Regulation of major histocompatibility complex class I expression by NF-kB-related proteins in breast cancer cells

Downregulation of MHC Class I antigens has been observed in many cancers and usually results from a decreased gene transcription. A reporter CAT gene dependent on the MHC Class I kB site or on a longer promoter is transactivated by NF-kB complexes contain- ing p65 or RelB. p100 as well as IkB-a are potent inhibitors of this transcription and p100 sequesters RelB and p65 complexes in the cytoplasm of breast cancer cells. However, although p100 is highly expressed in a number of breast cancer cell lines, MHC Class I antigen expression was observed on all the cell lines we analysed and could be further induced by stimulation with the cytokines IFN-g or TNF-a. Stable transfection of a unresponsive mutated IkB-a Ser 32-36 expression vector showed that TNF-a induced MHC Cl I expression in an NF-kB-dependent way while IFN-g did it independently of any NF-kB activation

Modulation of homing properties of primitive progenitor cells generated by ex vivo expansion.

BACKGROUND AND OBJECTIVES: The maintenance of adequate interactions with the bone marrow (BM) microenvironment is critical to ensure efficient homing of ex vivo-expanded hematopoietic cells. This study was intended to assess adhesion and migration properties of long-term culture-initiating cells (LTC-IC) harvested after self-renewal division in ex vivo culture and to determine their susceptibility to growth-inhibitory signals mediated by adhesion to BM stromal ligands. DESIGN AND METHODS: We used cell tracking to isolate primitive LTC-IC that had accomplished 1 or 2 divisions ex vivo. Adhesion, migration and growth inhibition of divided LTC-IC were determined in the presence of purified BM ligands, and compared to the properties of uncultured LTC-IC. RESULTS: As compared to undivided LTC-IC, adhesion and migration mediated by very late antigen (VLA)-4 integrin across both vascular cell adhesion molecule-1 (VCAM-1) and fibronectin (Fn) were downregulated in post-mitotic LTC-IC. Conversely, binding and motility via VLA-5 across Fn were stimulated. No changes occurred in LTC-IC interactions with intercellular adhesion molecule-1 (ICAM-1) or with E- or P-selectin. Proliferation of uncultured LTC-IC was inhibited by VLA-4-mediated binding to VCAM-1 and the CS-1 domain of Fn, as well as binding to P-selectin. Growth of ex vivo-generated LTC-IC became unresponsive to these 3 ligands but was suppressed through VLA-5 engagement by the cell binding domain of Fn. INTERPRETATION AND CONCLUSIONS: The generation of LTC-IC in expansion culture is associated with functional alterations of adhesion receptors, modulating not only binding and migration in the BM but also responsiveness to adhesion-mediated growth inhibitory signals. Such changes may limit homing and engraftment of expanded primitive stem/progenitor cells

Dendritic cell differentiation and immune tolerance to insulin-related peptides in Igf2-deficient mice

There is some evidence that insulin-like growth factor 2 (IGF-2) may intervene in the control of T cell differentiation. To further study the immunoregulatory function of this growth factor, we analyzed the immune system of Igf2(-/-) mice. Phenotypically, some immunological parameters such as lymphoid organ morphology and cellularity were unaltered in Igf2(-/-) mice, but an increase of CD8(+) cells and a decrease of B220(+) cells were observed in spleen. In vitro, the development of bone marrow-derived dendritic cells was affected by the absence of Igf2 expression. After maturation, a higher percentage of immature dendritic cells was observed in Igf2(-/-) population, together with a secondary decrease in allogenic T cell proliferation. Activation of T cells was also affected by the lack of expression of this growth factor. The profile of B cell response in mutant mice immunized with IGF-2 evidenced a T-dependent profile of anti-IGF-2 Abs that was absent in Igf2(+/+) mice. The influence of IGF-2 upon tolerance to insulin was also assessed in this model, and this showed that IGF-2 also intervenes in tolerance to insulin. The presence of a T-dependent response in Igf2-deficient mice should allow cloning of specific "forbidden" T CD4(+) lymphocytes directed against IGF-2, as well as further investigation of their possible pathogenic properties against insulin family

Open Implementation of DICOM for Whole-Slide Microscopic Imaging

peer reviewedThis paper introduces an open implementation of DICOM for whole-slide microscopic imaging, following Supplement 145 of the DICOM standard. The software is divided into two parts: (a) a command-line tool to convert an whole-slide image to the DICOM format, and (b) a zero-footprint Web interface to display such DICOM images. The software architecture leverages the DICOM server Orthanc. The entire framework is available as free and open-source software. The existence of this software supports the development of digital pathology and telepathology in clinical environments, featuring a smooth integration with existing EHR and PACS solutions

Cytofluorometric analysis of prostate cancer specimens: Histological and clinical correlations

DNA histograms were oblained by flow cytometry for 39 human prostate carcinomas (27 total proslatectomy specimens, 5 biopsies and 7 transuretral resections). The study was performed on formalin-fixed and paraffin-embedded. material. In this report, ploidy index did not seem to be a good marker of prognosis as no significant variation in ploidy was found neither among the different stages nor among the different Gleason categories. Proliferative index of the tumors seemed to be a more sensitive parameter; a significant relation was observed between proliferative index and stage of the tumor. The authors discuss these results under the light of previously reported observations

In Vitro Propagated Dendritic Cells from Patients with Human-Papilloma Virus-Associated Preneoplastic Lesions of the Uterine Cervix: Use of Flt3 Ligand

Dendritic cells (DC) are the most efficient antigen presenting cells. The clinical use of DC as vectors for antitumor and anti-infectious disease immunotherapy has been limited by their low level and accessibility in normal tissue. Substantial numbers of DC can be generated from peripheral blood cultured in the presence of interleukin-4 (IL-4) and granulocyte/macrophage-colony-stimulating factor (GM-CSF). We showed in this study that substantial numbers of DC can be obtained from the peripheral blood of patients with (pre)neoplastic lesions of the uterine cervix. The procedure required relatively small blood samples (10 ml) and the presence of 100 U/ml IL-4 and 800 U/ml GM-CSF in the culture medium. There was no significant difference in the morphology, yield, phenotype and function of generated DC between patients with cervical (pre)neoplastic lesions and healthy individuals. When the hematopoietic factor Flt3 ligand (Flt3L, 40 ng;ml) was added, there was an average increase in the DC population of 26% compared to cultures with GM-CSF and IL-4 alone. Approximately 1.2 x 10(6) cells with the characteristics of dendritic cells could be obtained when Flt3L was included in the medium. The addition of Flt3L did not modify the phenotypic profile of DC (HLA-DR+, CD1a+, CD4+, CD54+, CD80+, CD86+. CD40+, CD3- and CD14-). In addition, Flt3L generated functional DC capable of stimulating the proliferation of alloreactive T cells. These results suggest that Flt3L, in association with GM-CSF and IL-4, provides an advantageous tool for the large-scale generation of DC and that an immunotherapy based on the use of DC generated in vitro is possible in patients with (pre)neoplastic lesions of the uterine cervix

Maintenance of functional human cancellous bone and human hematopoiesis in NOD/SCID mice

Attempts were made to establish models to study interactions between marrow stromal cells and hematopoietic cells in vivo. The approach was to create a NOD-SCID-hu murine model of long-term human hematopoiesis by implantation of a human adult bone fragment. Nine to 12 weeks posuransplantation, human CD45(+) cells were detected in the blood and the spleen of some mice. The histology of the human transplant showed that human bone fragment was viable at 9 weeks. Moreover, vessels of human origin, as assessed by immunohistochemical detection of human beta(2)-microglobulin, were observed in the mouse tissue surrounding the transplanted human fragment

Further Characterization of Cytotoxic T Cells Generated by Short-Term Culture of Human Peripheral Blood Lymphocytes with Interleukin-2 and Anti-Cd3 Mab

In this study we have specifically investigated the participation of T cells in the cytotoxic activity of peripheral blood lymphocytes (PBL) activated by interleukin-2 (IL-2, 50 U/ml) alone or in combination with an anti-CD3 mAb (BMA030, 10 ng/ml, IgG2a). Purified CD3+ T cells, incubated in the presence of the anti-CD3 mAb for 4 days, mediated a cytotoxic activity against HL60 and U937 tumor cell lines. Several findings suggested the involvement of a redirected-cytotoxicity phenomenon, since the lytic process was restricted to target cell lines bearing the high-affinity Fc gamma receptor (Fc gamma RI) and T lymphocytes stimulated by IL-2 alone did not lyse these cell lines. Furthermore, anti-CD3 mAb F(ab')2, anti-CD3 IgG1 (UCHT1), phytohemagglutinin or staphylococcal enterotoxin A did not induce a similar cytotoxic activity in T lymphocytes. The cytotoxic process occurred in the presence of a very low level of anti-CD3 antibodies (in the nanomolar range). The cytotoxic activity of T cells stimulated by IL-2 or by IL-2 + BMA030, against OVCAR-3 cells (MOv18+ ovarian tumor cell line), was also compared in the presence of a bispecific antibody OC/TR, anti-CD3 x MOv18). The stimulation by IL-2 + BMA030 induced approximately a twofold higher cytotoxic activity than IL-2-activated T cells. This could be related to the state of activation of effector cells stimulated by IL-2 + BMA030, since the phenotypic analysis showed an increased proportion of T cells expressing several activation/differentiation markers (CD25, HLA-DR, CD45R0, adhesion molecules). These findings could be applied to the design of therapeutic protocols using anti-CD3 x antitumoral bispecific antibodies