stereotaxic coordinates of the mouse brain atlas . The sections were selected (9 per animal) by targeting the areas of interest (neocortex, striatum and hippocampus), which were left free-floating in a multi-well plate. To stain microglia, sections were incubated in polyclonal rabbit anti-Iba1 primary antibody overnight (1:2000; Wako Chemicals, Richmond, VA, USA), followed by a bio- tinylated anti-rabbit secondary antibody for 2 h (1:600, Sigma-Aldrich Corporation, St. Louis, EUA). After using an ABC kit (Avidin/Biotinylated enzyme Complex Vectastain Elite, Vector, Burlingame, CA, EUA) followed by a DAB reaction (3,3’-Diaminobenzidine, Sigma- Aldrich Corporation, St. Louis, EUA), the sections were mounted on slides and sealed with coverslips. The slides were examined using a light microscope (Nikon 80i), and images were captured and digitized using the Nikon ACT-1 v.2 system. In each section, nuclear profiles of Iba1 cells were counted by an observer blinded to the experimental condition of each animal. Cells stained for Iba1 were counted under 20× magnification in 18 ran- dom, non-overlapping fields of 0.02 mm 2 per structure of each animal.
The first works with tissue cultures were carried out with organic fluids of animals, such as lymph. When Eagle (1955) formulated a basic culture medium containing ami- no acids, carbohydrates, vitamins and minerals, he noted that the supplementation of the medium with organic flui- ds was still necessary, since such fluids contained factors that were undefined but essential for cell growth. The su- pplementation of basic culture medium with 20% animal serum was widely utilized. Due to the presence of growth factors and a small quantity of gammaglobulin, fetal bovine serum (FBS) is one of the most utilized. FBS is used at a concentration of 10%, but it can be increased or decreased depending on the culture (Gonçalves 2002).
improves healing (10). Studies have also linked increased cytokine production, as a result of specific signal transduction pathways, with BMSC differentiation, and in turn, bone fracture healing (11-13). Several studies have suggested that low- frequency vibration can effectively stimulate bone cell growth, increase the trabecular number and width, and prevent the loss of cortical bone strength (11, 14-17). Low-frequency signals propagate through the OPG pathway, stimulating the differentiation ofbone precursor cells into osteoblasts, thereby affecting bone reconstruction (12). However, the exact frequency best suited to stimulation of osteogenesis remains controversial. Further, a recent study showed that 30 Hz vibration could induce BMSC differentiation in vitro (18). In this study, we used rabbits as an in vivo bone defect small animal model system to test varying frequencies and their effect on BMSC differentiation and bone fracture healing.
Within the framework of this animal study, it could be demonstrated that the combined application of BMC+CPG+ PRP had a positive influence on bone defect healing after six weeks compared to the application of BMC+CPG and the CPG alone. This is backed by the outcome of the quantitative MDCT- and CBCT-evaluation, but also by the analysis of the histomorpho- metrical results. Radiologically and histomorphometrically (central defect zone) a significantly higher bone regeneration could be determined in the BMC+CPG+PRP group. In the cortical defect zone the BMC+CPG+PRP group showed a higher new bone formation by trend, although the differences were not significant. When comparing the total composite of BMC+CPG+PRP to the autograft group the quantitative radiological and histomorpho- metrical evaluations showed no significant differences between these two groups. Thus, within the limitations of our study the application of the composite BMC+CPG+PRP represents an comparable alternative to autologous bone grafting during the early phase ofbone healing after six weeks. In only very few histological slides of every treatment group an overgrowth of new bone above the cortical defect zone was observed. This overgrowing new bone formation was not quantified due to the rarity of this phenomenon and as it was located outside of the defect. This reaction was presumably part of the bone remodelling
The cells of the myeloid, lymphoid, and erythroid lineages of the bonemarrow were quantified in rats with hypo and hyperthyroidism. Fifteen Wistar rats were divided into three groups: hypothyroid (n=5), hyperthyroid (n=5), and control (n=5). Three months after the onset of the treatments, euthanasia was performed. Bonemarrow was aspirated from femurs of each animal to perform smears that were stained with Quick Panoptic. The percentages of rubroblast, prorubrocyte, metarubrocyte, myeloblast, promyelocytes, metamyelocytes, myelocytes, segmented, eosinophils, basophils, lymphocytes, plasma cells and monocytes in were determined a total of 500 cells. The bonemarrowof animals with hypothyroidism had hypoplasia. The myeloid:erythroid ratio was higher in animals with thyroid dysfunction. In hypo and hyperthyroidism, there was a significant reduction of the percentage of rubrocyte, metarubrocyte, and lymphocytes and increase of myelocytes and segmented cells. In hypothyroidism, there was a significant increase in the percentage of metamyelocytes. It is concluded that both hypo and hyperfunction of thyroid increase the myeloid:erythroid ratio by increasing the number of cells of the myeloid lineage and reducing the cells of the erythroid lineage.
The protocols for animal handling were previously approved by our institutional Animal Ethics Committee (CETEA/FMRP, protocol number 006/2008). Femurs were obtained from 6–12 week old C57BL/6 mice. After euthanasia, the mice were sprayed with 70% ethanol and the femurs were dissected using scissors, cutting through the tibia below the knee joints as well as through the pelvic bone close to the hip joint. Muscles connected to the bone were removed using clean gauze, and the femurs were placed into a polypropylene tube containing sterile PBS on ice. In a tissue culture hood, the bones were placed in 70% ethanol for 1 minute, washed in sterile RPMI 1640 and then both epiphyses were removed using sterile scissors and forceps. The bones were flushed with a syringe filled with RPMI 1640 to extrude bonemarrow into a 15 mL sterile polypropylene tube. A 5 ml plastic pipette was used to gently homogenize the bonemarrow. The cell suspension generated thereafter is called fresh bonemarrow cells.
Single morula were detected in peripheral blood smears on day 11 (animal 1) and on day 13 (animal 2) (Figure 1), while several E. canis inclusions were seen in the cytoplasm of monoblasts and mature monocytes in bonemarrow aspirates on day 15 PI (Figure 2).
Lateral amyotrophic sclerosis (ALS) is a neurodegenerative disease characterized by the selective loss of both lower and upper motoneurons in the spinal cord, brain stem and motor cortex. Loss of motoneurons leads to progressive paralysis, and death in 2 to 5 years. Treatment is based on symptomatic measures and pharmacological therapy available is not curative. Thus, it is of great importance to develop new therapeutic strategies to manage this disease. Alternatively, stem cells to replace could be used to replace degenerating neurons or to stop/delay neuronal death. The aim of this study was to verify if bonemarrow mononuclear cells (BMMC) have therapeutic potential in transgenic mice superexpressing SOD1 G93A . These animals’ phenotypic and physiopathological characteristics mimic those of ALS. Seventy-day-old or 110-day-old mice were administered with 10 7 BMMC in the tail vein. BMMC were obtained from C57BL/6-EGFP or SOD1 G93A donors. Control animals received saline. In order to determine disease onset and progression, animals were evaluated in the Rotarod test, by electromyography and had their body weight measured. Survival was also evaluated. Tissue samples were collected for PCR analysis of cell migration. Additional groups of animals were euthanized at 120-days-old for histological analysis and the alkaline comet assay. Our results indicate that EGFP BMMC transplantation in 70-day-old mice (pre-symptomatic) mice prolong survival, preserve motor function and preserve motoneurons in the ventral horn of the spinal cord. However, for mSOD1 BMMC the observed effect is intermediary. Furthermore, in 110-day-old (symptomatic) mice, only
The xenogenic graft has shown promising results. It is based on the abundance of the matrix, low cost of bovine bone and the proper techniques for mechanical and chemical preparation (Bigham et al., 2008). The lyophilized inorganic bovine bone (deproteinized) follows the same process of preparation of the organic matrix; however, it does not undergo the process of decalcification, in which all the mineral components ofbone are preserved and all the organic parts are eliminated (BMPs, collagen, proteins), i.e. the inorganic part is preserved (Sanada et al., 2003; Gerbi et al., 2005; Marin et al., 2007).
The desirable therapeutic radionuclide should have particulate emission (β - , conversion electron, Auger electron, or alpha emission) suited to the nature and stage of the disease, in sufficient abundance. A gamma emission component, in reasonable abundance, can be of advantage for low-dose imaging especially during staging, for dosimetry estimates, and for monitoring response to therapy. A physical half-life of 1-14 d (preferably with stable daughter products) is optimal; it should be matched with the in-vivo pharmacokinetics of the isotope-carrier vehicle combination. The radioelement’s chemistry should be amenable for its attachment with a broad class of compounds with high in-vivo stability. Finally, a free or chelated radionuclide should be excreted rapidly following metabolic processes, and should have little or no affinity for blood or normal body tissues. Although only few existing isotopes meet all of these criteria, there are a number of emerging “new” radionuclides that appear promising and versatile, and warrant continued investigations (Srivastava, 1996a; Srivastava, 1999). A list of current as well as future therapeutic radionuclides, chosen arbitrarily on the basis of particle emission and a few other parameters, is presented in Table 1. The nature of
Measurement of intracellular reduced protein thiol levels ThioGlo-1 (TG-1, [3H-Naphthol[2,1-b]pyran-s-carboxylic Ac- id, 10-(2,5-Dihydro-2,5-dioxo-1H-pyrrol-1-yl)-9-methoxy-3-oxo-, methyl ester]) (Calbiochem, San Diego, CA), a maleimide sulfhydryl-specific fluorescent probe, was used to monitor intra- cellular reduced protein thiols as reported earlier . BMCs, Lin(2) cells or BMDDCs were harvested, washed twice with PBS and then solubilized by ice-cold lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton, 1 mM EDTA, 1 mM EGTA, plus a protease inhibitor cocktail]. Cell lysates/superna- tants were collected from the cells after spinning at 16,000 g for 10 min at 4 uC, and low molecular weight (,6 kDa) compounds were eliminated from the cell lysates by using size-exclusion chromatography with a mirco Bio-Spin 6 column (BioRad). After measuring protein concentrations by BCA protein assay, 20 ul (,20–50 ug) cell lysate was diluted with 2 ml 20 mM sodium phosphate buffer, pH 7.4. The total intracellular reduced protein thiol levels were then determined by an immediate fluorescence increase upon the addition of 5 m M (final concentration) of TG-1. Briefly, fluorescence was measured by a QM-4 spectrofluorometer (PTI, Birmingham, NJ) in a 10610 mm quartz cuvette under constant stirring at 37 uC, controlled by TC 125 Temperature Controller (Quantum Northwest, Shoreline, WA), with excitation (Ex) at 379 nm and emission (Em) at 513 nm using standard kinetic mode with resolution of 0.1 sec. The emission of each sample was recorded for 100 seconds (background) before and until 500 seconds after the addition of TG-1. Saturated TG-1 fluorescence values were corrected for background emissions and normalized for protein content, and averaged using SigmaPlot 10.0 software (Systat Software, San Jose, CA).
Upon determining that early stages of cDC development were unperturbed in the bonemarrowof Pin1-null mice, we utilized established ex vivo bonemarrow culture protocols to examine later stages of cDC development. Because cDC in FL cultures do not express CD8, other cell surface markers have previously been used to distinguish between the CD8+ and CD8 2 cDC equivalents. We stained FLDC for two different sets of markers that have been described to specifically discriminate between the two cDC subsets. Both sets of markers clearly indicated a significant defect in the CD8+ cDC equivalent subset of cDC in Pin1-null cultures, which mirrored the defect identified in steady- state CD8+ cDC in vivo. Upon quantifying the CD82 cDC equivalent population in bonemarrow cultures, we encountered slightly more complex results. When cultured with FL and gated on Mac1 expression, Pin1-null Mac1+ (CD8 2 equivalent) cDC expressed less CD11c than WT Mac1+ cDC. To help clarify this result, bonemarrow was also cultured with GM-CSF to exclusively produce the Mac1+ (CD82 equivalent) subset of cDC. Under these conditions, Pin1-null Mac1+ cDC production was not impaired, nor was there any observable decrease in CD11c expression. Additionally, when FL cultured cells were alternately stained with CD24 and Sirpa, which have previously been used to discriminate between cDC subsets, we did not observe a decrease in the Pin1-null CD8 2 cDC equivalent cells, which are defined as CD24(lo)Sirpa+ cells. Taken together, these results indicate that Pin1 is unlikely to be required for the development of CD82 cDC. Conversely, Pin1 appears to modulate the development of CD8+ cDC, as indicated by the significant decrease in CD8+ equivalent cDC that was consis- tently observed in Pin1-null bonemarrow cultures.
Figure 3 - Immunohistochemical detection of DAPI-labeled BM-MSCs in injured retinal sections 8 weeks after intravitreal transplantation. The left panel of each row shows immunoreactivity for a particular marker in retinal sections with laser-induced injury without BM-MSC treatment (control). The right panel of each row shows immunoreactivity for a particular marker (green) in retinal sections with laser-induced injury and BM-MSC treatment. Eight weeks after transplantation, the grafted cells labeled with DAPI (blue) co-expressed the rod photoreceptor marker (green) (top) rhodopsin, the bipolar and amacrine cell marker (green) (top middle) parvalbumin and the Muller cell and astrocyte marker (green) (bottom middle) GFAP. No grafted cells coexpressed the RPE marker (green) (bottom) pan-cytokeratin. Note that only a small subset of grafted cells was GFAP-immunoreactive (dashed arrows). Arrowheads in the right panel (top) and (bottom middle) indicate the presence of grafted cells (blue) in the subretinal space. Micrographs of the control group did not show any nonspecific labeling or fluorescence expression. Scale bars, 20 µ µ µ µ µm.
Because both types ofbone processing were successful in eliminating the parasite in question, the best technique for the preservation ofbone depend on other factors, such as the mechanical resistance of the material, the type of collection from the donor (aseptic or not), the capacity of the material for osteogenesis, osteoinduction and osteoconduction and the cost of the technique. In this study, we chose the techniques of storage in glycerol and autoclave sterilization due to their low cost and ease of implementation along with the quality they provide to the graft. It is important to mention, though, that both have limitations, such as the reduction of mechanical strength in autoclaved tissues and reduction of osteogenic capacity in both techniques. Thus, other techniques for processing bone should also be studied in the future for the elimination of Leishmania, given that each has its advantages and limitations that must be analyzed to choose the best technique to be applied in each case, as described in literature (Fitch et al., 1997; Boyce et al., 1999; Giovani et al., 2006).
In addition to regulating monocyte/macrophage development, Slfn4 is likely to have some role in regulating macrophage activation, given the effect of TLR3 and TLR4 agonists on Slfn4 mRNA expression. TLR-inducible expression involves the auto- crine, type I IFN-dependent pathway, but there was some residual induction in IFNAR-1 2/2 macrophages (Figure 3B), and by agonists that act in a strictly MyD88-dependent manner (Figure 3A). The role of type I IFN in the induction of Slfn4 is consistent with the presence of appropriate response elements in the promoter (Figure 2). A major function of IFNs is to protect cells against viral infection by inducing a suite of genes involved in host defence . As a type I IFN-inducible gene, Slfn4 may play some role in responses to viral infections. Consistent with this, Slfn4 was induced in macrophages in response to adenovirus infection (AI, unpublished data), and microarray data published by others reported up-regulation of Slfn4 mRNA in BMM upon infection with Sendai virus  and in the lungs of influenza A/ PR/8/34 virus infected mice . Another link between Slfns and responses to viral infections is suggested by the similarity between these genes and the right inverted terminal repeat of several orthopoxviruses [5,35]. Somewhat surprisingly, Slfnlike protein 176 from Camelpox strain CMS apparently reduces virulence, because mice infected with recombinant vaccinia and variola viruses expressing v-Slfn recovered sooner than mice infected with non- recombinant virus . What functional role Slfn4 has in anti- viral responses remains to be determined. Unfortunately, we were not able to assess the impact of the Slfn4 over-expression on host defence because of the myelodysplastic phenotype of Slfn4 transgenic mice. This would render pathogen challenge experi- ments uninterpretable. Furthermore, given that our in vitro experiments assessing macrophage differentiation did not reveal a phenotype despite the fact that Slfn4 regulated monocytopoiesis in vivo, a yet to be identified in vivo factor may be required for Slfn4 function in vitro. Consistent with this, expression profiling of LPS- activated Slfn4 over-expressing macrophages versus controls did not reveal any consistent effect of constitutive Slfn4 expression on the TLR4 response (data not shown). Consequently, the role of Slfn4 in responses to TLR agonists and pathogen challenge in this in vitro setting may require the identification of such a factor.
The case of a 54-year-old man, who was referred to eval- uate a consumptive syndrome with anemia and fever of unknown origin, is reported. A physical examination revealed madarosis, nodular lesions on the ears, nasal soft tissue collapse, subcutaneous nodules on arms and generalized lymphadenopathy. Laboratory tests showed anemia (Hb 8.8 g/dL, mean corpuscular volume 82.4 fL, mean corpuscular hemoglobin 24.5 pg), normal reticulocyte count (0.5%), throm- bocytosis (482 × 10 9 /L), normal serum ferritin (268 ng/mL) and
Two-month-old male Swiss mice obtained from Universidade Federal de Santa Catarina were housed in individual metabolic cages. The undernourished group received a diet containing 4% casein (low-protein diet) and the control group received 20% casein (con- trol diet) for 15 days. The diet contained fibers, saline and balanced vitamin mixtures, supplemented with 0.2% choline and 0.15% methionine (11). The two groups were main- tained on a light/dark cycle of 12 h, with water and food supplied ad libitum. Body weight was monitored every 48 h and the animals were submitted to experimental as- says when the undernourished group attained a 20% loss of their original body weight. The differences between control and undernour- ished mice were analyzed by the unpaired Student t-test.
grade) osteoblastic osteosarcoma. Therefore, the lesion was excised, with tumor-free margins, and part of the adjacent dura mater was resected because of suspicion of neoplastic involvement. The patient presented clini- cal improvement after the tumor resection and was dis- charged to outpatient follow-up.
Recently it has been demonstrated that stem cells can originate from cardiac, neural, skeletal muscle, hepatic, and of course, bonemarrow tissues. These tissues can be grown in vitro and their clinical applications are obvious. Although the precise mechanisms or the fate determinants of stem cells as well as their origins are still far from being defined, there are clinical applications being tested with some success. 1,2 So, it seems like
The inclusion of stem cell technologies in the current approaches to bone recon- struction brings about novel challenges in the design and conception of scaffolds and carriers. Porosity of the material, tradition- ally seen as a necessary inherent characteris- tic, assumes a novel significance in this con- text, namely as the property whereby a uni- form access is granted to cells employed to generate a biotechnological construct. Little work has been done to determine the actual permeability of materials in use with respect to cells suspended in a fluid phase permeat- ing the micropores of the scaffold. More important, materials that are efficient in pro- viding physical support and appropriate ge- ometry for bone formation may not neces- sarily be endowed with the properties re- quired to ensure stem cell survival. This is particularly relevant when materials are con- sidered for the production of devices to be used in clinical procedures that have to last a very long time, such as those performed in infants or adolescents with skeletal disor- ders. Experience from separate fields, such as skin reconstruction using epidermal stem cells, has clearly indicated the critical role, in this specific context, of the supporting materials that are used (reviewed in Ref. 37). While the new perspectives opened by stem cell technologies have essentially employed materials conceived for a previous era of tissue engineering, in which autochthonous cells residing at the site of implantation would merely use a biomaterial scaffold, signifi- cant innovations have been introduced in the field ofbone biomaterial proper. These nota- bly include the design of injectable mineral phases capable of crystallization in vivo, or the design of biological matrices suited to direct cell growth and differentiation locally (reviewed in Ref. 37). Directing efforts to- wards the development of novel materials in