone marrow-derived MSC treatment for no-option patients with chronic severe refractory DCM.MethodsTen symptomatic patients with DCM and refractory cardiac function, despite maximum medical therapy, were selected. Five had ischemic DCM deemed unlikely to benefit from revascularization alone and underwent bypass operations with concurrent intramyocardial MSC injection (group A). Two patients had previous revascularization and three had non-ischemic DCM and received intracoronary MSC injection (group B).ResultsGroup A and B patients received 0.5–1.0 × 10⁶ and 2.0–3.0 × 10⁶ MSC/kg body weight, respectively. All patients remained alive at 1 year. There were significant improvements from baseline to 6 and 12 months in left ventricular ejection fraction and other left ventricular parameters. Scar reduction was noted in six patients by 12 months.ConclusionsAutologous bone marrow MSC treatment is safe and feasible for treating chronic severe refractory DCM effectively, via intracoronary or direct intramyocardial administration at prescribed doses.     

Mesenchymal stromal cells prolong the lifespan in a rat model of amyotrophic lateral sclerosis

Background aimsAmyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by the loss of brain and spinal cord motor neurons (MN). The intraspinal and systemic grafting of mesenchymal stromal cells (MSC) was used to treat symptomatic transgenic rats overexpressing human superoxide dismutase 1 (SOD1) in order to alleviate the disease course and prolong the animals’ lifespan.MethodsAt the age of 16 weeks (disease onset) the rats received two grafts of MSC expressing green fluorescent protein (GFP⁺ MSC) on the same day, intraspinally (10⁵ cells) and intravenously (2 × 10⁶ cells). Sham-treated animals were injected with phosphate-buffered saline (PBS). Motor activity, grip strength and body weight were tested, followed by immunohistochemical analysis.ResultsThe combined grafting of MSC into symptomatic rats had a significant effect on motor activity and grip strength starting 4 weeks after transplantation. The lifespan of animals in the treated group was 190 ± 3.33 days compared with 179 ± 3.6 days in the control group of animals. Treated rats had a larger number of MN at the thoracic and lumbar levels; these MN were of larger size, and the intensity of terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining in the somas of apoptotic MN at the thoracic level was much lower than in sham-treated animals. Transplanted GFP⁺ MSC survived in the spinal cord until the end stage of the disease and migrated both rostrally and caudally from the injection site.ConclusionsIntraspinal and intravenous transplantation of MSC has a beneficial and possibly synergistic effect on the lifespan of ALS animals.     

Investigation of porcine circovirus contamination in human vaccines

DNA from porcine circovirus type 1 (PCV1) and 2 (PCV2) has recently been detected in two vaccines against rotaviral gastroenteritis from manufacturers A and B. We investigated if PCV1 sequences are present in other viral vaccines. We screened seeds, bulks and final vaccine preparations from ten manufacturers using qRT-PCR. We detected 3.8 × 10³ to 1.9 × 10⁷ PCV1 DNA copies/milliliter in live poliovirus seeds for inactivated polio vaccine (IPV) from manufacturer A, however, following inactivation and purification, the finished IPV was PCV1-negative. PCV1 DNA was not detectable in live polio preparations from other vaccine producers. There was no detectable PCV1 DNA in the measles, mumps, rubella and influenza vaccines analysed including material supplied by manufacturer A. We confirmed that the PCV1 genome in the rotavirus vaccine from manufacturer A is near full-length. It contains two mutations in the PCV cap gene, which may result from viral adaptation to Vero cells. Bulks of this vaccine contained 9.8 × 10¹⁰ to 1.8 × 10¹¹ PCV1 DNA copies/millilitre and between 4.1 × 10⁷ and 5.5 × 10⁸ DNA copies were in the final doses. We found traces of PCV1 and PCV2 DNA in the rotavirus vaccine from manufacturer B. This highlights the issue of vaccine contamination and may impact on vaccine quality control.     

Gene expression profiles following intracoronary injection of mesenchymal stromal cells using a porcine model of chronic myocardial infarction

Background aimsWe evaluated the therapeutic potential of injection of in vitro differentiated bone marrow mesenchymal stromal cells (MSC) using a swine model.Methods and ResultsMyocardial infarction was induced by coronary occlusion. Three groups (n = 5 each) were analyzed: one group received an injection of 17.8 ± 9.3 × 10⁶ 5-azacytidine-treated allogeneic MSC 1 month after infarction; a placebo group received an injection of medium; and controls were kept untreated. After 4 weeks, heart samples were taken from three infarcted areas, interventricular septa, ventricles and atria. Gene expression profiles of genes related to contractility (Serca2a), fibrosis (Col1a1), cardiomyogenesis (Mef2c, Gata4 and Nkx2.5) and mobilization of stem cells (Sdf1, Cxcr4 and c-kit) were compared by quantitative real-time PCR (qRT-PCR). Gene expression profiles varied in different heart areas. Thus Serca2a expression was reduced in infarcted groups in all heart regions except for the left ventricles, where Col1a1 was overexpressed. The expression of genes related to cardiomyogenesis decreased in the infarcted zones and left atria compared with healthy hearts. Interestingly, increased expression of Cxcr4 was detected in infarcted regions of MSC-treated pigs compared with the placebo groupConclusionsInfarction induced changes in expression of genes involved in various biologic processes. Genes involved in cardiomyogenesis were downregulated in the left atrium. The intracoronary injection of MSC resulted in localized changes in the expression of Cxcr4.     

Cryopreserved mobilized autologous blood progenitors stored for more than 2 years successfully support blood count recovery after high-dose chemotherapy

Background aimsThe ability of hematopoietic progenitor cells–apheresis (HPC-A) that have been stored for many years after cryopreservation to reconstitute hematopoiesis following high-dose chemo/radiotherapy has not been well-documented.MethodsIn this retrospective study, eight Canadian centers contributed data from 53 autologous stem cell transplants (ASCT) performed using HPC-A that had undergone long-term storage (>2 years, range 2–7 years) and 120 ASCT using HPC-A stored for <6 months (short-term storage).ResultsThe doses of nucleated and CD34⁺ cells per kilogram recipient weight were similar between the short- (mean ± SD, 4.7 ± 4.9 × 10⁸ and 6.8 ± 4.3 × 10⁶, respectively) and long- (4.0 ± 4.9 × 10⁸ and 6.1 ± 3.4 × 10⁶, respectively) term storage groups. The median days to neutrophils (absolute neutrophil count; ANC) >0.5 × 10⁹/L (median 11 days for both short- and long-term storage) and platelets >20 × 10⁹/L (median 12 and 11 for short- and long-term storage, respectively) post-ASCT were not significantly different between the two groups. When ASCT performed with <5 × 10⁶/kg CD34⁺ cells was compared there was also no difference in ANC or platelet recovery (median 12 days for both after short-term storage, and 12 and 11 days, respectively, after long-term storage). Fourteen HPC-A products stored for >5 years also showed similar count recoveries as the entire long-term storage group (median 11 days for both ANC and platelets).ConclusionsCryopreserved HPC-A can be stored for at least 5 years with no apparent loss in their ability to support hematopoietic reconstitution after high-dose chemotherapy.     

Clinical analysis of the treatment of spinocerebellar ataxia and multiple system atrophy-cerebellar type with umbilical cord mesenchymal stromal cells

Background aimsThe aims of this study were to observe the safety and effectiveness of umbilical cord mesenchymal stromal cells (UC-MSC) in the treatment of spinocerebellar ataxia (SCA) and multiple system atrophy-cerebellar type (MSA-C).MethodsFrom October 2009 to September 2010, 14 cases of SCA and 10 cases of MSA-C were given UC-MSC by weekly intrathecal injection, at a dose of 1 × 10⁶/kg four times as one course. All the patients received one course of treatment, except three patients who received two courses. The movement ability and quality of daily life were evaluated with the International Cooperative Ataxia Rating Scale (ICARS) and Activity of Daily Living Scale (ADL) and the scores compared with those before cell therapy. A follow-up of 6–15 months was carried out for all of the patients.ResultsThe results showed that the ICARS and ADL scores were significantly decreased 1 month after treatment (P < 0.01). The symptoms, including unstable walking and standing, slow movement, fine motor disorders of the upper limbs, writing difficulties and dysarthria, were greatly improved except for one patient, who had no response. The observed side-effects included dizziness (four patients), back pain (two cases) and headache (one case), which disappeared within 1–3 days. During the follow-up, 10 cases remained stable for half a year or longer, while 14 cases had regressed to the status prior to the treatment within 1–14 months (an average of 3 months).ConclusionsIntrathecal injection of UC-MSC is safe and can delay the progression of neurologic deficits for SCA and MSA-C patients.     

Influence of related donor age on outcomes after peripheral blood stem cell transplantation

Background aimsThe rising use of allogeneic transplantation in older recipients necessitates considering older related donors. The effect of related donor age for peripheral blood stem cell allografts (PBSC) on graft maintenance and outcomes, independent of CD34⁺cell dose, has not been well-characterized.MethodsHLA-related donors (98% siblings) underwent a uniform filgrastim-based mobilization regimen aiming to collect and infuse 5 × 10⁶ CD34⁺ cells/recipient kg. Donor and recipient age were modeled in multiple ways to account for the correlation, and outcomes reported by decade of donor age.ResultsThe median donor and recipient ages were 52 years and 54 years, respectively. The mean CD34⁺ cell dose infused was 5.6 × 10⁶ CD34⁺/kg and 75% of patients received a narrow range between 4.4 and 6.6 × 10⁶ CD34⁺ cells/kg. Neither better PBSC mobilization nor higher CD34⁺ content of allografts was significantly associated with engraftment or transplant outcomes. After adjusting for recipient age and other prognostic factors, older donor age by decade conferred a lower risk of non-relapse mortality (NRM) [hazard ratio (HR) = 0.64, 95% confidence interval (CI) 0.45–0.91, P = 0.013] and borderline improvement in overall survival (OS) (HR = 0.76, 95% CI 0.58–0.99, P = 0.045) without altering progression-free survival (PFS) (HR = 0.85, 95% CI 0.66–1.07, P = 0.18).ConclusionsOlder donor age does not worsen outcome after matched related donor PBSC transplantation in patients receiving a narrow range CD34⁺ cells. The relatively small sample size mandates that the finding of similar to improved outcomes for older related donor age must be confirmed in larger studies.     

Single-platform quality control assay to quantify multipotential stromal cells in bone marrow aspirates prior to bulk manufacture or direct therapeutic use

Background aimsThe manufacture of multipotential stromal cell (MSC)-based products is costly; therefore, a rapid evaluation of bone marrow (BM) ‘quality’ with respect to MSC content is desirable. The aim of this study was to develop a rapid single-platform assay to quantify MSC in BM aspirates.MethodsAspirated MSC were enumerated using the CD45^?/low CD271^bright phenotype and AccuCheck counting beads and compared with a classic colony-forming unit–fibroblast (CFU-F) assay. The phenotype of CD45^?/low CD271^bright cells was defined using a range of MSC (CD73, CD105, CD90) and non-MSC (CD31, CD33, CD34, CD19) markers. The effect of aspirated BM volume on MSC yield was also determined.ResultsCD45^?/low CD271^bright cells had a classic MSC phenotype (CD73⁺ CD105⁺ CD90⁺ ). Their numbers correlated positively with CFU-F counted manually (R = 0.81, P < 0.001) or using automatic measurements of surface area occupied by colonies (R = 0.66, P < 0.001). Simultaneous enumeration of CD34 ⁺ cells revealed donor variability ranges compatible with standard International Society of Hematotherapy and Graft Engineering (ISHGE) protocols. Aspirating larger marrow volumes gave a significant several-fold reduction in the frequency of CFU-F and CD45^?/low CD271^bright cells per milliliter. Therefore aspirated MSC yields can be maximized through a standardized, low-volume harvesting technique.ConclusionsAbsolute quantification of CD45^?/low CD271^bright cells was found to be a reliable method of predicting CFU-F yields in BM aspirates. This rapid (< 40 min) procedure could be suitable for intra-operative quality control of BM aspirates prior to volume reduction/direct injection in orthopedics. In the production of culture-expanded MSC, this assay could be used to exclude samples containing low numbers of MSC, resulting in improved consistency and quality of manufactured MSC batches.     

A clinically adaptable method to enhance the cytotoxicity of natural killer cells against B-cell malignancies

Background aimsRetroviral transduction of anti-CD19 chimeric antigen receptors significantly enhances the cytotoxicity of natural killer (NK) cells against B-cell malignancies. We aimed to validate a more practical, affordable and safe method for this purpose.MethodsWe tested the expression of a receptor containing CD3ζ and 4-1BB signaling molecules (anti-CD19-BB-ζ) in human NK cells after electroporation with the corresponding mRNA using a clinical-grade electroporator. The cytotoxic capacity of the transfected NK cells was tested in vitro and in a mouse model of leukemia.ResultsMedian anti-CD19-BB-ζ expression 24 h after electroporation was 40.3% in freshly purified (n =18) and 61.3% in expanded (n = 31) NK cells; median cell viability was 90%. NK cells expressing anti-CD19-BB-ζ secreted interferon (IFN)-γ in response to CD19-positive target cells and had increased cytotoxicity. Receptor expression was detectable 6 h after electroporation, reaching maximum levels at 24–48 h; specific anti-CD19 cytotoxicity was observed at 96 h. Levels of expression and cytotoxicities were comparable with those achieved by retroviral transduction. A large-scale protocol was developed and applied to expanded NK cells (median NK cell number 2.5 × 10⁸, n = 12). Median receptor expression after 24 h was 82.0%; NK cells transfected under these conditions exerted considerable cytotoxicity in xenograft models of B-cell leukemia.ConclusionsThe method described here represents a practical way to augment the cytotoxicity of NK cells against B-cell malignancies. It has the potential to be extended to other targets beyond CD19 and should facilitate the clinical use of redirected NK cells for cancer therapy.     

Characterization of Paecilomycescinnamomeus from the camellia whitefly, Aleurocanthus camelliae (Hemiptera: Aleyrodidae), infesting tea in Japan

The whitefly, Aleurocanthus camelliae Kanmiya and Kasai (Hemiptera: Aleyrodidae), is an invasive species in Japan that was first discovered in 2004 on tea in Kyoto. Soon after its arrival epizootics of an entomopathogenic fungus were observed in populations of the whitefly in many tea-growing regions. Here we identify this fungus as Paecilomyces cinnamomeus (Petch) Samson and W. Gams (Hypocreales: Clavicipitaceae) based on morphological characteristics and molecular analyses. This is the first record of P. cinnamomeus in Japan and also the first time it has been recorded from the genus Aleurocanthus. A isolate of P. cinnamomeus caused greater than 50% and 90% infection in whitefly nymphs at 1 × 10⁶ and 1 × 10⁷ conidia/ml respectively, while the commercial mycoinsecticides Preferd® (Isaria fumosorosea) and Mycotal® (Lecanicillium muscarium) caused <10% infection at their recommended field rates (5 × 10⁶ and 9 × 10⁶ conidia/ml, respectively), suggesting that P. cinnamomeus may be more useful as a control agent than the currently available mycoinsecticides. Optimum and upper limit temperatures for in vitro growth of P. cinnamomeus isolates were 22.5–25 °C and 32.5 °C, respectively. At field rates, the fungicide thiophanate-methyl caused some inhibition of in vitro growth of P. cinnamomeus isolates, and the bactericide copper oxychloride and the insecticides tolfenpyrad and methidathion were strongly inhibitory. The findings obtained in this study will be useful in the development of microbial control programs using P. cinnamomeus against A. camelliae.     

Development and validation of a procedure to isolate viable bone marrow cells from the vertebrae of cadaveric organ donors for composite organ grafting

Background aimsDonor-derived vertebral bone marrow (BM) has been proposed to promote chimerism in solid organ transplantation with cadaveric organs. Reports of successful weaning from immunosuppression in patients receiving directed donor transplants in combination with donor BM or blood cells and novel peri-transplant immunosuppression has renewed interest in implementing similar protocols with cadaveric organs.MethodsWe performed six pre-clinical full-scale separations to adapt vertebral BM preparations to a good manufacturing practice (GMP) environment. Vertebral bodies L4–T8 were transported to a class 10 000 clean room, cleaned of soft tissue, divided and crushed in a prototype bone grinder. Bone fragments were irrigated with medium containing saline, albumin, DNAse and gentamicin, and strained through stainless steel sieves. Additional cells were eluted after two rounds of agitation using a prototype BM tumbler.ResultsThe majority of recovered cells (70.9 ± 14.1%, mean ± SD) were eluted directly from the crushed bone, whereas 22.3% and 5.9% were eluted after the first and second rounds of tumbling, respectively. Cells were pooled and filtered (500, 200 μm) using a BM collection kit. Larger lumbar vertebrae yielded about 1.6 times the cells of thoracic vertebrae. The average product yielded 5.2 ± 1.2 × 10¹⁰ total cells, 6.2 ± 2.2 × 10⁸ of which were CD45⁺ CD34⁺. Viability was 96.6 ± 1.9% and 99.1 ± 0.8%, respectively. Multicolor flow cytometry revealed distinct populations of CD34⁺ CD90⁺ CD117^dim hematopoietic stem cells (15.5 ± 7.5% of the CD34 ⁺ cells) and CD45^? CD73⁺ CD105⁺ mesenchymal stromal cells (0.04 ± 0.04% of the total cells).ConclusionsThis procedure can be used to prepare clinical-grade cells suitable for use in human allotransplantation in a GMP environment.     

Adoptive immunotherapy with cytokine-induced killer cells generated with a new good manufacturing practice-grade protocol

Background aimsWe have recently shown that thymoglobulin (TG) efficiently expands cytokine-induced killer (CIK) cells in combination with interferon (IFN)-γ and interleukin (IL)-2 (ITG2 protocol). It is presently unknown whether the infusion of autologous immune effector cells generated by TG, IFN-γ and IL-2 is feasible and safeMethodsFive patients with advanced and/or refractory solid tumors were enrolled in the present phase I/II study. Peripheral blood mononuclear cells (PBMC) collected by leukapheresis were stimulated under good manufacturing practice (GMP)-conditions with IFN-γ, followed by TG and IL-2. After 2–3 weeks in culture, a median of 4.65 × 10⁶ immune effector cells per kilogram of recipient's body weight was obtained and infused intravenously. The median time from enrollment into the study to infusion of the expanded CIK cells was 30 daysResultsITG2 efficiently expanded immune effector cells that comprised both conventional natural killer (NK) cells and CD3⁺ CD16⁺ CD56⁺ CIK cells. One patient with advanced melanoma died because of disease progression before the infusion of CIK cells. The target dose of at least 2.5 × 10⁶ CIK cells/kg of recipient's body weight was reached in four out of five evaluable patients. CIK cells were administered intravenously without any measurable toxicity. In vitro, CIK cells exerted lytic activity against cervical cancer cells. The median survival was 4.5 months (range 1–13) from the first infusion of CIK cells.ConclusionsThis study has highlighted the feasibility and safety of the administration of CIK cells generated with the ITG2 protocol. Whether CIK cells can help control disease burden in patients with advanced malignancies will be determined in future clinical trials.     

Mesenchymal stromal cell-conditioned medium prevents radiation-induced small intestine injury in mice

Background aimsEffective therapy for radiation-induced intestinal injury is currently unavailable. Mesenchymal stromal cells (MSC) are expected to be useful in repairing intestinal damage caused by irradiation. We determined whether the MSC-derived bioactive components could protect radiation-induced small intestine injury in miceMethodsHuman umbilical cord (UC)-derived MSC were isolated, expanded and exposed to hypoxic conditions in vitro. The hypoxia-conditioned medium was ultrafiltrated with a 3-kDa molecular weight cut-off to prepare the high molecular weight fraction (HMWF). The effect of HMWF on the viability of irradiated rat intestinal epithelial cells (IEC-6) was examined by MTT(methyl thiazolyl tetrazolium) assay. HMWF was also delivered to BALB/C male mice by tail intravenous injection immediately after receiving local abdominal irradiation at a selected dose of 10 Gy. Animal body weight, survival and diarrhea were monitored for 30 days. The improvement of mice intestine structure, including epithelium thickness and villus height, was examined by histologyResultsHMWF enhanced the viability of irradiated IEC-6 cells in vitro. Repeated infusion of HMWF for 7 days immediately after abdominal irradiation of 10 Gy (⁶⁰Coγ-ray) increased the survival rate, decreased diarrhea occurrence and improved the small intestinal structural integrity of irradiated miceConclusionsMSC-derived bioactive components could be a novel therapeutic approach for the treatment of radiation-induced injury.     

Canine bone marrow mesenchymal stromal cells with lentiviral mHCN4 gene transfer create cardiac pacemakers

Background aimsThe study objective was to test the ability of canine mesenchymal stromal cells (cMSC) transfected with the mouse hyperpolarization-activated cyclic nucleotide-gated channel 4 (mHCN4) gene to deliver a biologic pacemaker to the canine heart.Methods and ResultscMSC that were transfected by lentiviral vector with the cardiac pacemaker gene mHCN4 expressed high levels of Cs⁺ -sensitive current (26.4 ± 1.8pA/pF at –140 mV; (n = 17) and were activated in the diastolic potential range with a reversal potential of –29.7 ± 2.5 mV (n = 14), confirming that the expressed current was Funny current (I_f)-like. Next, 3 × 10⁶ cMSC transfected with either control plasmid or the mHCN4 gene construct were injected subepicardially into the canine right ventricular wall in situ. During sinus arrest, all control hearts had spontaneous atrioventricular node rhythms [rate = 21 ± 5beats per minute (b.p.m.)]. In the mHCN4 group, six of eight animals developed spontaneous ventricular rhythms of right-sided origin (rate = 45 ± 9b.p.m.; P < 0.01). Moreover, immunohistochemical analysis of the injected regions demonstrated neither apoptosis nor cellular or humoral rejection at 2 weeks.ConclusionsThese results demonstrate that genetically modified cMSC can express functional HCN4 channels in vitro and in vivo and represent a novel delivery system for pacemaker genes into the heart.     

Feasibility and safety of adoptive immunotherapy with ex vivo-generated autologous, cytotoxic T lymphocytes in patients with solid tumor

Background aimsAdoptive T-cell therapy with tumor-specific T cells has emerged as a potentially useful approach for treating patients with advanced malignancies. We have demonstrated previously the feasibility of obtaining large numbers of autologous anti-tumor-specific cytotoxic T lymphocytes (CTL) generated by stimulation of patients' peripheral blood mononuclear cells with dendritic cells pulsed with apoptotic tumor cellsMethodsSix patients with progressing metastatic solid tumors (one renal cell carcinoma, two ovarian cancers, two extraosseous peripheral neuroectodermal tumors, one soft tissue sarcoma) not eligible for conventional therapies were treated with adoptive immunotherapy. Anti-tumor CTL, proven to be reactive in vitro against patient tumor cells, but not against normal cells, were infused following lymphodepleting chemotherapy administered to favor T-cell proliferation in vivo.ResultsPatients received a median of nine CTL infusions (range 2–19). The median number of CTL administered per infusion was 11 × 10⁸ (range 1–55 × 10⁸). No patient experienced acute or late adverse events related to CTL infusion, even when large numbers of cells were given. Post-infusion laboratory investigations demonstrated an increase in the frequency of circulating anti-tumor T-cells and, in patients with a longer follow-up receiving two CTL infusions/year, a stabilization of these values.ConclusionsOur study demonstrates that autologous ex vivo-generated anti-tumor CTL can be administered safely in patients with advanced solid tumors and can improve the immunologic reactivity of recipients against tumor. These preliminary results provide a rationale for evaluating the clinical efficacy of this immunotherapeutic approach in phase I/II studies.     

Extracellular alkalinization induces endothelium-derived nitric oxide dependent relaxation in rat thoracic aorta

AimTo investigate the mechanism through which the extracellular alkalinization promotes relaxation in rat thoracic aorta.MethodsThe relaxation response to NaOH-induced extracellular alkalinization (7.4–8.5) was measured in aortic rings pre-contracted with phenylephrine (Phe, 10^?6 M). The vascular reactivity experiments were performed in endothelium-intact and -denuded rings, in the presence or and absence of indomethacin (10^?5 M), NG-nitro-l-arginine methyl ester (L-NAME, 10^?4 M), N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide/HCl (W-7, 10^?7 M), 2,5-dimethylbenzimidazole (DMB, 2 × 10^?5 M) and methyl-β-cyclodextrin (10^?2 M). In addition, the effects of NaOH-induced extracellular alkalinization (pH 8.0 and 8.5) on the intracellular nitric oxide (NO) concentration was evaluated in isolated endothelial cells loaded with diaminofluorescein-FM diacetate (DAF-FM DA, 5 μM), in the presence and absence of DMB (2 × 10^?5 M).ResultsThe extracellular alkalinization failed to induce any change in vascular tone in aortic rings pre-contracted with KCl. In rings pre-contracted with Phe, the extracellular alkalinization caused relaxation in the endothelium-intact rings only, and this relaxation was maintained after cyclooxygenase inhibition; completely abolished by the inhibition of nitric oxide synthase (NOS), Ca²⁺/calmodulin and Na⁺/Ca²⁺ exchanger (NCX), and partially blunted by the caveolae disassembly.ConclusionsThese results suggest that, in rat thoracic aorta, that extracellular alkalinization with NaOH activates the NCX reverse mode of endothelial cells in rat thoracic aorta, thereby the intracellular Ca²⁺ concentration and activating the Ca²⁺/calmodulin-dependent NOS. In turn, NO is released promoting relaxation.     

Root health evaluation of three alfalfa varieties in Kerquin sandy land

Without a robust and healthy root system, establishment, productivity, and persistence are compromised. Consequently, research on alfalfa root morphology and health is very important in development of technology for efficient improvement and production of alfalfa. The objectives of this study were to evaluate the root morphology and health of three alfalfa varieties, Algonquin, Golden Queen, and Yellow Flower and to determine relationships among root morphology traits and root health. Yields from these varieties ranged from 5.83 to 43.93 t/ha, total root length ranged from 215.17 to 708.89 mm, root surface area from 124.95 to 468.37 cm², volume from 3.24 to 57.72 cm³, and forks from 1.25 × 10³ to 10.54 × 10³, and tips from 0.65 × 10³ to 3.17 × 10³. Root infestation score was negatively correlated with yield (r = ?0.997, P < 0.01), and was positively correlated with all root morphology traits (r = 0.466–0.997, P < 0.01), and yield was negatively associated with root morphology traits (r = ?0.755 to ?0.998, p < 0.01) with the exception of root tips (r = 0.448, P < 0.01). Results from these analyses indicated that root infestation score was the lowest averaged over age of alfalfa stand in Algonquin. Yield in 2-year old stands was greater in Golden Queen compared to the other two cultivars.