Effects of composite films of silk fibroin and graphene oxide on the proliferation, cell viability and mesenchymal phenotype of periodontal ligament stem cells.

In regenerative dentistry, stem cell-based therapy often requires a scaffold to deliver cells and/or growth factors to the injured site. Graphene oxide (GO) and silk fibroin (SF) are promising biomaterials for tissue engineering as they are both non toxic and promote cell proliferation. On the other hand, periodontal ligament stem cells (PDLSCs) are mesenchymal stem cells readily accessible with a promising use in cell therapy. The purpose of this study was to investigate the effects of composite films of GO, SF and GO combined with fibroin in the mesenchymal phenotype, viability, adhesion and proliferation rate of PDLSCs. PDLSCs obtained from healthy extracted teeth were cultured on GO, SF or combination of GO and SF films up to 10 days. Adhesion level of PDSCs on the different biomaterials were evaluated after 12 h of culture, whereas proliferation rate of cells was assessed using the MTT assay. Level of apoptosis was determined using Annexin-V and 7-AAD and mesenchymal markers expression of PDLSCs were analyzed by flow cytometry. At day 7 of culture, MTT experiments showed a high rate of proliferation of PDLSCs growing on GO films compared to the other tested biomaterials, although it was slightly lower than in plastic (control). However PDLSCs growing in fibroin or GO plus fibroin films showed a discrete proliferation. Importantly, at day 10 of culture it was observed a significant increase in PDLSCs proliferation rate in GO films compared to plastic (P < 0.05), as well as in GO plus fibroin compared to fibroin alone (P < 0.001). Flow cytometry analysis showed that culture of PDLSCs in fibroin, GO or GO plus fibroin films did not significantly alter the level of expression of the mesenchymal markers CD73, CD90 or CD105 up to 168 h, being the cell viability in GO even better than obtained in plastic. Our findings suggest that the combination of human dental stem cells/fibroin/GO based-bioengineered constructs have strong potential for their therapeutic use in regenerative dentistry.

Immunophenotyping of lymphocyte subsets in the third eyelid tissue in dogs (Canis familiaris): morphological, microvascular, and secretory aspects of this ocular adnexa.

The third eyelid is an important adnexa of the eye. The objective of this study was to evaluate (i) morphological aspects (ii) vascularization, and (iii) the immunophenotype of lymphocyte subsets in the third eyelid of dogs. Flow cytometric analysis revealed the presence of three patterns concerning the immunophenotype of the third eyelid tissue. Dogs without ocular insult or deficient tear production might belong to one of the following immunophenotype patterns: I--the number of T-cells that expressed CD3(+) CD8(+) was higher than the number of cells that expressed CD3(+)CD4(+). II--the number of cells CD3(+)C4(+) was higher than the number of cells CD3(+)CD8(+) and in this case a higher number of cells that expressed CD19 were identified. III--Proximity of values of the cells that expressed CD3(+)CD4(+) and CD3(+)CD8(+). These data might suggest that the number of lymphocyte T cells alone should not be considered a direct indicator of the presence of an immune-based inflammation. Besides, a particular population of T-cells does not indicate a particular inflammatory state. The morphological study of the third eyelid revealed a rather uncommon angioarchitecture. The artery that irrigates the eyelid crosses almost the entire length of this structure to achieve its free border, and only then, ramificates deeply towards an inner segmental level. This spatial microvascular arrangement probably results from an adaptation to the fact that the third eyelid, in the medial cantus of the eye, is inwardly compressed into a tiny space. Efficient vascularization is achieved by allowing the first ramifications of the third eyelid artery to run straight to the top. Accini secretor cells of the third eyelid show a mucin content while tubuloacinar cells are mainly serous.

Immunophenotypic analysis of the TCR-Vbeta repertoire in 98 persistent expansions of CD3(+)/TCR-alphabeta(+) large granular lymphocytes: utility in assessing clonality and insights into the pathogenesis of the disease.

At present, a major challenge in the initial diagnosis of leukemia of large granular lymphocytes (LGLs) is to establish the clonal nature of the expanded population. In the present study we have analyzed by flow cytometry immunophenotyping the TCR-Vbeta repertoire of 98 consecutive cases of persistent expansions of CD4(+) or CD8(+bright) CD3(+)/TCR-alphabeta(+) LGLs and compared the results with those obtained in molecular studies of TCR-beta gene rearrangements. Fifty-eight cases were considered to be monoclonal in molecular studies whereas in the remaining 40 cases there was no evidence for monoclonality (11 cases were considered oligoclonal and 29 polyclonal). The TCR-Vbeta repertoire was biased to the preferential use of one or more TCR-Vbeta families in 96% of cases, a total of 124 TCR-Vbeta expansions being diagnosed: one TCR-Vbeta expansion in 71 cases and two or more TCR-Vbeta expansions in 23 cases. The highest TCR-Vbeta expansion observed in each case was higher among monoclonal (74 +/- 19%) as compared to nonmonoclonal cases (24 +/- 14%) (P = 0.001), as did the fraction of LGLs that exhibited a TCR-Vbeta-restricted pattern (86 +/- 16% and 42 +/- 23%, respectively; P = 0.0001); by contrast, the proportion of cases displaying more than one TCR-Vbeta expansion was higher in the latter group: 7% versus 48%, respectively (P = 0.001). Results obtained in oligoclonal cases were intermediate between those obtained in polyclonal and monoclonal cases and similar results were observed for CD4(+) as for CD8(+bright) T-cell expansions. TCR-Vbeta families expressed in CD8(+bright) T-cell-LGL proliferations showed a pattern of distribution that mimics the frequency at which the individual TCR-Vbeta families are represented in normal peripheral blood T cells. Assuming that a given proliferation of LGLs is monoclonal whenever there is an expansion of a given TCR-Vbeta family of at least 40% of the total CD4(+) or CD8(+bright) T-cell compartment, we were able to predict clonality with a sensitivity of 93% and a specificity of 80%. By increasing the cut-off value to 60%, sensitivity and specificity were of 81% and 100%. In summary, our results suggest that flow cytometry immunophenotypic analysis of the TCR-Vbeta repertoire is a powerful screening tool for the assessment of T-cell clonality in persistent expansions of TCR-alphabeta(+) LGLs.

Comparative analysis of the morphological, cytochemical, immunophenotypical, and functional characteristics of normal human peripheral blood lineage(-)/CD16(+)/HLA-DR(+)/CD14(-/lo) cells, CD14(+) monocytes, and CD16(-) dendritic cells.

Human peripheral blood (PB) CD14(lo)/HLA-DR(+) cells were initially described as a subset of mature monocytes. Recently, it has been suggested that these represent a part of a new subset of dendritic cells (DC), characterized by the coexpression of MDC-8/HLA-DR/CD16. The aim of the present paper was to analyze the morphological, cytochemical, phenotypical, and functional characteristics of PB CD16(+)/HLA-DR(+) cells compared to both PB CD14(+) monocytes and CD16(-) DC. In contrast to CD14(+) monocytes, purified CD16(+)/HLA-DR(+) cells displayed cytoplasmic veils and lacked cytoplasmic myeloperoxidase and alpha-naphthyl acetate esterase. Normal human PB CD16(+)/HLA-DR(+) cells also displayed phenotypic characteristics different from those of CD14(+) monocytes: they lacked the CD64 Fcgamma receptor, showed lower levels of CD32, and expressed higher amounts of CD16 compared to CD14(+) monocytes. They also displayed a different pattern of expression of other antigens, including CD14, HLA-DR, CD45RA, CD45RO, complement receptors and complement regulatory surface proteins, adhesion and costimulatory molecules, and cytokine receptors, among others. When compared to CD16(-) DC, CD16(+)/HLA-DR(+) cells showed reactivity for CD16, dim positivity for CD14, higher expression of both Ig- and complement-receptors and lower reactivity for HLA-DR, adhesion, and costimulatory molecules (with the exception of CD86). The CD16(+)/HLA-DR(+) cell subset displayed a higher Ig/complement-mediated phagocytic/oxidative activity than CD16(-) DC, although this activity was significantly lower than that of mature monocytes. Regarding cytokine production at the single cell level, LPS plus IFN-gamma-stimulated PB CD16(+)/HLA-DR(+) cells produced significant amounts of IL1beta, IL6, IL12, TNFalpha, and IL8; however, the percentage of cytokine-producing cells and the amount of cytokine/cell were lower in CD16(+)/HLA-DR(+) cells than in CD14(+) monocytes. In addition, upon comparing CD16(+)/HLA-DR(+) cells with CD33(+++)/CD16(-) DC, we found that the percentage of cytokine-producing cells and the amount of cytokine/cell were significantly different in both cell subsets. In summary, our results show that CD16(+)/HLA-DR(+) cells clearly display different morphologic, cytochemical, immunophenotypical, and functional characteristics compared to both mature monocytes and CD16(-) DC. Interestingly, these cells are more frequent than other DC in normal human adult PB and cord blood samples, while they are less represented in normal bone marrow.

Flow cytometric analysis of cytokine production by normal human peripheral blood dendritic cells and monocytes: comparative analysis of different stimuli, secretion-blocking agents and incubation periods.

In this paper, we comparatively analyze the effects of the following different stimuli on the production and intracellular accumulation of the interleukin (IL)-1 beta, IL-6, IL-12, tumor necrosis factor-alpha (TNF-alpha), and IL-8 inflammatory cytokines in both normal human peripheral blood (PB) dendritic cell (DC) subsets and monocytes: lipopolysaccharide (LPS) versus Staphylococcus aureus cowan I (SAC) in the presence or absence of interferon-(IFN)-gamma-, cytokine secretion-blocking agents (brefeldin A alone versus brefeldin A plus monensin), and incubation periods (6, 12, and 24 h). For this purpose, a four-color multiple-staining direct immunofluorescence technique analyzed by flow cytometry was systematically used in all experiments (n = 19). Our results show that after stimulation, an important proportion of each of the two CD33(+) myeloid DC subsets as well as the monocytes produce significant amounts of all cytokines analyzed under each of the experimental conditions assayed. In contrast, CD33(-/+lo) lymphoplasmocytoid DC failed to produce detectable levels of any of the above-mentioned cytokines under the same stimulatory conditions. Upon comparing the different stimuli used, LPS was associated with higher percentages of cytokine-producing cells compared with SAC, especially within the CD33(hi) DC subset; interestingly, the addition of IFN-gamma enhanced the response of monocytes to both LPS and SAC. As regards the secretion-blocking agents, brefeldin A alone was superior to the combination of brefeldin A and monensin. This is because it was frequently associated with both a higher percentage of cytokine-positive cells and greater amounts of detectable cytokines per cell. Sequential analysis of cytokine production by PB DC and monocytes after 6, 12, and 24 h of cell culture showed that after 6 h, an increased cell death rate existed among DC, which became even undetectable at 24 h, in the absence of a significant increase in cytokine secretion. In summary, our results show that from the experimental conditions assayed in this paper, to induce cytokine production by normal human DC and monocytes, maximum response is obtained once PB samples are stimulated for 6 h with LPS (with or without IFN-gamma) in the presence of brefeldin A alone.

Extensive characterization of the immunophenotype and pattern of cytokine production by distinct subpopulations of normal human peripheral blood MHC II+/lineage- cells.

Dendritic cells (DC) represent the most powerful professional antigen-presenting cells (APC) in the immune system. The aim of the present study was to analyse, on a single-cell basis by multiparametric flow cytometry with simultaneous four-colour staining and a two-step acquisition procedure, the immunophenotypic profile and cytokine production of DC from 67 normal whole peripheral blood (PB) samples. Two clearly different subsets of HLA-II+/lineage- were identified on the basis of their distinct phenotypic characteristics: one DC subset was CD33strong+ and CD123dim+ (0.16 +/- 0.06% of the PB nucleated cells and 55.9 +/- 11. 9% of all PB DC) and the other, CD33dim+ and CD123strong+ (0.12 +/- 0.04% of PB nucleated cells and 44.53 +/- 11.5% of all PB DC). Moreover, the former DC subpopulation clearly showed higher expression of the CD13 myeloid-associated antigen, the CD29 and CD58 adhesion molecules, the CD2, CD5 and CD86 costimulatory molecules, the CD32 IgG receptor and the CD11c complement receptor. In addition, these cells showed stronger HLA-DR and HLA-DQ expression and a higher reactivity for the IL-6 receptor alpha-chain (CD126) and for CD38. In contrast, the CD123strong+/CD33dim+ DC showed a stronger reactivity for the CD4 and CD45RA molecules, whereas they did not express the CD58, CD5, CD11c and CD13 antigens. Regarding cytokine production, our results show that while the CD33strong+/CD123dim+ DC are able to produce significant amounts of inflammatory cytokines, such as IL-1beta (97 +/- 5% of positive cells), IL-6 (96 +/- 1.1% of positive cells), IL-12 (81.5 +/- 15.5% of positive cells) and tumour necrosis factor-alpha (TNF-alpha) (84 +/- 22.1% of positive cells) as well as chemokines such as IL-8 (99 +/- 1% of positive cells), the functional ability of the CD123strong+/CD33dim+ DC subset to produce cytokines under the same conditions was almost null. Our results therefore clearly show the presence of two distinct subsets of DC in normal human PB, which differ not only in their immunophenotype but also in their functionality, as regards cytokine production.