The bone marrow compartment is modified in the absence of galectin-3

Galectin-3 (gal-3) is a β-galactoside binding protein present in multivalent complexes with an extracellular matrix and with cell surface glycoconjugates. In this context, it can deliver a variety of intracellular signals to modulate cell activation, differentiation and survival. In the hematopoietic system, it was demonstrated that gal-3 is expressed in myeloid cells and surrounding stromal cells. Furthermore, exogenous and surface gal-3 drive the proliferation of myeloblasts in a granulocyte–macrophage colony-stimulating factor (GM-CSF)-dependent manner. Here, we investigated whether gal-3 regulates the formation of myeloid bone marrow compartments by studying galectin-3−/− mice (gal-3−/−) in the C57BL/6 background. The bone marrow histology of gal-3−/− mice was significantly modified and the myeloid compartments drastically disturbed, in comparison with wild-type (WT) animals. In the absence of gal-3, we found reduced cell density and diaphyseal disorders containing increased trabecular projections into the marrow cavity. Moreover, myeloid cells presented limited capacity to differentiate into mature myeloid cell populations in gal-3−/− mice and the number of hematopoietic multipotent progenitors was increased relative to WT animals. In addition, bone marrow stromal cells of these mice had reduced levels of GM-CSF gene expression. Taken together, our data suggest that gal-3 interferes with hematopoiesis, controlling both precursors and stromal cells and favors terminal differentiation of myeloid progenitors rather than proliferation

The Role of Galectin-1 and Galectin-3 in the Mucosal Immune Response to Citrobacter rodentium Infection

Despite their abundance at gastrointestinal sites, little is known about the role of galectins in gut immune responses. We have therefore investigated the Citrobacter rodentium model of colonic infection and inflammation in Galectin-1 or Galectin-3 null mice. Gal-3 null mice showed a slight delay in colonisation after inoculation with C. rodentium and a slight delay in resolution of infection, associated with delayed T cell, macrophage and dendritic cell infiltration into the gut mucosa. However, Gal-1 null mice also demonstrated reduced T cell and macrophage responses to infection. Despite the reduced T cell and macrophage response in Gal-1 null mice, there was no effect on C. rodentium infection kinetics and pathology. Overall, Gal-1 and Gal-3 play only a minor role in immunity to a gut bacterial pathogen

Visualization of Gli Activity in Craniofacial Tissues of Hedgehog-Pathway Reporter Transgenic Zebrafish

The Hedgehog (Hh)-signaling pathway plays a crucial role in the development and maintenance of multiple vertebrate and invertebrate organ systems. Gli transcription factors are regulated by Hh-signaling and act as downstream effectors of the pathway to activate Hh-target genes. Understanding the requirements for Hh-signaling in organisms can be gained by assessing Gli activity in a spatial and temporal fashion.We have generated a Gli-dependent (Gli-d) transgenic line, Tg(Gli-d:mCherry), that allows for rapid and simple detection of Hh-responding cell populations in both live and fixed zebrafish. This transgenic line expresses a mCherry reporter under the control of a Gli responsive promoter, which can be followed by using fluorescent microscopy and in situ hybridization. Expression of the mCherry transgene reporter during embryogenesis and early larval development faithfully replicated known expression domains of Hh-signaling in zebrafish, and abrogating Hh-signaling in transgenic fish resulted in the suppression of reporter expression. Moreover, ectopic shh expression in Tg(Glid:mCherry) fish led to increased transgene production. Using this transgenic line we investigated the nature of Hh-pathway response during early craniofacial development and determined that the neural crest skeletal precursors do not directly respond to Hh-signaling prior to 48 hours post fertilization, suggesting that earlier requirements for pathway activation in this population of facial skeleton precursors are indirect.We have determined that early Hh-signaling requirements in craniofacial development are indirect. We further demonstrate the Tg(Gli-d:mCherry) fish are a highly useful tool for studying Hh-signaling dependent processes during embryogenesis and larval stages

Galectin-1 Is Part of Human Trophoblast Invasion Machinery - A Functional Study In Vitro

Interactions of glycoconjugates with endogenous galectins, have been long proposed to participate in several reproductive processes including implantation. In human placenta gal-1, gal-3, gal-8, and gal-13 proteins are known to be present. Each of them has been proposed to play multiple functions, but so far no clear picture has emerged. We hypothesized that gal-1 participates in trophoblast invasion, and conducted Matrigel invasion assay using isolated cytotrophoblast from first trimester placenta and HTR-8/SVneo cell line to test it.<0.001) by Ox-gal-1 at 1 µg/ml. Both sets of results confirmed involvement of gal-1 in trophoblast invasion. Galectin profile of isolated cytotrophoblast and HTR-8/SVneo cells was established using RT-PCR and real-time PCR and found to consist of gal-1, gal-3 and gal-8 for both cell types. Only gal-1 was located at the trophoblast cell membrane, as determined by FACS analysis, which is consistent with the results of the functional tests.These findings qualify gal-1 as a member of human trophoblast cell invasion machinery

Long term survival following the detection of circulating tumour cells in head and neck squamous cell carcinoma

Background Techniques for detecting circulating tumor cells in the peripheral blood of patients with head and neck cancers may identify individuals likely to benefit from early systemic treatment. Methods Reconstruction experiments were used to optimise immunomagnetic enrichment and RT-PCR detection of circulating tumor cells using four markers (ELF3, CK19, EGFR and EphB4). This method was then tested in a pilot study using samples from 16 patients with advanced head and neck carcinomas. Results Seven patients were positive for circulating tumour cells both prior to and after surgery, 4 patients were positive prior to but not after surgery, 3 patients were positive after but not prior to surgery and 2 patients were negative. Two patients tested positive for circulating cells but there was no other evidence of tumor spread. Given this patient cohort had mostly advanced disease, as expected the detection of circulating tumour cells was not associated with significant differences in overall or disease free survival. Conclusion For the first time, we show that almost all patients with advanced head and neck cancers have circulating cells at the time of surgery. The clinical application of techniques for detection of spreading disease, such as the immunomagnetic enrichment RT-PCR analysis used in this study, should be explored further

Interplay of Nkx3.2, Sox9 and Pax3 Regulates Chondrogenic Differentiation of Muscle Progenitor Cells

Muscle satellite cells make up a stem cell population that is capable of differentiating into myocytes and contributing to muscle regeneration upon injury. In this work we investigate the mechanism by which these muscle progenitor cells adopt an alternative cell fate, the cartilage fate. We show that chick muscle satellite cells that normally would undergo myogenesis can be converted to express cartilage matrix proteins in vitro when cultured in chondrogenic medium containing TGFß3 or BMP2. In the meantime, the myogenic program is repressed, suggesting that muscle satellite cells have undergone chondrogenic differentiation. Furthermore, ectopic expression of the myogenic factor Pax3 prevents chondrogenesis in these cells, while chondrogenic factors Nkx3.2 and Sox9 act downstream of TGFß or BMP2 to promote this cell fate transition. We found that Nkx3.2 and Sox9 repress the activity of the Pax3 promoter and that Nkx3.2 acts as a transcriptional repressor in this process. Importantly, a reverse function mutant of Nkx3.2 blocks the ability of Sox9 to both inhibit myogenesis and induce chondrogenesis, suggesting that Nkx3.2 is required for Sox9 to promote chondrogenic differentiation in satellite cells. Finally, we found that in an in vivo mouse model of fracture healing where muscle progenitor cells were lineage-traced, Nkx3.2 and Sox9 are significantly upregulated while Pax3 is significantly downregulated in the muscle progenitor cells that give rise to chondrocytes during fracture repair. Thus our in vitro and in vivo analyses suggest that the balance of Pax3, Nkx3.2 and Sox9 may act as a molecular switch during the chondrogenic differentiation of muscle progenitor cells, which may be important for fracture healing