Quantitative analysis of the phase volume of agarose-canola oil gels in comparison to blending law predictions using 3D imaging based on confocal laser scanning microscopy

Studying the phase behaviour of composite gels facilitates understanding of their structural and textural properties at low and intermediate levels of solids. In this work, the phase behaviour of a model system of agarose including various concentrations of canola oil was studied. This was pursued using a variety of techniques including SEM, FTIR, microDSC and dynamic oscillation in-shear. The structural studies recorded strong, continuous agarose networks supporting soft, discontinuous canola oil inclusions, with increasing levels of canola oil strengthening the composite system. A novel confocal laser scanning microscopy (CLSM) method for quantitative in situ examination of the oil phase volume was developed using three-dimensional (3D) imaging and image analysis software - FIJI and Imaris. Microscopic observations were assessed in relation to theoretical predictions from rheology-based blending-law analysis. Quantitative outcomes from the combined 3D imaging and image analysis are in close agreement with the volume predictions for the oil phase obtained from the isostrain blending law indicating the suitability of this approach in quantifying the phase behaviour of composite materials. The results of this work indicate that the developed microscopic method shows promise and could be used in the determination of phase volume in more complex and industrially relevant systems

Expression profile and clonality of T-Cell receptor beta variable genes in normal human endometrium

Problem: In spite of their key immunological role, αβ+ T cells residing in endometrium have not been extensively explored. We analyzed here expression profile of TCRBV genes in normal human endometrium compared with peripheral blood. Methods: Samples were taken from normal reproductive women. RT-PCR using BV-gene specific primers was performed on blood and endometrial samples. After blotting, hybridization with radiolabelled probe and autoradiography, relative expression of each TCRBV family was determined. Clonal expansions of the over-expressed genes were assessed by CDR3 length polymorphism. Results: Only one gene (TCRBV7) was expressed significantly and two other genes marginally more in the endometrium compared with blood. All three TCRBV genes examined showed a rather restricted pattern in the endometrium in contrast to polyclonal patterns in the blood. Conclusion: Our results stress the similarities between T cells residing in different mucosal tissues and provide a basis for future investigations about endometrial T cells and their antigen specificities in gynecological problems. © 2006 The Authors Journal compilation 2006 Blackwell Munksgaard

Microenvironment of the feto-maternal interface protects the semiallogenic fetus through its immunomodulatory activity on dendritic cells

Objective: To investigate the immunomodulatory activity of decidual culture supernatant on dendritic cell (DC) functions. Design: In vivo and in vitro experimental study using mice. Setting: Academic research laboratory. Animal(s): C57BL/6-mated female Balb/c mice. Intervention(s): Culture supernatants of decidual cells obtained from the uteri of allogenic pregnant mice (Balb/c � C57BL/6) were collected. Dendritic cells were purified from Balb/c mice spleens and pulsed with antigen during overnight culture. In some cultures, decidual supernatant was added at 5, 10, or 20 final concentration. Endometrial culture supernatant-treated DCs served as a control. Antigen-pulsed DCs were injected into the front footpads of syngeneic mice. Main Outcome Measure(s): Lymph nodes of primed mice were removed 5 days after DC injection. Antigen-specific proliferation and interleukin-10 and interferon gamma production by lymphocytes were measured by 3H-Thymidine incorporation and ELISA, respectively. Result(s): The results showed that decidual culture supernatant markedly blocked in vivo antigen presentation by DCs and inhibited their capacity to induce interferon gamma (but not interleukin-10) production by primed lymphocytes. Conclusion(s): It seems that soluble factors produced by decidual cells are important mediators of immunoregulation at the feto-maternal interface, which provide the two fundamental requirements for protection of the semiallogenic fetus, namely immunologic tolerance and predominance of T helper 2 immunity, through modulation of DCs function. © 2008 American Society for Reproductive Medicine

The efficient isolation of murine splenic dendritic cells and their cytochemical features

Despite their importance in professional antigen presentation and their ubiquitous presence, dendritic cells (DCs) are usually found in such trace amounts in tissues that their isolation with high purity is a difficult task. Because of their scarcity, accurate determination of the purity of isolated dendritic cells is very important. In this study, we purified murine splenic dendritic cells by a three-step enrichment method and evaluated their morphological, cytochemical and functional characteristics. Purity of the isolated cells was determined by established methods such as flow cytometry (FC) and immunocytochemistry (ICC) using anti-CD11c monoclonal antibody. In order to test purified DC functional properties, we used in vivo antigen presentation assay. Our results showed that antigen-pulsed DCs are potent stimulators of antigen-specific lymphocyte proliferation. We studied myeloperoxidase (MPO) and non-specific esterase (NSE) activity in isolated cells to determine the purity of dendritic cells compared to more conventional methods. Our results showed that murine splenic dendritic cells were deficient in both MPO and NSE activity and the percentage of purity obtained by NSE staining on isolated cells was comparable to the results obtained by either FC or ICC. To our knowledge, this is the first report on using NSE activity for determination of the purity of isolated murine splenic dendritic cells. We, therefore, recommend that NSE activity be employed as a simple, inexpensive and yet accurate method for evaluation of the purity of isolated murine splenic dendritic cells. © Springer-Verlag 2006

Evaluation of thyroglobulin expression in murine reproductive organs during pregnancy

Problem: In pregnant women with antithyroglobulin antibody, prevalence of abortion is 2-4 fold higher compared to normal controls. Direct effect of such harmful autoantibodies on female reproductive organs may serve a role in pregnancy loss. Method of study: Expression of thyroglobulin in decidua, placenta, and ovary of pregnant Balb/c mice ((Balb/c�Balb/c and Balb/c�C57BL/6) during early, middle, and late stages of pregnancy was evaluated. Expression of thyroglobulin was investigated in these tissues by semi-quantitative RT-PCR. In addition, polyclonal antithyroglobulin antibody was produced, and expression of thyroglobulin protein in aforesaid tissues was evaluated by immunohistochemistry and dot-blot analysis. Results: The results showed that thyroglobulin message is not expressed in placenta, decidua, or ovary in any stages of pregnancy. The same results were obtained at the protein level. Conclusion: It is likely that antithyroglobulin antibodies have no direct detrimental effect on such organs in patients with thyroid autoimmunity suffering from recurrent abortion. © 2010 John Wiley & Sons A/S

Repertoire and clonality of t-cell receptor beta variable genes expressed in endometrium and blood t cells of patients with recurrent spontaneous abortion

Problem: Recurrent spontaneous abortion (RSA) is a relatively common disorder, the underlying causes of which are thought to be immunological in most cases. Method of Study: Expression profile and clonality pattern of T-cell receptor beta variable (TCRBV) genes in endometrium and blood of patients with RSA were investigated by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) using BV gene-specific primers. Relative expression of each BV family was determined and clonal expansion of the over-expressed genes was assessed by analysis of CDR3 length polymorphism. Results: Compared to blood, relative expression of four TCRBV genes was significantly higher in the endometrium of RSA group. Over-expressed genes, except for TCRBV3, all had restricted and oligoclonal patterns of expression in the endometrium. Conclusion: Endometrial T cells have a skewed TCRBV repertoire with restricted transcript heterogeneity, which is shared by both groups and minor variations observed in this pattern in RSA patients may reflect more recent and/or repeated exposure to nominal antigens or superantigens. © 2008 The Authors Journal compilation © 2008 Blackwell Munksgaard