Marie Follo

Chronic lymphocytic leukaemia is driven by antigen-independent cell-autonomous signalling

B-cell antigen receptor (BCR) expression is an important feature of chronic lymphocytic leukaemia (CLL), one of the most prevalent B-cell neoplasias in Western countries. The presence of stereotyped and quasi-identical BCRs in different CLL patients suggests that recognition of specific antigens might drive CLL pathogenesis. Here we show that, in contrast to other B-cell neoplasias, CLL-derived BCRs induce antigen-independent cell-autonomous signalling, which is dependent on the heavy-chain complementarity-determining region (HCDR3) and an internal epitope of the BCR. Indeed, transferring the HCDR3 of a CLL-derived BCR provides autonomous signalling capacity to a non-autonomously active BCR, whereas mutations in the internal epitope abolish this capacity. Because BCR expression was required for the binding of secreted CLL-derived BCRs to target cells, and mutations in the internal epitope reduced this binding, our results indicate a new model for CLL pathogenesis, with cell-autonomous antigen-independent signalling as a crucial pathogenic mechanism.

The Nlrp3 inflammasome regulates acute graft-versus-host disease.

Conditioning therapies before transplantation induce the release of uric acid, which triggers the NLRP3 inflammasome and IL-1β production contributing to graft-versus-host disease.

Visualization of adherent micro-organisms using different techniques

The visualization and quantification of adherent bacteria is still one of the most relevant topics in microbiology. Besides electron microscopic techniques such as transmission electron microscopy, scanning electron microscopy and environmental scanning electron microscopy, modern fluorescence microscopic approaches based on fluorogenic dyes offer detailed insight into bacterial biofilms. The aim of the present review was to provide an overview of the advantages and disadvantages of different methods for visualization of adherent bacteria with a special focus on the experiences gained in dental research.

Epithelial Tissue Chimerism after Human Hematopoietic Cell Transplantation Is a Real Phenomenon

Bone marrow transplantation in animals has been shown to generate epithelial populations, a phenomenon that has also recently been suggested to take place after human hematopoietic cell transplantation. However, reports in humans are not conclusive because they still leave open the possibility that the identified donor-derived cells are not epithelial cells but intraepithelial lymphocytes. Here, we demonstrate that donor-derived CD45(+) hematopoietic cells in close contact with epithelial tissue may be falsely characterized as donor-derived epithelial cells if the three-dimensional structure of the tissue is not considered and the hematopoietic markers are not examined. By using a rigorous three-dimensional analysis on single sections of colon biopsies triple stained with donor-specific, epithelial-specific, and hematopoietic-specific markers we demonstrate that chimerism of colon epithelium is a real phenomenon occurring constantly after human hematopoietic cell transplantation. We exclude horizontal DNA transfer or cell fusion as the underlying mechanism of our findings. Tissue damage enhances the engraftment of the donor-derived epithelial cells. The physiological and therapeutical role of the donor-derived epithelial cells after human hematopoietic cell transplantation needs further investigation. However, their identification requires stringent and unequivocal detection systems.

Definition and characterization of the systemic T-cell dysregulation in untreated indolent B-cell lymphoma and very early CLL

Epidemiologic data show that the immune system may control or promote the emergence and growth of neoplastic lymphomatous clones. Conversely, systemic lymphomas, especially myeloma and chronic lymphocytic leukemia (CLL), are associated with clinical immunodeficiency. This prospective controlled study demonstrates substantially reduced circulating T helper cells, predominantly naive CD4(+) cells, in patients with nonleukemic follicular lymphoma and extranodal marginal zone lymphoma, but not in monoclonal gammopathy and early CLL. These changes were correlated with a preactivated phenotype, hyperreactivity in vitro, pre-senescence, and a T helper 2 shift of peripheral T helper cells. No prominent alterations existed in the regulatory T-cell compartment. Gene expression profiling of in vitro-stimulated CD4(+) cells revealed an independent second alteration of T helper cell physiology, which was most pronounced in early CLL but also detectable in follicular lymphoma/extranodal marginal zone lymphoma. This pattern consisted of down-regulation of T-cell receptor signaling cascades and globally reduced cytokine secretion. Both types of T-cell dysfunction may contribute to significant immunodeficiency in nonleukemic indolent B-cell lymphomas as demonstrated by unresponsiveness to hepatitis B vaccination. The precise definition of systemic T-cell dysfunction serves as the basis to study its prognostic impact, its relationship to the established influence of the lymphoma microenvironment, and its therapeutic manipulation.

BM cells giving rise to MSC in culture have a heterogeneous CD34 and CD45 phenotype

BACKGROUND Mesenchymal stromal cells (MSC) isolated from adult human BM are characterized by their fibroblast-like morphology, adherent growth and capacity to differentiate into adipocytes, osteocytes, chondrocytes, cardiomyocytes and neuroprogenitors. After culturing these cells in vitro, they express the cell-surface molecules CD44, CD90, SH2 and SH3, and are negative for CD34 and the hematopoietic marker CD45. The aim of this study was to characterize the in vivo phenotype of MSC relative to the expression of CD34 and CD45. METHODS BM mononuclear cells were stained with Ab against both molecules and separated into the CD34(+), CD34(-), CD45(+) CD34(+), CD45(high+) CD34(-), CD45(med,low+) CD34(-) and CD45(-) CD34(-) subpopulations, which were then cultured under the same conditions and analyzed for growth of MSC. RESULTS A small population of MSC arose from the CD45(+) CD34(+) fraction, although the majority was obtained from the CD45(-) CD34(-) subpopulation. MSC from all fractions could be differentiated into adipocytes and osteocytes. In addition, MSC from the CD34(+) and CD34(-) fractions were shown to differentiate into chondrocytes. After in vitro culture, MSC from both fractions possessed the same phenotype, which was negative for CD34 and CD45. DISCUSSION MSC from the CD45(+) CD34(+) fraction change their phenotype under in vitro conditions.

The IL-33/ST2 axis augments effector T cell responses during acute GVHD

Interleukin (IL)-33 binding to the receptor suppression of tumorigenicity 2 (ST2) produces pro-inflammatory and anti-inflammatory effects. Increased levels of soluble ST2 (sST2) are a biomarker for steroid-refractory graft-versus-host disease (GVHD) and mortality. However, whether sST2 has a role as an immune modulator or only as a biomarker during GVHD was unclear. We show increased IL-33 production by nonhematopoietic cells in the gastrointestinal (GI) tract in mice post-conditioning and patients during GVHD. Exogenous IL-33 administration during the peak inflammatory response worsened GVHD. Conversely, GVHD lethality and tumor necrosis factor-α production was significantly reduced in il33(-/-) recipients. ST2 was upregulated on murine and human alloreactive T cells and sST2 increased as experimental GVHD progressed. Concordantly, st2(-/-) vs wild-type (WT) donor T cells had a marked reduction in GVHD lethality and GI histopathology. Alloantigen-induced IL-18 receptor upregulation was lower in st2(-/-) T cells, and linked to reduced interferon-γ production by st2(-/-) vs WT T cells during GVHD. Blockade of IL-33/ST2 interactions during allogeneic-hematopoietic cell transplantation by exogenous ST2-Fc infusions had a marked reduction in GVHD lethality, indicating a role of ST2 as a decoy receptor modulating GVHD. Together, these studies point to the IL-33/ST2 axis as a novel and potent target for GVHD therapy.

Transgenic expression of human cathepsin B promotes progression and metastasis of polyoma-middle-T-induced breast cancer in mice.

Elevated expression of the cysteine protease cathepsin B (CTSB) has been correlated with a poor prognosis for cancer patients. In order to model high CTSB expression in mammary cancer, transgenic mice expressing human CTSB were crossed with transgenic polyoma virus middle T oncogene breast cancer mice (mouse mammary tumor virus-PymT), resulting in a 20-fold increase in cathepsin B activity in the tumors of double-transgenic animals. CTSB expression did not affect tumor onset, but CTSB transgenic mice showed accelerated tumor growth with significant increase in weight for end-stage tumors, as well as an overall worsening in their histopathological grades. Notably, the lung metastases in the CTSB transgenic animals were found to be both significantly larger and to occur at a significantly higher frequency. Ex vivo analysis of primary PymT tumor cells revealed no significant effects from elevated CTSB levels on tumor cell characteristics, that is, the formation of tumor cell colonies and the sprouting of invasive strands from PymT cell spheroids. However, tumors from CTSB-overexpressing mice showed increased numbers of tumor-associated B cells and mast cells. In addition, more CD31+ endothelial cells were detected in these tumors, correlating with higher levels of vascular endothelial growth factor (VEGF) being present in the tumor and serum. We conclude that elevated proteolytic CTSB activity facilitates progression and metastasis of PymT-induced mammary carcinomas, and is associated with increased immune cell infiltration, enhanced VEGF levels and the promotion of tumor angiogenesis.

Biofilm formation and composition on different implant materials in vivo

Biofilm formation was evaluated on the following titanium and zirconia implants in vivo: machined titanium (Ti-m), modified titanium (TiUnite), modified zirconia (ZiUnite), machined alumina-toughened zirconia (ATZ-m), sandblasted alumina-toughened zirconia (ATZ-s), and machined zirconia (TZP-A-m). Bovine enamel slabs were used as controls. Surface morphologies were examined by atomic force (AFM) and scanning electron microscopy (SEM). The surface wettability was also determined. Twelve healthy volunteers wore a splint system with the tested materials. After 3 and 5 days the materials were examined by fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). The levels of Streptococcus spp., Veillonella spp., Fusobacteriaum nucleatum, and Actinomyces naeslundii were quantitatively determined. The biofilm thickness was found to be between 19.78 and 36.73 μm after 3 days and between 26.11 and 32.43 μm after 5 days. With the exception of Ti-m the biofilm thickness after 3 days was correlated with surface roughness. In addition to Streptococcus spp. as the main component of the biofilm (11.23-25.30%), F. nucleatum, A. naeslundii, and Veillonella spp. were also detected. No significant differences in biofilm composition on the implant surfaces could be observed. In total, the influence of roughness and material on biofilm formation was compensated by biofilm maturation.

CCL19 is a specific ligand of the constitutively recycling atypical human chemokine receptor CRAM-B

The human chemokine receptor CRAM (chemokine receptor on activated macrophages), encoded by the gene CCRL2, is a new candidate for the atypical chemokine receptor family that includes the receptors DARC, D6 and chemocentryx chemokine receptor (CCX‐CKR). CRAM is maturation‐stage‐dependently expressed on human B lymphocytes and its surface expression is up‐regulated upon short‐term CCL5 exposure. Here, we demonstrate that the homeostatic chemokine CCL19 is a specific ligand for CRAM. In radioactive labelling studies CCL19 bound to CRAM‐expressing cells with an affinity similar to the described binding of its other receptor CCR7. In contrast to the known CCL19/CCR7 ligand/receptor pair, CRAM stimulation by CCL19 did not result in typical chemokine‐receptor‐dependent cellular activation like calcium mobilization or migration. Instead, we demonstrate that CRAM is constitutively recycling via clathrin‐coated pits and able to internalize CCL19 as well as anti‐CRAM antibodies. As this absence of classical chemokine receptor responses and the recycling and internalization features are characteristic for non‐classical chemokine receptors, we suggest that CRAM is the newest member of this group. As CCL19 is known to be critically involved in lymphocyte and dendritic cell trafficking, CCL19‐binding competition by CRAM might be involved in modulating these processes.