Expression of PIM kinases in Reed-Sternberg cells fosters immune privilege and tumor cell survival in Hodgkin lymphoma.

Reed-Sternberg (RS) cells of classical Hodgkin lymphoma (cHL) express multiple immunoregulatory proteins that shape the cHL microenvironment and allow tumor cells to evade immune surveillance. Expression of certain immunoregulatory proteins is modulated by prosurvival transcription factors, such as NFκB and STATs. Because these factors also induce expression of the oncogenic PIM1/2/3 serine/threonine kinases, and as PIMs modulate transcriptional activity of NFκB and STATs, we hypothesized that these kinases support RS cell survival and foster their immune privilege. Here, we investigated PIM1/2/3 expression in cHL and assessed their role in developing RS cell immune privilege and survival. PIM1/2/3 were ubiquitously expressed in primary and cultured RS cells, and their expression was driven by JAK-STAT and NFκB activity. Genetic or chemical PIM inhibition with a newly developed pan-PIM inhibitor, SEL24-B489, induced RS cell apoptosis. PIM inhibition decreased cap-dependent protein translation, blocked JAK-STAT signaling, and markedly attenuated NFκB-dependent gene expression. In a cHL xenograft model, SEL24-B489 delayed tumor growth by 95.8% (P = .0002). Furthermore, SEL24-B489 decreased the expression of multiple molecules engaged in developing the immunosuppressive microenvironment, including galectin-1 and PD-L1/2. In coculture experiments, T cells incubated with SEL24-B489-treated RS cells exhibited higher expression of activation markers than T cells coincubated with control RS cells. Taken together, our data indicate that PIM kinases in cHL exhibit pleiotropic effects, orchestrating tumor immune escape and supporting RS cell survival. Inhibition of PIM kinases decreases RS cell viability and disrupts signaling circuits that link these cells with their niches. Thus, PIM kinases are promising therapeutic targets in cHL.

Early severe impairment of hematopoietic stem and progenitor cells from the bone marrow caused by CLP sepsis and endotoxemia in a humanized mice model.

INTRODUCTION: An effective immune response to severe bacterial infections requires a robust production of the innate immunity cells from hematopoietic stem and progenitor cells (HSPCs) in a process called emergency myelopoiesis. In sepsis, an altered immune response that leads to a failure of bacterial clearance is often observed. In this study, we aimed to evaluate the impact of sepsis on human HSPCs in the bone marrow (BM) microenvironment of humanized mice subjected to acute endotoxemia and polymicrobial sepsis. METHODS: Humanized mice (hu-NSG) were generated by transplanting NOD.Cg-Prkdc/scidIL2rγ (NSG) mice with the human cord blood CD34(+) cells. Eight weeks after the transplantation, hu-NSG mice were subjected to sepsis induced by endotoxemia-Escherichia coli lipopolysaccharide (LPS)-or by cecal ligation and puncture (CLP). Twenty-four hours later, HSPCs from BM were analyzed by flow cytometry and colony-forming unit (CFU) assay. CLP after inhibition of Notch signaling was also performed. The effects of LPS on the in vitro proliferation of CD34(+) cells from human BM were tested by CellTrace Violet dye staining. RESULTS: The expression of Toll-like receptor 4 receptor was present among engrafted human HSPCs. Both CLP and endotoxemia decreased (by 43 % and 37 %) cellularity of the BM. In addition, in both models, accumulation of early CD34(+) CD38(-) HSCs was observed, but the number of CD34(+) CD38(+) progenitors decreased. After CLP, there was a 1.5-fold increase of proliferating CD34(+) CD38(-)Ki-67(+) cells. Moreover, CFU assay revealed a depressed (by 75 % after LPS and by 50 % after CLP) production of human hematopoietic colonies from the BM of septic mice. In contrast, in vitro LPS stimulated differentiation of CD34(+) CD38(-) HSCs but did not induce proliferation of these cells in contrast to the CD34(+) CD38(+) progenitors. CLP sepsis modulated the BM microenvironment by upregulation of Jagged-1 expression on non-hematopoietic cells, and the proliferation of HSCs was Notch-dependent. CONCLUSIONS: CLP sepsis and endotoxemia induced a similar expansion and proliferation of early HSCs in the BM, while committed progenitors decreased. It is suggestive that the Notch pathway contributed to this effect. Targeting early hematopoiesis may be considered as a viable alternative in the existing arsenal of supportive therapies in sepsis.

Growth factors and their receptors derived from human amniotic cells in vitro.

In vitro studies have shown that amnion-produced growth factors participated in angiogenesis, re-epithelialization, and immunomodulation. The aim of our study was to investigate the growth factors and receptors produced by human amnion tissue and amniotic cells. Human amnions (hAM) were isolated, and amnion circles were dissected for in vitro analysis. Some amnion fragments were digested by the use of different methods to obtain two cell fractions, which were analysed for mesenchymal and epithelial cell markers. Amniotic circles and human amniotic cell fractions were cultured in a protein-free medium. Proteins secreted into the culture medium were analysed with a human growth factor antibody array. Conditioned culture media were added to human umbilical vein epithelial cells (HUVECs) to test for stimulation of migration (scratch test) and proliferation (Ki67 expression). Fraction 1 cells expressed both cytokeratin and mesenchymal cell markers which indicated that it was composed of a mixture of human amnion epithelial cells (hAECs) and mesenchymal stromal cells (hAMSCs). Fraction 2 cells mainly expressed cytokeratin and, therefore, were designed as hAECs. Secretion of proteins by the cultured cells increased with time. The hAM cultures secreted EGF-R, IGF, and IGFBP-2,-3 and -6; Cell Fraction 1 secreted NT-4, whereas Cell Fraction 2 secreted G-CSF, M-CSF, and PDGF. Conditioned media of hAM cultures stimulated HUVECs migration. We have showed for the first time that human amnions and amniotic cells secreted IGFBP-6, MCSF-R, PDGF-AB, FGF-6, IGFBP-4, NT-4, and VEGF-R3. We found that Cell Fraction 1, Cell Fraction 2, and the whole amnion secreted different proteins, possibly due to different proportions of amnion-derived cells and different cell-cell interactions. The hAM cell factors remained functional in vitro and induced intensified migration of HUVECs. The growth factors and receptors found in amnion or amniotic cell media might be used for regenerative medicine.

Monitoring cell proliferation in vitro with different cellular fluorescent dyes.

There are few methods for quantifying cell proliferation. Those tests describe the proliferation kinetics of a cell population, but they do not report the history of single cells, the number and frequency of cell divisions, or the precursor cell frequency. Cell-tracking assays based on dilution of the green fluorescent protein labelling dye, CFSE, has become the standard for monitoring cell proliferation. Other labelling dyes, e.g. CellTrace Violet and CellVue Claret, are also used for the same purpose. This study aimed to compare these three cell labelling methods for analysing the kinetics of cell viability, proliferation, and precursor cell frequency. Human peripheral blood mononuclear cells stimulated with Concanavalin A (ConA) were used as a model system. After labelling with a cell-tracking dye cells were divided into groups with and without ConA stimulation. From the 5th to 8th day, cells were collected and analysed with flow cytometry. Cell viability was not significantly different between labelled and unlabelled cells that received ConA stimulation. The proliferative fraction, proliferation index, and nonproliferative fraction were not significantly different among lymphocytes labelled with different dyes. Precursor cell frequency was also similar among cells labelled with the three cell-tracing dyes. The practical conclusion from our observations is that the results from cells labelled with different tracers may be compared directly and discussed jointly.

Induced death of Escherichia coli encapsulated in a hollow fiber membrane as observed in vitro or after subcutaneous implantation.

The encapsulation of bacteria may be used to harness them for longer period of time in order to make them viable, while antibiotic treatment would result in controlled release of therapeutic molecules. Encapsulated bacteria Escherichia coli GFP (green fluorescent protein) (E. coli GFP) were used here as a model for therapeutic substance - GFP fragments release (model of bioactive substances). Our aim was to evaluate the performance of bacteria encapsulated in hollow fibers (HF) treated with antibiotic for induction of cell death. The polypropylene surface modified HF was applied for E. coli encapsulation. The encapsulated bacteria were treated with tetracycline in vitro or in vivo during subcutaneous implantation into mice. The HF content was evaluated in flow cytometer, to assess the bacteria cell membrane permeability changes induced by tetracycline treatment. It was observed that applied membranes prevent release of bacteria through the HF wall. The encapsulated in HF E. coli GFP culture in vitro proves the tetracycline impact on bacteria viability and allows recognition sequence of events within process of bacteria death. Treatment with tetracycline of the SCID mice for 8 hours proves the tetracycline impact on bacteria viability in vivo, rising the necrotic bacteria releasing GFP fragments. It was concluded, that the bacteria may be safely enclosed within the HF at site of implantation, and when the animal is treated with antibiotic bacteria may act as a local source of bioactive factor.

Participation of stem cells from human cord blood in skeletal muscle regeneration of SCID mice.

OBJECTIVE: In this report, we demonstrate the participation of human cord blood (HUCB) stem cells in the skeletal muscle regeneration of SCID (severe combined immunodeficient) mice. MATERIALS AND METHODS: The HUCB cells were labeled with the PKH26 fluorescent marker or recognized by an anti-HLA-ABC or anti-beta-2-microglobulin antibody. The HUCB cells were implanted directly into the damaged mouse muscle. The regeneration process and the implanted HUCB cells were traced each day after the damage, throughout a period of 7 days, and additionally at day 30 with the use of flow cytometry and confocal microscopy. RESULTS: The PKH26-labeled cells isolated from the regenerating muscle were positive for the anti-HLA-ABC antibody. The percentage of the PKH26(+) and HLA-ABC(+) cells decreased from day 1 to day 5. In the regenerating muscle, the percentage of the HLA-ABC(+) cells increased, as measured on days 7 and 30. Moreover, myofibers containing fragments of the PKH26-labeled sarcolemma were noticed. Labeling with the anti-human beta(2)-microglobulin antibody showed the presence of positive cells and myofibers at day 7 of the regeneration, suggesting fusion of human and mouse cells. CONCLUSIONS: We suggest that the HUCB cells implanted into the damaged muscle are present there for at least 30 days and that they participate in the muscle regeneration. Moreover, our study shows that the implanted HUCB cells form human muscle precursor cells residing in the repaired mouse muscle. We suggest that the HUCB cell circulation after transplantation depends on SDF-1 (stromal-derived factor-1) expression in regenerating muscle.

Contribution of stem cells to skeletal muscle regeneration.

Stem cells for skeletal muscle originate from dermomyotome of the embryo. The early marker of these cells is expression of both transcription factors Pax3 and Pax7 (Pax3+/Pax7+ cells). The skeletal muscles in the adult organism have a remarkable ability to regenerate. Skeletal muscle damage induces degenerative phase, followed by activation of inflammatory and satellite cells. The satellite cells are quiescent myogenic precursor cells located between the basal membrane and the sarcolemma of myofiber and they are characterized by Pax7 expression. Activation of the satellite cells is regulated by muscle growth and chemokines. Apart from the satellite cells, a population of adult stem cells (muscle side population--mSP) exists in the skeletal muscles. Moreover, the cells trafficking from different tissues may be involved in the regeneration of damaged muscle. Trafficking of cells in the process of damaged muscle regeneration may be traced in the SCID mice.

NK cell depletion and recovery in SCID mice treated with anti-NK1.1 antibody.

The anti-NK1.1 antibody produced by PK136 hybridoma cell line administered subcutaneously to SCID mice effectively decreased the level of peripheral blood NK cells and weight of the spleen for 3-4 days. The antibody treatment did not harm the general state of the animal, and may be practically applied in xenograft experiments.

Expression of Fas receptor on peripheral blood lymphocytes from patients with non-small cell lung cancer.

In recent years many data indicate that lymphocytes from cancer patients undergo increased apoptosis. The objective of this study was to evaluate the expression of Fas receptor on lymphocytes obtained from patients with lung cancer. Eighteen patients with non-small cell lung cancer and 18 healthy volunteers were investigated. Expression of Fas (CD95) on CD4+ and CD8+ blood lymphocytes was evaluated by flow cytometry. The proportion of blood Fas+ lymphocytes was significantly higher in lung cancer patients when compared with healthy individuals and in smokers when compared with nonsmokers.