Nonprofit Business Plan Development: From Vision, Mission and Values to Implementation

Describes steps for nonprofit planning, with sections that cover organizational assessment, vision and mission statements, goal-setting, and plan implementation

Revista complutense de educación

Resumen basado en el de la publicaciónSe lleva a cabo una revisión general del procedimiento cloze, procedimiento que es ampliamente conocido y utilizado como instrumento de evaluación de la lectura en los países de habla inglesa pero que apenas es conocido y empleado en España. Dicha revisión hace referencia tanto a los aspectos metodológicos relacionados con dicho procedimiento como a los distintos usos para los que puede emplearse en el campo de la evaluación de la lectura.ES

Cloning and Characterization of a Bovine Adeno-Associated Virus

To better understand the relationship between primate adeno-associated viruses (AAVs) and those of other mammals, we have cloned and sequenced the genome of an AAV found as a contaminant in two isolates of bovine adenovirus that was reported to be serologically distinct from primate AAVs. The bovine AAV (BAAV) genome has 4,693 bp, and its organization is similar to that of other AAV isolates. The left-hand open reading frame (ORF) and both inverted terminal repeats (ITRs) have the highest homology with the rep ORF and ITRs of AAV serotype 5 (AAV-5) (89 and 96%, respectively). However, the right-hand ORF was only 55% identical to the AAV-5 capsid ORF; it had the highest homology with the capsid ORF of AAV-4 (76%). By comparing the BAAV cap sequence with a model of an AAV-4 capsid, we mapped the regions of BAAV VP1 that are divergent from AAV-4. These regions are located on the outside of the capsid and are partially located in exposed loops. BAAV was not neutralized by antisera raised against recombinant AAV-2, AAV-4, or AAV-5, and it demonstrated a unique cell tropism profile in four human cancer cell lines, suggesting that BAAV might have transduction activity distinct from that of other isolates. A murine model of salivary gland gene transfer was used to evaluate the in vivo performance of recombinant BAAV. Recombinant BAAV-mediated gene transfer was 11 times more efficient than that with AAV-2. Overall, these data suggest that vectors based on BAAV could be useful for gene transfer applications

Optimal initial cell density that yields the highest number of primary synovial mesenchymal stem cells in a clinical setting

Synovial mesenchymal stem cell (MSC) transplantation improved clinical outcomes in knees. In clinical situations, nucleated cells were cultured with 10% autologous serum for 14 days. Nucleated cell number and serum volume practically varied by patient, and initial cell density and dish number affected yield/donor. Our purpose was to estimate the MSC yield/donor from the fold increase obtained in this experiment in several patterns. We obtained the data of the autologous serum volume, nucleated cell number, and initial plating cell density from 10 patients who participated in our previous clinical studies. We used these previous clinical data and the current experimental data. Experimentally, fold increase significantly decreased between 700 and 1,900 cells/cm². In cases where the nucleated cell number was low and the serum volume was high, the estimated MSC yield/donor decreased along with the initial plating density. In cases where the nucleated cell number was high and the serum volume was low, the estimated MSC yield/donor increased along with the initial plating density. In cases where some nucleated cells were discarded, the maximum MSC yield/donor were obtained when the initial cell density was the highest cell density where some nucleated cells were discarded

High-sensitivity virus and mycoplasma screening test reveals high prevalence of parvovirus B19 infection in human synovial tissues and bone marrow

Abstract Background Latent microorganism infection is a safety concern for the clinical application of mesenchymal stem cells (MSCs). The aim of this study is to investigate the frequencies and sensitivities of the latent virus and mycoplasma infections in synovium, bone marrow, peripheral blood cells, and blood plasma and cultured synovial MSCs. Methods Total DNA and RNA of the synovium (n = 124), bone marrow (n = 123), peripheral blood cells (n = 121), plasma (n = 121), and 14-day cultured synovial MSCs (n = 63) were collected from patients who underwent total knee arthroplasty or anterior ligament reconstruction after written informed consents were obtained. The multiplex polymerase chain reaction (PCR) primers were designed to quantitatively measure the representative genomes of 13 DNA viruses, 6 RNA viruses, and 9 mycoplasmas. Multi-spliced mRNA detection and virus spike test were also performed to demonstrate the sensitivity of synovial MSCs to the candidate pathogens. Results In synovium and bone marrow, the positive rates of parvovirus B19 genome were significantly higher than in peripheral blood cells (18.7% and 22% vs. 0.8%, respectively). Multi-alignment analysis of amplified and sequenced viral target genes showed the proximity of the parvovirus B19 gene from different tissue in the same patients. Synovial MSCs cultured for 14 days were positive for virus infection only in two patients (2/62 = 3%). Parvovirus B19 multi-spliced mRNAs were not detected in these two samples. Virus spike test demonstrated the sensitivity of synovial MSCs to herpes simplex virus (HSV)1 and cytomegalovirus (CMV), but not to parvovirus B19. Conclusion This study revealed a relatively high incidence of latent parvovirus B19 in synovium and bone marrow tissue

Optimal Pore Size of Honeycomb Polylactic Acid Films for In Vitro Cartilage Formation by Synovial Mesenchymal Stem Cells

Background. Tissue engineering of cartilage requires the selection of an appropriate artificial scaffold. Polylactic acid (PLA) honeycomb films are expected to be highly biodegradable and cell adhesive due to their high porosity. The purpose of this study was to determine the optimal pore size of honeycomb PLA films for in vitro cartilage formation using synovial mesenchymal stem cells (MSCs). Methods. Suspensions of human synovial MSCs were plated on PLA films with different pore sizes (no pores, or with 5 μm or 20 μm pores) and then observed by scanning electron microscopy. The numbers of cells remaining in the film and passing through the film were quantified. One day after plating, the medium was switched to chondrogenic induction medium, and the films were time-lapse imaged and observed histologically. Results. The 5 μm pore film showed MSCs with pseudopodia that extended between several pores, while the 20 μm pore film showed MSC bodies submerged into the pores. The number of adhered MSCs was significantly lower for the film without pores, while the number of MSCs that passed through the film was significantly higher for the 20 μm pore film. MSCs that were induced to form cartilage peeled off as a sheet from the poreless film after one day. MSCs formed thicker cartilage at two weeks when growing on the 5 μm pore films than on the 20 μm pore films. Conclusions. Honeycomb PLA films with 5 μm pores were suitable for in vitro cartilage formation by synovial MSCs

The environmental risk assessment of cell-processing facilities for cell therapy in a Japanese academic institution.

Cell therapy is a promising treatment. One of the key aspects of cell processing products is ensuring sterility of cell-processing facilities (CPFs). The objective of this study was to assess the environmental risk factors inside and outside CPFs. We monitored the temperature, humidity, particle number, colony number of microorganisms, bacteria, fungi, and harmful insects in and around our CPF monthly over one year. The temperature in the CPF was constant but the humidity fluctuated depending on the humidity outside. The particle number correlated with the number of entries to the room. Except for winter, colonies of microorganisms and harmful insects were detected depending on the cleanliness of the room. Seven bacterial and two fungal species were identified by PCR analyses. Psocoptera and Acari each accounted for 41% of the total trapped insects. These results provide useful data for taking the appropriate steps to keep entire CPFs clean

Canine mesenchymal stem cells from synovium have a higher chondrogenic potential than those from infrapatellar fat pad, adipose tissue, and bone marrow.

Osteoarthritis (OA), a common chronic joint disorder in both humans and canines, is characterized by a progressive loss of articular cartilage. Canines can serve as an animal model of OA for human medicine, and this research can simultaneously establish effective veterinary treatments for canine OA. One attractive treatment that can lead to cartilage regeneration is the use of mesenchymal stem cells (MSCs). However, for canine OA, little information is available regarding the best source of MSCs. The purpose of this study was to identify a promising MSC source for canine cartilage regeneration. We collected synovial, infrapatellar fat pad, inguinal adipose, and bone marrow tissues from six canines and then conducted a donor-matched comparison of the properties of MSCs derived from these four tissues. We examined the surface epitope expression, proliferation capacity, and trilineage differentiation potential of all four populations. Adherent cells derived from all four tissue sources exhibited positivity for CD90 and CD44 and negativity for CD45 and CD11b. The positive rate for CD90 was higher for synovium-derived than for adipose-derived and bone marrow-derived MSCs. Synovium-derived and infrapatellar fat pad-derived MSCs displayed substantial proliferation ability, and all four populations underwent trilineage differentiation. During chondrogenesis, the wet weight was heavier for cartilage pellets derived from synovium MSCs than from the other three sources. The synovium is therefore a promising source for MSCs for canine cartilage regeneration. Our findings provide useful information about canine MSCs that may be applicable to regenerative medicine for treatment of OA

Comparison of mesenchymal stem cells obtained by suspended culture of synovium from patients with rheumatoid arthritis and osteoarthritis

Abstract Background Mobilization of mesenchymal stem cells (MSCs) from the synovium was revealed using a “suspended synovium culture model” of osteoarthritis (OA). The pathology of rheumatoid arthritis (RA) differs from that of OA. We investigated whether mobilization of MSCs from the synovium also occurred in RA, and we compared the properties of synovial MSCs collected from suspended synovium culture models of RA and OA. Methods Human synovium was harvested during total knee arthroplasty from the knee joints of patients with RA (n = 8) and OA (n = 6). The synovium was suspended in a bottle containing culture medium and a culture dish at the bottom. Cells were harvested from the dish and analyzed. Results No significant difference was observed between RA and OA in the harvested cell numbers per g of synovium. However, the variation in the number of cells harvested from each donor was greater for RA than for OA. The harvested cells were multipotent and no difference was observed in the cartilage pellet weight between RA and OA. The surface epitopes of the cells in RA and OA were similar to those of MSCs. Conclusion Mobilization of MSCs from the synovium was demonstrated using a suspended synovium culture model for RA. The harvested cell numbers, chondrogenic potentials, and surface epitope profiles were comparable between the RA and OA models

Microgravity and Signaling Molecules in Rat Osteoblasts: Downstream of Receptor Tyrosine Kinase, G-Protein-Coupled Receptor, and Small GTP-Binding Proteins

Rat osteoblasts were cultured for 4 and 5 days aboard Space Shuttle and solubilized on board. The mRNA levels of the post-receptor signaling molecules were analyzed by quantitative RT-PCR. The G-protein alpha subunit G(alpha)q mRNA levels were elevated 3-fold by microgravity. G(alpha)q stimulates PLC(beta), and then PKC. PKC(delta) and PKC(theta) mRNA levels were increased 2- to 5-fold by microgravity The mRNA levels of SOS and Ras GRF were increased 4 to 5-fold by microgravity, while Ras GAP was not altered. Spaceflight-induced bone loss might be attributed to microgravity modulation of the signaling pathway in osteoblasts