Characterization of Adherent Umbilical Cord Blood Stromal Cells Regarding Passage, Cell Number, and Nano-biomarking Utilization

Adherent umbilical cord blood stromal cells (AUCBSCs) are multipotent cells with differentiation capacities. Therefore, these cells have been investigated for their potential in cell-based therapies. Quantum Dots (QDs) are an alternative to organic dyes and fluorescent proteins because of their long-term photostability. In this study we determined the effects of the cell passage on AUCBSCs morphology, phenotype, and differentiation potential. QDs labeled AUCBSCs in the fourth cell passage were differentiated in the three mesodermal lineages and were evaluated using cytochemical methods and transmission electron microscopy (TEM). Gene and protein expression of the AUCBSCs immunophenotypic markers were also evaluated in the labeled cells by real-time quantitative PCR and flow cytometry. In this study we were able to define the best cellular passage to work with AUCBSCs and we also demonstrated that the use of fluorescent QDs can be an efficient nano-biotechnological tool in differentiation studies because labeled cells do not have their characteristics compromised.

Glycosaminoglycan chains from alpha(5)beta(1) integrin are involved in fibronectin-dependent cell migration

alpha(5)beta(1) integrin from both wild-type CHO cells (CHO-K1) and deficient in proteoglycan biosynthesis (CHO-745) is post-translationally modified by glycosaminoglycan chains. We demonstrated this using [(35)S]sulfate metabolic labeling of the cells, enzymatic degradation, immunoprecipitation reaction with monoclonal antibody, fluorescence microscopy, and flow cytometry. The alpha(5)beta(1) integrin heterodimer is a hybrid proteoglycan containing both chondroitin and heparan sulfate chains. Xyloside inhibition of sulfate incorporation into alpha(5)beta(1) integrin also supports that integrin is a proteoglycan. Also. cells grown with xyloside adhered on fibronectin with no alteration in alpha(5)beta(1) integrin expression. However, haptotactic motility on fibronectin declined in cells grown with xyloside or chlorate as compared with controls. Thus, alpha(5)beta(1) integrin is a proteoglycan and the glycosaminoglycan chains of the integrin influence cell motility on fibronectin. Similar glycosylation of alpha(5)beta(1) integrin was observed in other normal and malignant cells, suggesting that this modification is conserved and important in the function of this integrin. Therefore, these glycosaminoglycan chains of alpha(5)beta(1) integrin are involved in cellular migration on fibronectin.

In vitro study of CD133 human stem cells labeled with superparamagnetic iron oxide nanoparticles

Superparamagnetic iron oxide nanoparticles (SPIONs) are applied in stem cell labeling because of their high magnetic susceptibility as compared with ordinary paramagnetic species, their low toxicity, and their ease of magnetic manipulation. The present work is the study of CD133(+) stem cell labeling by SPIONs coupled to a specific antibody (AC133), resulting in the antigenic labeling of the CD133+ stem cell, and a method was developed for the quantification of the SPION content per cell, necessary for molecular imaging optimization. Flow cytometry analysis established the efficiency of the selection process and helped determine that the CD133 cells selected by chromatographic affinity express the transmembrane glycoprotein CD133. The presence of antibodies coupled to the SPION, expressed in the cell membrane, was observed by transmission electron microscopy. Quantification of the SPION concentration in the marked cells using the ferromagnetic resonance technique resulted in a value of 1.70 x 10 (13) mol iron (9.5 pg) or 7.0 x 10 (6) nanoparticles per cell ( the measurement was carried out in a volume of 2 mu L containing about 6.16 x 10 5 pg iron, equivalent to 4.5 x 10 (11) SPIONs). (c) 2008 Elsevier Inc. All rights reserved.

Analysis of cell cycle phases and proliferative capacity in mice bearing melanoma maintained on different dietary proteins

Background Diet seems to represent, directly or indirectly, 35% of all cancer reports. In this study, the influence of dietary protein on the growth of melanoma B16F10 was evaluated through analyses of cell cycle phases and proliferative capacity. Methods Flow cytometry and argyrophilic nucleolar organizer regions (AgNORs) technique were applied in mice bearing B16F10 melanoma cells fed on different dietary proteins. All data were submitted to statistical analyses. Results The G0/G1 phase increased for the animal groups fed bovine collagen hydrolysate (BCH) or BCH-P1 + whey protein isolate (WPI), compared with mice receiving only WPI, for all dietary groups treated and nontreated with paclitaxel. Mice that received BCH + WPI treated with paclitaxel showed the highest percentage of apoptosis compared with WPI group. AgNORs, total nucleolar organizer regions (NORs)/cells and dot number/cell for all dietary protein groups nontreated with paclitaxel were higher than for the WPI. The only two dietary protein groups treated with paclitaxel that presented higher total NORs and dot number/cell than the WPI group were BCH + WPI and BCH-P1 + WPI. Conclusions A significantly lower proliferative capacity and larger number of cells in the G0/G1 phase were observed for the dietary protein groups combining the two collagen hydrolysates, BCH or BCH-P1 with WPI, treated with paclitaxel. Castro GA, Maria DA, Rodrigues CJ, Sgarbieri VC. Analysis of cell cycle phases and proliferative capacity in mice bearing melanoma maintained on different dietary proteins.

Wave expansion of CD(34+) progenitor cells in the spleen in rodent malaria

Defense against malaria depends upon amplification of the spleen structure and function for the clearance of parasitized red blood cells (pRBC). We studied the distribution and amount of CD(34+) cells in the spleens of mice infected with rodent malaria. We sought to identify these cells in the spleen and determine their relationship to infection. C57BL/6J mice were infected with self-resolving, Plasmodium chabaudi CR, or one of the lethal rodent malaria strains, P. chabaudi AJ and P. berghei ANKA. We then recorded parasitemia, mortality, and the presence of CD(34+) cells in spleen, as determined by immunohistochemistry and flow cytometry. In the non-lethal strain, the spleen structure was maintained during amplification, but disrupted in lethal models. The abundance of CD(34+) cells increased in the red pulp on the 4th and 6th days p.i. in all models, and subsided on the 8th day p.i. Faint CD(34+) staining on the 8th day p.i., was probably due to differentiation of committed cell lineages. In this work, increase of spleen CD(34+) cells did not correlate with infection control. (c) 2009 Published by Elsevier Inc.

Activated status of basophils in chronic urticaria leads to interleukin-3 hyper-responsiveness and enhancement of histamine release induced by anti-IgE stimulus

Background Basophils and mast cells are the main target cells in chronic idiopathic urticaria (CIU). Besides the basopenia, intrinsic defects of the anti-IgE cross-linking signalling pathway of basophils have been described in CIU. Objectives We sought to investigate the profile of expression of activation markers on basophils of patients with CIU and to explore the effect of interleukin (IL)-3 priming upon anti-IgE cross-linking stimuli through expression of activation markers and basophil histamine releasability. Methods Evaluation of the surface expression of Fc epsilon RI alpha, CD63, CD203c and CD123 on whole blood basophils of patients with CIU undergoing autologous serum skin test (ASST) was performed by flow cytometry. The effect of pretreatment with IL-3 in the anti-IgE response was analysed by the expression of basophil activation markers and histamine release using enzyme-linked immunosorbent assay. Results Blood basophils of patients with CIU were reduced in number and displayed increased surface expression of Fc epsilon RI alpha, which was positively correlated with the IgE serum levels. Upregulation of expression of both surface markers CD203c and CD63 was verified on basophils of patients with CIU, regardless of ASST response. High expression of IL-3 receptor on basophils was detected only in ASST+ patients with CIU. Pretreatment with IL-3 upregulated CD203c expression concomitantly with the excreting function of blood basophils and induced a quick hyper-responsiveness to anti-IgE cross-linking on basophils of patients with CIU compared with healthy controls. Conclusions Basophils of patients with CIU showed an activated profile, possibly due to an in vivo priming. Functionally, basophils have high responsiveness to IL-3 stimulation, thereby suggesting that defects in the signal transduction pathway after IgE cross-linking stimuli are recoverable in subjects with chronic urticaria.

Increased Levels of Circulating Endothelial Progenitor Cells in Human T-cell Lymphotropic Virus Type I Carriers

Background and Aims. HTLV-I-transformed T cells secrete biologically active forms of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF). In addition, HTLV-I-transformed cells have a high capacity of adhesion to endothelial cells. Methods. We measured the circulating endothelial progenitor cells (EPCs) and mature endothelial cells (MECs) by flow cytometry in 27 HTLV-I carriers in comparison to 30 healthy, age- and gender-matched subjects. All subjects had HTLV-I positivity confirmed by Western blot and/or polymerase chain reaction (PCR). The numbers of different subpopulations of EPCs and MECSs were evaluated by four-color flow cytometry using a panel of monoclonal antibodies. All reactions were done in duplicate to confirm reproducibility of the results. Results. The median age of all 27 HTLV-I carriers enrolled in this study was 45 years (range: 27-65 years); 11(41%) were male and 16 (59%) were female. The median age of the 30 healthy subjects in the control group was 45.5 years (range: 20-63 years); 11 (36.6%) were male and 19 (63.4%) were female. The number of EPCs was significantly higher in HTLV-I carriers (median 0.8288 cells/mu L, range: 0.0920-3.3176 cells/mu L) as compared to control group (median 0.4905 cells/mu L, range: 0.0000-1.5660 cells/mu L) (p = 0.035). In contrast, the median of the MECs in the HTLV-I carriers was 0.6380 cells/mu L (range: 0.0473-5.7618 cells/mu L) and 0.4950 cells/mu L (range: 0.0000-4.0896 cells/mu L) in the control group, with no statistical difference (p = 0.697). Conclusions. We demonstrated that EPCs, but not MECs, are increased in the peripheral blood of HTLV-I carriers. (C) 2011 IMSS. Published by Elsevier Inc.

Systemic lupus erythematosus exhibits a dynamic and continuum spectrum of effector/regulatory T cells

Objective: The identification of regulatory T cells (Treg cells) as CD4(+)CD25(high) cells may be upset by the increased frequency of activated effector T cells (Teff cells) in inflammatory diseases such as systemic lupus erythematosus (SLE). This study aimed to evaluate the frequency of T-cell subsets according to the expression of CD25 and CD127 in active (A-SLE) and inactive SLE (I-SLE). Methods: Peripheral blood mononuclear cells (PBMCs) from 26 A-SLE patients (SLE Disease Activity Index (SLEDAI) = 10.17 +/- 3.7), 31 I-SLE patients (SLEDAI = 0), and 26 healthy controls (HC) were analysed by multicolour flow. cytometry. Results: CD25(high) cell frequency was increased in A-SLE (5.2 +/- 5.7%) compared to I-SLE (3.4 +/- 3.4%) and HC (1.73 +/- 0.8%) (p < 0.01). However, the percentage of FoxP3(+) cells in the CD25(high) subset was decreased in A-SLE (24.6 +/- 16.4%) compared to I-SLE (33.7 +/- 16) and HC (45 +/- 25.1%) (p < 0.01). This was partly due to the increased frequency of Teff cells (CD25(high)CD127(+)FoxP3(empty set)) in A-SLE (10.7 +/- 7.3%) compared to I-SLE (8.5 +/- 6.5) and HC (6.1 +/- 1.8%) (p = 0.02). Hence the frequency of Treg cells (CD25(+/high)CD127(low/empty set)FoxP3(+)) was equivalent in A-SLE (1.4 +/- 0.8%), I-SLE (1.37 +/- 1.0%), and HC (1.13 +/- 0.59%) (p = 0.42). A-SLE presented an increased frequency of CD25(+)CD127(+)FoxP3(+) and CD25(empty set)FoxP3(+)CD127(low/empty set) T cells, which may represent intermediate phenotypes between Treg and Teff cells. Conclusions: The present study has provided data supporting normal Treg cell frequency in A-SLE and I-SLE as well as increased frequency of Teff cells in A-SLE. This scenario reflects a Treg/Teff ratio imbalance that may favour the inflammatory phenotype of the disease. In addition, the increased frequency of T cells with putative intermediate phenotypes may be compatible with a highly dynamic immune system in SLE.

Lower cytokine secretion ex vivo by natural killer T cells in HIV-infected individuals is associated with higher CD161 expression

Objective: Natural killer T (NKT) cells are efficiently targeted by HIV and severely reduced in numbers in the circulation of infected individuals. The functional capacity of the remaining NKT cells in HIV-infected individuals is poorly characterized. This study measured NKT cell cytokine production directly ex vivo and compared these responses with both the disease status and NKT subset distribution of individual patients. Methods: NKT cell frequencies, subsets, and ex-vivo effector functions were measured in the peripheral blood mononuclear cells of HIV-infected patients and healthy controls by flow cytometry. We measured cytokines from NKT cells after stimulation with either a-galactosyl ceramide-loaded CD1d dimers (DimerX-alpha GalCer) or phorbol myristate acetate and ionomycin. Results: The frequencies of NKT cells secreting interferon-gamma and tumor necrosis factor-alpha were significantly lower in HIV-infected patients than healthy controls after DimerX-alpha GalCer treatment, but responses were similar after treatment with phorbol myristate acetate and ionomycin. The magnitude of the interferon-gamma response to DimerX-alpha GalCer correlated inversely with the number of years of infection. Both interferon-gamma and tumor necrosis factor-alpha production in response to DimerX-alpha GalCer correlated inversely with CD161 expression. Conclusion: The ex-vivo Th1 responses of circulating NKT cells to CD1d-glycolipid complexes are impaired in HIV-infected patients. NKT cell functions may be progressively lost over time in HIV infection, and CD161 is implicated in the regulation of NKT cell responsiveness. (C) 2009 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins

Interferon and Alternative Activation of Monocyte/Macrophages in Systemic Sclerosis-Associated Pulmonary Arterial Hypertension

Objective. To explore the relationship between biomarkers of pulmonary arterial hypertension (PAH), interferon (IFN)-regulated gene expression, and the alternative activation pathway in systemic sclerosis (SSc). Methods. Peripheral blood mononuclear cells (PBMCs) were purified from healthy controls, patients with idiopathic PAH, and SSc patients (classified as having diffuse cutaneous SSc, limited cutaneous SSc [lcSSc] without PAH, and lcSSc with PAH). IFN-regulated and ""PAH biomarker"" genes were compared after supervised hierarchical clustering. Messenger RNA levels of selected IFN-regulated genes (Siglec1 and MX1), biomarker genes (IL13RA1, CCR1, and JAK2), and the alternative activation marker gene (MRC1) were analyzed on PBMCs and on CD14- and CD14+ cell populations. Interleukin-13 (IL-13) and IL-4 concentrations were measured in plasma by immunoassay. CD14, MRC1, and IL13RA1 surface expression was analyzed by flow cytometry. Results. Increased PBMC expression of both IFN-regulated and biomarker genes distinguished SSc patients from healthy controls. Expression of genes in the biomarker cluster, but not in the IFN-regulated cluster, distinguished lcSSc with PAH from lcSSc without PAH. The genes CCR1 (P < 0.001) and JAK2 (P < 0.001) were expressed more highly in lcSSc patients with PAH compared with controls and mainly by CD14+ cells. MRC1 expression was increased exclusively in lcSSc patients with PAH (P < 0.001) and correlated strongly with pulmonary artery pressure (r = 0.52, P = 0.03) and higher mortality (P = 0.02). MRC1 expression was higher in CD14+ cells and was greatly increased by stimulation with IL-13. IL-13 concentrations in plasma were most highly increased in lcSSc patients with PAH (P < 0.001). Conclusion. IFN-regulated and biomarker genes represent distinct, although related, clusters in lcSSc patients with PAH. MRC1, a marker for the effect of IL-13 on alternative monocyte/macrophage activation, is associated with this severe complication and is related to mortality.

Mesenchymal Stem Cell Properties of Periodontal Ligament Cells From Deciduous and Permanent Teeth

Background: Human postnatal stem cells have been identified in periodontal ligaments (PDLs). In this study, the in vitro biologic properties of CD105(+) enriched cell subsets from PDLs harvested from deciduous (DePDL) and permanent (PePDL) teeth are comparatively assessed. Methods: PDL tissue was obtained from 12 teeth (six primary and six permanent) from which CD105(+) CD34(-) CD45(-) cells were isolated by magnetic cell sorting. To identify and quantitatively compare the stem cell markers, DePDL and PePDL cells were assessed for CD166 surface antigen expression by flow cytometry, real-time polymerase chain reaction, and immunostaining for Stro-1 and Oct-4, osteogenic and adipogenic differentiation, and proliferation rate by trypan blue method. Results: Magnetic cell sorting isolated cell populations containing 23.87% (+/- 11.98%) and 11.68% (+/- 6.27%) of CD105(+) expressing cells from PePDL and DePDL, respectively. Flow cytometric analysis demonstrated a higher proportion of CD105(+) cells coexpressing CD166 surface antigen in PePDL, whereas immunostaining and real-time polymerase chain reaction analysis demonstrated that both cell subsets expressed Stro-1 and Oct-4. DePDL-CD105(+) subsets were more proliferative compared to PePDL subsets, and both cell populations showed multipotential capabilities to differentiate in vitro to osteoblast/cementoblast- and adipocyte-like cells. However, a higher expression of adipogenic-related genes was observed in DePDL cells, whereas PePDL-CD105(+) cell subset presented a more homogeneous osteoblast/cementoblast response. Conclusion: These findings demonstrate that highly purified mesenchymal progenitor cell subsets can be obtained from the PDLs of both deciduous and permanent teeth, and further indicate phenotype dissimilarities that may have an impact on their clinical applications. J Periodontol 2010;81:1207-1215.

Application of recombinant antibody library for screening specific antigens in a bovine sperm cell subpopulation

Antibody phage display libraries are a useful tool in proteomic analyses. This study evaluated an antibody recombinant library for identification of sex-specific proteins on the sperm cell surface. The Griffin.1 library was used to produce phage antibodies capable of recognizing membrane proteins from Nelore sperm cells. After producing soluble monoclonal scFv, clones were screened on Simental sperm cells by flow cytometry and those that bound to 40-60% of cells were selected. These clones were re-analyzed using Nelore sperm cells and all clones bound to 40-60% of cells. Positive clones were submitted to a binding assay against male and female bovine leukocytes by flow cytometry and one clone preferentially bound to male cells. The results indicate that phage display antibodies are an alternative method for identification of molecules markers on sperm cells. (C) 2007 Elsevier B.V. All rights reserved.

Phenotypic and functional characterization of pulmonary macrophages subpopulations after intratracheal injection of Paracoccidioides brasiliensis cell wall components

A shift in the activation of pulmonary macrophages characterized by an increase of IL-1, INF-alpha and IL-6 production has been induced in mice infected with Paracoccidioides brasiliensis. It is still unclear whether a functional shift in the resident alveolar macrophage population would be responsible for these observations due to the expression of cell surface molecules. We investigated pulmonary macrophages by flow cytometry from mice treated with P. brasiliensis derivatives by intratracheal route. In vivo labeling with the dye PKH26GL was applied to characterize newly recruited pulmonary macrophages from the bloodstream. Pulmonary macrophages from mice inflamed with P. brasiliensis derivatives showed a high expression of the surface antigens CD11b/CD18 and CD23 among several cellular markers. The expression of these markers indicated a pattern of activation of a subpopulation characterized as CD11b(+) or CD23(+), which was modulated in vitro by IFN-gamma and IL-4. Analysis of monocytes labelled with PKH26GL demonstrated that CD11b(+) cells did infiltrate the lung exhibiting a proinflammatoni pattern of activation, whereas CD23(+) cells were considered to be resident in the lung. These findings may contribute to better understand the pathology of lung inflammation caused by P. brasiliensis infection. (C) 2010 Elsevier GmbH. All rights reserved.

Monoclonal B-cell lymphocytosis in first-degree relatives of patients with sporadic (non-familial) chronic lymphocytic leukaemia

Although biological similarities have been described among monoclonal B-cell lymphocytosis (MBL) and chronic lymphocytic leukaemia (CLL), the relationships between these two conditions are not fully understood, and new epidemiological studies in different populations and different countries continue to be reported. Here, we investigated 167 first-degree relatives from 42 families of patients with non-familial (sporadic) CLL, using four-colour flow cytometry. MBL was found in seven of 167 subjects (4.1%). Monoclonality was detected in all cases either by light-chain restriction or by polymerase chain reaction. Fluourescence in situ hybridization did not show any chromosomal abnormality. The prevalence of MBL according to age was 0 (0/54) in individuals aged less than 40 years, 2.5% (2/81) between 40 and 60 years, and 15.6% (5/32) in individuals over 60 years. The prevalence of MBL cases in individuals over 60 years was similar to that found in familial CLL relatives at the same age group. This suggests that in older first-degree relatives of patients with sporadic CLL, the risk of MBL detection is as high as in older first-degree relatives from CLL families, which could render these individuals belonging to `sporadic CLL families` as susceptible as individuals from `familial CLL` to the development of clinical CLL.

Transcriptional Response of Peripheral Lymphocytes to Early Fibrosarcoma: A Model System for Cancer Detection Based on Hybridization Signatures

Since circulating leukocytes, mainly B and T cells, continuously maintain vigilant and comprehensive immune surveillance, these cells could be used as reporters for signs of infection or other pathologies, including cancer. Activated lymphocyte clones trigger a sensitive transcriptional response, which could be identified by gene expression profiling. To assess this hypothesis, we conducted microarray analysis of the gene expression profile of lymphocytes isolated from immunocompetent BALB/c mice subcutaneously injected with different numbers of tumorigenic B61 fibrosarcoma cells. Flow cytometry demonstrated that the number of circulating T (CD3(+)CD4(+) or CD3(+)CD8(+)) or B (CD19(+)) cells did not change. However, the lymphocytes isolated from tumor cell-injected animals expressed a unique transcriptional profile that was identifiable before the development of a palpable tumor mass. This finding demonstrates that the transcriptional response appears before alterations in the main lymphocyte subsets and that the gene expression profile of peripheral lymphocytes can serve as a sensitive and accurate method for the early detection of cancer. Exp Biol Med 234:802-812, 2009