Lack of evidence of rotavirus-dependent molecular mimicry as a trigger of coeliac disease.

New data suggest the involvement of rotavirus (RV) in triggering autoimmunity in coeliac disease (CD) by molecular mimicry between the human-transglutaminase protein and the dodecapeptide (260-271 aa) of the RV protein VP7 (pVP7). To assess the role of RV in the onset of CD, we measured anti-pVP7 antibodies in the sera of children with CD and of control groups. We analysed serum samples of 118 biopsy-proven CD patients and 46 patients with potential CD; 32 children with other gastrointestinal diseases; 107 no-CD children and 107 blood donors. Using enzyme-linked immunosorbent assay (ELISA) assay, we measured immunoglobulin (Ig)A-IgG antibodies against the synthetic peptides pVP7, the human transglutaminase-derived peptide (476-487 aa) which shows a homology with VP7 protein and a control peptide. The triple-layered RV particles (TLPs) containing the VP7 protein and the double-layered RV-particles (DLPs) lacking the VP7 protein were also used as antigens in ELISA assay. Antibody reactivity to the RV-TLPs was positive in 22 of 118 (18%) CD patients and in both paediatric (17 of 107, 16%) and adult (29 of 107, 27%) control groups, without showing a statistically significant difference among them (P = 0·6, P = 0·1). Biopsy-proven CD patients as well as the adult control group demonstrated a high positive antibody reactivity against both pVP7 (34 of 118, 29% CD patients; 66 of 107, 62% adult controls) and control synthetic peptides (35 of 118, 30% CD patients; 56 of 107, 52% adult controls), suggesting a non-specific response against RV pVP7. We show that children with CD do not have higher immune reactivity to RV, thus questioning the molecular mimicry mechanism as a triggering factor of CD.

Different expression of CD41 on human lymphoid and myeloid progenitors from adults and neonates.

The glycoprotein (Gp) IIb/IIIa integrin, also called CD41, is the platelet receptor for fibrinogen and several other extracellular matrix molecules. Recent evidence suggests that its expression is much wider in the hematopoietic system than was previously thought. To investigate the precise expression of the CD41 antigen during megakaryocyte (MK) differentiation, CD34(+) cells from cord blood and mobilized blood cells from adults were grown for 6 days in the presence of stem cell factor and thrombopoietin. Two different pathways of differentiation were observed: one in the adult and one in the neonate cells. In the neonate samples, early MK differentiation proceeded from CD34(+)CD41(-) through a CD34(-)CD41(+)CD42(-) stage of differentiation to more mature cells. In contrast, in the adult samples, CD41 and CD42 were co-expressed on a CD34(+) cell. The rare CD34(+)CD41(+)CD42(-) cell subset in neonates was not committed to MK differentiation but contained cells with all myeloid and lymphoid potentialities along with long-term culture initiating cells (LTC-ICs) and nonobese diabetic/severe combined immune-deficient repopulating cells. In the adult samples, the CD34(+)CD41(+)CD42(-) subset was enriched in MK progenitors, but also contained erythroid progenitors, rare myeloid progenitors, and some LTC-ICs. All together, these results demonstrate that the CD41 antigen is expressed at a low level on primitive hematopoietic cells with a myeloid and lymphoid potential and that its expression is ontogenically regulated, leading to marked differences in the surface antigenic properties of differentiating megakaryocytic cells from neonates and adults. (Blood. 2001;97:2023-2030)

Stromal cells retard the differentiation of CD34(+)CD38(low/neg) human primitive progenitors exposed to cytokines independent of their mitotic history.

Stem cell proliferation induced by potent cytokines usually leads to a loss of primitive potential through differentiation. In this study, the ability of cytokines and murine MS5 stromal cells to independently regulate the proliferation and long-term culture-initiating cell (LTC-IC) activity of primitive CD34(+)CD38(low/neg) human bone marrow cells was evaluated. To compare populations with identical proliferation histories, cells were labeled with carboxy fluorescein diacetate succinimidyl ester, and LTC-IC activity was assessed 4 days later in cells that had accomplished the same number of divisions with or without MS5 cells. MS5 cells counteracted dramatically the loss of LTC-IC activity observed in the presence of cytokines alone. Thus, in the presence of MS5 cells, means of 1233 (n = 5) and 355 (n = 9) LTC-IC-derived colony-forming cells (CFCs) were generated by 1000 cells that performed 3 and 4 divisions respectively, whereas 311 (n = 5) and 64 (n = 5) CFCs were generated by 1000 cells cultured without MS5 cells. Interestingly, MS5 cells had no detectable effect on the LTC-IC activity of cells that divided only twice in 4 days-1606 CFCs (n = 6) and 1993 (n = 6) CFCs, respectively, without and with MS5 cells-and a 48 additional hours of coculture were necessary to unmask changes in the LTC-IC activity mediated by stromal cells. These results indicate that cytokines and stroma-derived signals can regulate independently the proliferation and differentiation of primitive cells and that these stroma-derived extracellular factors act directly on their target cells.

G10.3 monoclonal antibody identifies novel functional cell surface structures expressed by normal B lymphocytes and various malignant cell lines.

G10.3, a unique monoclonal antibody (mAb), was produced to better characterize lymphocyte subsets. In the present study, we show that this mAb identifies 118, 83 and 51 kDa cell surface sialylated glycoproteins on the immunizing cell line YTindi. The reactivity of G10.3 mAb is restricted in normal cells to B lymphocytes, whereas within tumoral cell lines various lymphoid and non-lymphoid cells were found positive. Interestingly, functional studies revealed that triggering G10.3 mAb reactive molecules with soluble antibody led to an inhibition of growth and to an induction of programmed cell death in tumor cell lines expressing high levels of reactive molecules.

Lymphocytes subsets in normal individuals: analysis by four color immunofluorescence and flow cytometry on whole blood.

In order to precisely define human lymphocytes subsets, we used four color immunofluorescence analysis and flow cytometry. We report here the results of systematic studies performed on whole blood from 20 normal volunteers. This was performed using monoclonal antibodies recognizing 18 different CD molecules and analyzed here into 14 different 4-color immunofluorescence combinations. This showed that among T cells, the two major CD3+CD8+ and CD3+CD8- subsets differed greatly in the percentage of cells expressing CD26, CD27, CD28, CD38, CD45RA, CD45RO, CD57, S6F1, BY55, CD101 and their respective combination representing an array of subsets. Furthermore the minor NK subsets, i.e. CD2+CD3-CD8+, CD2-CD3-CD8+, CD2+CD3-CD8-, CD2-CD3-CD8- also differed in percentages of cells expressing CD16, CD56, CD57, S6F1, BY55 and their combinations.

BY55 monoclonal antibody delineates within human cord blood and bone marrow lymphocytes distinct cell subsets mediating cytotoxic activity.

We had previously reported, using BY55 monoclonal antibody, a cell surface 80-kDa protein restricted to human functional peripheral blood cytotoxic lymphocytes with either natural killer CD3- or cytotoxic T lymphocyte CD3+CD8+ phenotype. In the present report, we studied the cytotoxic lymphocytes in adult bone marrow and newborn cord blood as these organs are commonly used as sources of hematological stem cells for allogeneic transplantation. Our results showed that BY55 mAb labeled only 5-10% of the bone marrow lymphocytes, which included a major proportion of CD3+ CD8+ cytotoxic T lymphocytes. Interestingly, within cord blood cells, BY55+ lymphocytes represented 20-35% of the lymphocytes corresponding exclusively to a CD3- cell subset. Furthermore, we detected in cord blood no cytotoxic T lymphocyte activity but we demonstrated that the CD3-BY55+ cell subset contained the whole natural killer activity.

Significant enlargement of a specific subset of CD3+CD8+ peripheral blood leukocytes mediating cytotoxic T-lymphocyte activity during human immunodeficiency virus infection.

We have obtained a monoclonal antibody termed BY55 that defines an 80-kDa cell-surface structure on a subset of circulating peripheral blood mononucleocytes. This structure, which was not detected on most cell lines or activated lymphocytes, was expressed exclusively on 15-25% of CD2+ circulating lymphocytes, including a major subset within the CD3- and the T-cell receptor gamma delta + lymphocytes and a small percentage of the CD3+CD8+ cells. Moreover, we have shown that the BY55 molecule delineated the competent killer circulating lymphocytes. In the present report, additional two- and three-color immunofluorescence studies of peripheral blood lymphocytes were done to precisely determine BY55 expression within the T-cell population. In normal individuals, peripheral blood CD3+CD8+BY55+ cells represented only 5-6% of the lymphocytes, and these cells possessed cytolytic activity. Interestingly, we found that the percentage of total BY55+ lymphocytes as well as the percentage of CD3+CD8+BY55+ was significantly increased in peripheral blood lymphocytes of human immunodeficiency virus-seropositive individuals.

A novel 80-kD cell surface structure identifies human circulating lymphocytes with natural killer activity.

Human lymphocytes with natural killer (NK) activity, including most activated gamma/delta+ T lymphocytes, recognize and lyse tumor target cells without requiring recognition of major histocompatibility complex antigen. However, unlike gamma/delta+ T lymphocytes, NK cells do not express CD3/T cell receptor (TCR) molecules, and the receptors involved in cell-mediated cytotoxicity are unknown. To further delineate circulating NK cells, we developed monoclonal antibodies (mAbs) against the human NK leukemia YT2C2. We report the isolation of a mAb termed BY55, recognizing at the cell surface a novel 80-kD protein with restricted expression. In addition to the immunizing cell line, this mAb binds to circulating NK cells, gamma/delta+ cells, and a minor subset of alpha/beta+ T lymphocytes. Expression of the BY55 mAb-reactive epitope/molecule is regulated by activation, as short-term culture of peripheral blood lymphocytes (PBL) with phorbol ester induced its downmodulation. Furthermore, BY55 mAb reactivity was found neither with the NK nor with the TCR alpha/beta+ gamma/delta+ clones tested. Biochemical studies as well as phenotypic analysis revealed that this structure is different from all previously identified molecules on the lymphocyte cell surface. Interestingly, we found that BY55+ cells exert most NK activity obtained with fresh circulating lymphocytes. We report that within fresh E rosette-positive PBL only a subset of the CD16+, CD56+, and CD57+ cells coexpressed BY55 molecule, indicating that BY55 mAb defines a unique subset mediating NK activity of circulating PBL.