Identification of compounds responsible for the anthelmintic effects of chicory (Cichorium intybus) by molecular networking and bio-guided fractionation

[EN] Increasing resistance towards anthelmintic drugs has necessitated the search for alternative treatments for the control of gastrointestinal nematode parasites. Animals fed on chicory (Cichorium intybus L.), a temperate (pasture) crop, have reduced parasite burdens, hence making C. intybus a potentially useful source for novel anthelmintic compounds or a diet-based preventive/therapeutic option. Here, we utilized in vitro bioassays with the parasitic nematode Ascaris suum and molecular networking techniques with five chicory cultivars to identify putative active compounds. Network analysis predicted sesquiterpene lactones (SL) as the most likely group of anthelmintic compounds. Further bioassay-guided fractionation supported these predictions, and isolation of pure compounds demonstrated that the SL 8-deoxylactucin (8-DOL) is the compound most strongly associated with anti-parasitic activity. Furthermore, we showed that 8-DOL acts in a synergistic combination with other SL to exert the anti-parasitic effects. Finally, we established that chicory-derived extracts also showed activity against two ruminant nematodes (Teladorsagia circumcincta and Cooperia oncophora) in in vitro assays. Collectively, our results confirm the anti-parasitic activity of chicory against a range of nematodes, and pave the way for targeted extraction of active compounds or selective breeding of specific cultivars to optimize its future use in human and veterinary medicineSIWe are very grateful for the guidance and support by Dr. Thomas Ostenfeld Larsen and Christopher Phippen, Technical University of Denmark, Natural product discovery, and the laboratory assistance of Mette Schjelde, University of Copenhagen. This work was funded by the Danish Council for Independent Research (Grant DFF–6111-00394). Fractionation and purification of compounds were further supported by the Green Development and Demonstration Program (GUDP) (Project No. 34009-17-1220). MPE was supported by CONICYT Chile (FONDECYT Postdoctorado #3170875

ES-Cell Derived Hematopoietic Cells Induce Transplantation Tolerance

Background: Bone marrow cells induce stable mixed chimerism under appropriate conditioning of the host, mediating the induction of transplantation tolerance. However, their strong immunogenicity precludes routine use in clinical transplantation due to the need for harsh preconditioning and the requirement for toxic immunosuppression to prevent rejection and graft-versus-host disease. Alternatively, embryonic stem (ES) cells have emerged as a potential source of less immunogenic hematopoietic progenitor cells (HPCs). Up till now, however, it has been difficult to generate stable hematopoietic cells from ES cells. Methodology/Principal Findings: Here, we derived CD45 + HPCs from HOXB4-transduced ES cells and showed that they poorly express MHC antigens. This property allowed their long-term engraftment in sublethally irradiated recipients across MHC barriers without the need for immunosuppressive agents. Although donor cells declined in peripheral blood over 2 months, low level chimerism was maintained in the bone marrow of these mice over 100 days. More importantly, chimeric animals were protected from rejection of donor-type cardiac allografts. Conclusions: Our data show, for the first time, the efficacy of ES-derived CD45 + HPCs to engraft in allogenic recipient

Tolerance induction is accompanied by the presence of CD4FoxP3⁺ T cells in the tolerated allografts but not in the syngeneic control grafts.

<p>A) & B) Frozen sections of tolerated cardiac allografts were stained for CD4 on day 40 post-transplantation using a peroxidase-conjugated antibody (A). A section from an acutely rejected graft was used as control (B). Positively stained cells are stained brown. C)–H) To determine whether any of the CD4⁺ T cells were Tregs, frozen sections were co-stained for CD4 (FITC) and FoxP3 (PE). In the tolerant allograft, FoxP3⁺ cells (C) and CD4⁺ (D) cells were detected, which overlapped when merged (E) suggesting that the CD4⁺ cells expressed FoxP3. In contrast, none of the syngeneic or rejected allografts showed any FoxP3-expressing cells, as reflected by the merged stains of both FoxP3 and CD4 stains in F and G, respectively. (Magnification for C–E: ×600; magnification for F and G: ×200.)H) Lastly, splenic CD4⁺ T cells from these tolerant animals were used as responder cells in an ELISPOT assay that measured IL-2 production. Controls were animals pre-sensitized with two separate IP injections of donor splenocytes and non-transplanted control animals. The sensitized animals showed a higher number of IL-2 spots against the donor splenocytes, as expected. However, the control non-sensitized and tolerant animals showed equal numbers of spots, suggesting that indeed the tolerant animals had no activated alloreactive T cells. All three groups of animals responded equally to third-party Balb/c splenocytes.</p

Cell fusion of bone marrow cells and somatic cell reprogramming by embryonic stem cells

Bone marrow transplantation is a curative treatment for many diseases, including leukemia, autoimmune diseases, and a number of immunodeficiencies. Recently, it was claimed that bone marrow cells transdifferentiate, a much desired property as bone marrow cells are abundant and therefore could be used in regenerative medicine to treat incurable chronic diseases. Using a Cre/loxP system, we studied cell fusion after bone marrow transplantation. Fused cells were chiefly Gr-1+, a myeloid cell marker, and found predominantly in the bone marrow; in parenchymal tissues. Surprisingly, fused cells were most abundant in the kidney, Peyer’s patches, and cardiac tissue. In contrast, after cell fusion with embryonic stem cells, bone marrow cells were reprogrammed into new tetraploid pluripotent stem cells that successfully differentiated into beating cardiomyocytes. Together, these data suggest that cell fusion is ubiquitous after cellular transplants and that the subsequent sharing of genetic material between the fusion partners affects cellular survival and function. Fusion between tumor cells and bone marrow cells could have consequences for tumor malignancy.—Bonde, S., Pedram, M., Stultz, R., Zavazava, N. Cell fusion of bone marrow cells and somatic cell reprogramming by embryonic stem cells

HPC-induced mixed chimerism preserves organ architecture post-transplantation.

<p>Transplanted syngeneic and allogenic animals were sacrificed at either 40 or 100 days post-transplantation and the histology of the grafts studied after H & E staining. These grafts showed preservation of the muscle architecture and lack of mononuclear cell infiltration in the syngeneic and tolerant grafts, but massive tissue destruction in the acutely rejected control grafts.</p

Poor T and B cell development in HPC-derived hematopoietic cells.

<p>Using flow cytometry, chimeric 129SvJ, MRL, and Rag2^−/−γ_c^−/− mice were analyzed for donor cells at 28 days post-transplantation. In all three strains, Gr-1⁺ cells were the predominant sub-population with lower cell numbers of B220- and CD3-expressing B and T cells, respectively. The percentages are calculated within the GFP⁺ population.</p

Tolerant mice maintain bone marrow mixed chimerism post-transplantation.

<p>The percentage of donor-derived hematopoietic cells in bone marrow cells of recipient tolerant mice was monitored using flow cytometry. A robust CD45⁺ cell population was detected (A) of which a large portion was Gr-1⁺ (B). Interestingly a small proportion of these cells was CD117-expressing, suggesting the presence of a truly bone marrow-resident stem cell population (C). At day 100 post-transplantation, the percentage of donor cells had decreased (D). Altogether, at day 100 post-transplantation, donor cells were hardly detectable in peripheral blood, 0.5–1% in the spleen, and 1–2% in the bone marrow (E, n = 6).</p

ES-derived HOXB4-expressing HPCs induce immunological tolerance to donor type cardiac allografts.

<p>A) Treatment protocol: MRL recipient mice were transplanted HPCs 14 days prior to transplantation of cardiac allografts which was performed between days 0 and 7. Cardiac function and survival were monitored by daily abdominal palpations. B) Graft survival: Chimeric MRL recipient mice tolerated donor type cardiac allografts, but not Balb/c third party allografts. Non-chimeric MRL mice acutely rejected allografts within 13 days. Syngeneic grafts were additionally used as controls.</p