Low-Dose Pesticides Alter Primary Human Bone Marrow Mesenchymal Stem/Stromal Cells through ALDH2 Inhibition

(1) Background: The impact of occupational exposure to high doses of pesticides on hematologic disorders is widely studied. Yet, lifelong exposure to low doses of pesticides, and more particularly their cocktail effect, although poorly known, could also participate to the development of such hematological diseases as myelodysplastic syndrome (MDS) in elderly patients. (2) Methods: In this study, a cocktail of seven pesticides frequently present in water and food (maneb, mancozeb, iprodione, imazalil, chlorpyrifos ethyl, diazinon and dimethoate), as determined by the European Food Safety Authority, were selected. Their in vitro effects at low-doses on primary BM-MSCs from healthy volunteers were examined. (3) Results: Exposure of normal BM-MSCs to pesticides for 21 days inhibited cell proliferation and promoted DNA damage and senescence. Concomitantly, these cells presented a decrease in aldehyde dehydrogenase 2 (ALDH2: mRNA, protein and enzymatic activity) and an increase in acetaldehyde levels. Pharmacological inhibition of ALDH2 with disulfiram recapitulated the alterations induced by exposure to low doses of pesticides. Moreover, BM-MSCs capacity to support primitive hematopoiesis was significantly altered. Similar biological abnormalities were found in primary BM-MSCs derived from MDS patients. (4) Conclusions: these results suggest that ALDH2 could participate in the pathophysiology of MDS in elderly people long exposed to low doses of pesticides.

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Acrylamide-induced changes of granulopoiesis in porcine bone marrow

Journal of Veterinary Research ◽

10.2478/jvetres-2021-0040 ◽

2021 ◽

Vol 0 (0) ◽

Author(s):

Dominika Grzybowska ◽

Anna Snarska

Keyword(s):

Bone Marrow ◽

Cell Morphology ◽

Light Microscope ◽

Low Dose ◽

Low Doses ◽

Cytological Evaluation ◽

High Doses ◽

Induced Changes ◽

The Impact ◽

Experimental Group

Abstract Introduction Due to the widely documented and diverse toxic effects of acrylamide, the authors decided to evaluate the impact of high and low doses of this compound on the process of granulopoiesis in porcine bone marrow. Material and Methods The experiment was conducted on 15 Danish Landrace pigs at the age of 8 weeks. The animals were randomly assigned into three equal groups (n = 5). Control animals received empty gelatine capsules as placebo. Animals in the first experimental group (the LD group) received a low dose of acrylamide of 0.5 μg/kg b.w./day, and animals in the second experimental group (the HD group) received a tenfold higher dose of acrylamide of 5 μg/kg b.w./day. Placebo and acrylamide capsules were administered with feed every morning for 28 days. Bone marrow was collected into tubes without an anticoagulant twice – before the first capsule administration (day 0) and on the 28th day of the study. After drying and staining, bone marrow smears were subjected to detailed cytological evaluation under a light microscope. Results Changes in cell morphology, i.e. degenerative changes in the cellular nuclei, were observed in both experimental groups. Both low and high doses of acrylamide decreased the number of segmented eosinophils, neutrophilic and segmented metamyelocytes, neutrophils, as well as basophils and basophilic metamyelocytes. Conclusion Acrylamide at doses of 0.5 μg/kg b.w./day and 5 μg/kg b.w./day clearly influences porcine granulopoiesis.

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Interference with Bone Marrow Stroma Derived Factors CXCL12 and TGFBI Increases Apoptosis in ALL Cells and Enhances Antileukemia Effects of Dexamethasone, Methotrexate, Vincristine and 6-Mercaptopurine

Blood ◽

10.1182/blood.v126.23.4774.4774 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4774-4774

Author(s):

Sana Usmani ◽

Olena Tkachencko ◽

Leti Nunez ◽

Craig A. Mullen

Keyword(s):

Bone Marrow ◽

Cell Death ◽

Stromal Cells ◽

Stromal Cell ◽

Low Dose ◽

Marrow Stromal Cells ◽

Bone Marrow Stroma ◽

Marrow Stroma ◽

The Impact

Abstract Background: Bone marrow stroma provides a favorable microenvironmental niche for ALL cell survival. We and others have demonstrated that bone marrow stromal cells contribute to prevention of apoptosis in ALL cells. Objective: Identify potentially "drug-able" molecules derived from marrow stromal cells that contribute to prevention of ALL cell apoptosis. Methods: We have developed an in vitro system to identify stromal gene products that deliver antiapoptotic signals to ALL cells. Primary human ALL cells are co-cultured with human bone marrow stromal cells. We manipulate stromal cells with siRNA directed against candidate stromal cell genes. Two days later the siRNA is washed out of culture and primary ALL cells are added to the stromal cells. Controls include irrelevant siRNA. Five days later we measure viability and apoptosis in ALL cells by flow cytometry. Results: (1) Knockdown of stroma cell CXCL12 or TGFBI reduces ALL survival. We performed global gene expression analysis upon human marrow stromal cells using RNASeq technology. Using bioinformatic approaches we are selecting some of the expressed stromal genes as candidates for the molecular mechanisms by which stromal cells prevent ALL apoptosis. We present preliminary results for two of our early candidates. (A) CXCL12 is a paracrine chemokine known to have activity in the marrow microenvironment upon hematopoietic cells and we hypothesized it may participate in the effect. Knockdown of CXCL12 with siRNA increased ALL cell death in the co-culture system. As measured by quantitative reverse transcriptase PCR stromal cell CXCL12 mRNA was reduced approximately 75% by siRNA treatment. Figure 1 displays representative results of the impact of CXCL12 knockdown in stromal cell on the survival of ALL cells in the coculture. The magnitude of effect was ~40% increase in ALL cell death. (B) TGFBI (transforming growth factor beta induced) is expressed by stromal cells. The gene is involved in cell-collagen interactions and we hypothesized it played a role. siRNA reduced stromal gene expression by about 90%. Figure 2 displays representative results in which ALL cell death increased by about 50%. (2) Validation of results using inhibitors to CXCL12. The gene knockdown experiments suggested a potential role for CXCL12 in prevention of ALL cell apoptosis. To further test this we tested the effect of plerixafor, a specific inhibitor of CXCL12/CXCR4 interactions, on survival of ALL. ALL cells express CXCR4. In a dose dependent manner (25 - 400 micromolar) we observed a 31-39% reduction in ALL survival in stromal co-cultures including plerixafor. Figure 3 depicts representative results with plerixafor 200 micromolar. We are evaluating small molecules to block TGFBI. (3) Potential augmentation of chemotherapy drug effects on ALL. We hypothesize that interference with stromal cell molecules that prevent apoptosis in ALL cells may increase the effectiveness of conventional antileukemia drugs. In our stromal cell/ALL coculture system we have identified the effective in vitro concentrations of the most commonly used ALL drugs. We measured the impact of combination of low dose plerixafor (LD10) and these individual drugs (used at approximately the LD50 concentrations). Figure 4 demonstrates increased antileukemia effects related to plerixafor for dexamethasone, vincristine, and 6-mercaptopurine. Results are plotted as a percentage of ALL cells surviving in the absence of any drugs. The low dose plerixafor alone control did not produce a statistically significant reduction in ALL survival. Conclusions: Marrow stromal cell-produced CXCL12 may contribute to prevention of apoptosis in human ALL cells. Pharmacological interference with its effect may enhance the effectiveness of some conventional chemotherapy drugs. Marrow stromal cell-produced TGFBI may also contribute to prevention of apoptosis in human ALL cells. Disclosures No relevant conflicts of interest to declare.

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Proteins as targets for high dilutions of drugs: Interaction between ?-amylase and mercuric chloride.

International Journal of High Dilution Research - ISSN 1982-6206 ◽

10.51910/ijhdr.v17i2.920 ◽

2021 ◽

Vol 17 (2) ◽

pp. 24-25

Author(s):

Nirmal Chandra Sukul ◽

Tandra Sarkar ◽

Atheni Konar ◽

Anirban Sukul

Keyword(s):

Mercuric Chloride ◽

Binding Sites ◽

Low Dose ◽

Soluble Starch ◽

Low Doses ◽

Substrate Interaction ◽

Enzyme Substrate ◽

High Dilutions ◽

High Doses ◽

Mother Tincture

Background: High dilutions of drugs, used in homeopathy, are usually applied by oral route or foliar spray. These dilutions first come in contact with membrane or circulating proteins. Ultra low doses of mercuric chloride, called potencies, promote activity of diastase or ?-amylase in terms of breakdown of starch, a polysaccharide into a disaccharide maltose in a cell-free medium in test tubes. Merc cor or HgCl2 in high doses inhibits the enzyme activity. Aims: To see (i) whether the high and ultra low dose effects of HgCl2 involve different binding sites of the enzyme and (ii) to find an explanation for the low dose effect of HgCl2 in spite of absence of its original molecules. Methodology: Merc cor mother tincture (147 mM HgCl2) in distilled water was used undiluted in this experiment. Merc cor 200c and 1000c were prepared from the mother tincture (MT) by successive dilution with water 1:100 followed by succussion in 200 and 1000 steps, respectively, and finally preserved in 90% EtOH. These potencies and blank 90% ethanol, were diluted with deionized, distilled (DD) water 1:1000 to minimize ethanol content in test solutions. Each test solution or control was mixed with the enzyme 1:10 just before experiment. The control consisted of DD water. An isothermal calorimetry (ITC) instrument was used to measure the interaction between soluble starch and ?-amylase mixed with each potency (200c/1000c) of Merc cor, its mother tincture, ethanol and control. ITC is a thermodynamic technique which helps in measuring directly very small amount of heat evolved during chemical reaction. Soluble starch 90 µM was injected into 300 µl of 15µM ?-amylase at 2 µl / injection. Twenty injections, one every 2 min, were given. The enzyme substrate interaction in terms of heat released (exothermic) or absorbed (endothermic) were monitored by the ITC instrument. All ITC measurements were calculated and analyzed statistically by an in-built software Origin 7. Results and discussion: The data are presented in figures. While Merc cor MT shows endothermic reaction, all its potencies, ethanol and water control show exothermic reactions. There is wide variation in enthalpy (?H), entropy (?S), binding constant (K) and Gibbs free energy change (?G) among the treatments with Merc cor MT, potencies, ethanol and also control. The results indicate that Merc cor MT and its potencies act on different binding sites of the enzyme. The variation in thermodynamic parameters suggest difference in binding interaction between the drug solutions and the enzyme. This in turn influences the enzyme substrate interaction as reported in earlier studies. The potencies are virtually water modified by the starting substance HgCl2. Conclusion: The mother tincture and potencies of mercuric chloride produce different effects on the enzyme substrate interaction. Potencies show wide variation in ?H, ?S, K and ?G values. It appears from the results that the drugs used in homeopathy produce dual action on proteins. At high doses they act on a binding site(s) but at ultra low doses they act on a different binding site(s). Proteins in an organism may serve as targets for initiation of action of homeopathic potencies.

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Toll-Like Receptor Signaling in Airborne Burkholderia thailandensis Infection

Infection and Immunity ◽

10.1128/iai.00618-09 ◽

2009 ◽

Vol 77 (12) ◽

pp. 5612-5622 ◽

Cited By ~ 17

Author(s):

T. Eoin West ◽

Thomas R. Hawn ◽

Shawn J. Skerrett

Keyword(s):

Low Dose ◽

Inflammatory Responses ◽

High Dose ◽

Tlr Signaling ◽

Necrosis Factor Alpha ◽

Airborne Infection ◽

Essential Components ◽

High Doses

ABSTRACT Melioidosis is a tropical disease endemic in southeast Asia and northern Australia caused by the gram-negative soil saprophyte Burkholderia pseudomallei. Although infection is often systemic, the lung is frequently involved. B. thailandensis is a closely related organism that at high doses causes lethal pneumonia in mice. We examined the role of Toll-like receptors (TLRs), essential components of innate immunity, in vitro and in vivo during murine B. thailandensis pneumonia. TLR2, TLR4, and TLR5 mediate NF-κB activation by B. thailandensis in transfected HEK293 or CHO cells. In macrophages, TLR4 and the adaptor molecule MyD88, but not TLR2 or TLR5, are required for tumor necrosis factor alpha production induced by B. thailandensis. In low-dose airborne infection, TLR4 is needed for early, but not late, bacterial containment, and MyD88 is essential for control of infection and host survival. TLR2 and TLR5 are not necessary to contain low-dose infection. In high-dose airborne infection, TLR2 deficiency confers a slight survival advantage. Lung and systemic inflammatory responses are induced by low-dose inhaled B. thailandensis independently of individual TLRs or MyD88. These findings suggest that redundancy in TLR signaling or other MyD88-dependent pathways may be important in pneumonic B. thailandensis infection but that MyD88-independent mechanisms of inflammation are also activated. TLR signaling in B. thailandensis infection is substantially comparable to signaling induced by virulent B. pseudomallei. These studies provide additional insights into the host-pathogen interaction in pneumonic Burkholderia infection.

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Decidualization of human endometrial stromal cells in vitro: effects of progestin and relaxin on the ultrastructure and production of decidual secretory proteins*

Human Reproduction ◽

10.1093/oxfordjournals.humrep.a138492 ◽

1994 ◽

Vol 9 (2) ◽

pp. 259-266 ◽

Cited By ~ 53

Author(s):

Bernard Lane ◽

William Oxberry ◽

James Mazella ◽

Linda Tseng

Keyword(s):

Stromal Cells ◽

Secretory Proteins ◽

Endometrial Stromal Cells ◽

In Vitro Effects

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Interaction of allogeneic adipose tissue-derived stromal cells and unstimulated immune cells in vitro: the impact of cell-to-cell contact and hypoxia in the local milieu

Cytotechnology ◽

10.1007/s10616-017-0144-x ◽

2017 ◽

Vol 70 (1) ◽

pp. 299-312 ◽

Cited By ~ 4

Author(s):

Aleksandra N. Gornostaeva ◽

Elena R. Andreeva ◽

Polina I. Bobyleva ◽

Ludmila B. Buravkova

Keyword(s):

Adipose Tissue ◽

Immune Cells ◽

Stromal Cells ◽

Cell Contact ◽

The Impact

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Interleukin-2 (hrIL2) Primed T cells display Reduced Alloproliferative Capacity In Vitro Due To Induction Of FOXP3+ Regulatory Cells

Blood ◽

10.1182/blood.v122.21.5433.5433 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5433-5433

Author(s):

Dimitra Kokkinou ◽

Panagiota Stamou ◽

Angeliki Vittoraki ◽

Anne-Lise De Lastic ◽

Spyros Chondropoulos ◽

...

Keyword(s):

T Cells ◽

Low Dose ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Interleukin 2 ◽

Regulatory Cells ◽

Transplant Tolerance ◽

Immune Exhaustion ◽

The Impact

Abstract Introduction Prophylactic donor lymphocyte infusions (pDLI) after allogeneic transplantation contribute to immune restoration and reduce viral infections. Furthermore, we have recently shown that pDLI in patients with high risk leukemia significantly reduces the relapse rate, however, they were associated with a relatively high incidence of Graft versus Host Disease (GvHD)(BBMT 2013;19:75-81). Strategies to minimize GvHD without compromising the effect of pDLI against leukemia are needed. IL2 plays dual role in immune responses, contributing to both the generation of effector T cells and the maintenance of regulatory T cells (Tregs). Recently, low dose interleukin-2 (IL-2) therapy has been advanced as a potential immune modulator able to modulate the immune response to aid transplant tolerance and to suppress GvHD through expansion of Tregs (N Engl J Med. 2011; 2055-66). We investigated the impact of priming DLI with low dose IL2 on the proliferative responses to allo-stimulation in vitro. Methods CD3+ T cells purified from healthy individuals by MACS negative selection were primed (p-T cells) or unprimed (np-T cells), with or without (control) 100 U/ml hrIL2 (Proleukin, Novartis) for 7 days. Composition of T-cell cultures was analyzed by flow cytometry for: a) the percentage of T regulatory cells (CD4+/CD25high/Foxp3+/Helios+, b) their differentiation (CD28/CD27), c) their immune exhaustion (Programmed cell death 1, PD1). In vitro alloproliferative capacity of the p-T cells was analyzed with CFSE cell proliferation assay by using them as responder cells in mixed lymphocyte cultures (MLC), with irradiated allo-PB mononuclear cells as stimulators. Results In vitro priming of T-cells with IL-2 (p-T cells) in contrast to np-T or control cells: 1) increase the numbers of CD4+CD25highFoxp3+/Helios+ cells (n=8, 3.3%±0.7 mean±SEM vs 1.01%±0.22, p=0.004 και 1.4%±0.42, p=0.006). Increased levels of Foxp3 expression was also confirmed by Real Time PCR (n=2,1.25AU±0.15 vs 0.29AU±0.04, p=0.028 και 0.26AU±0.07, p=0.024). 2) did not affect the proportion of CD28+/CD27+ non late-differentiated cells (n=3, 60%±0.15 vs61%±0.04, p=0.91 και 59%±0.08, p=0.024). 3) did not cause immune exhaustion through PD1 expression (n=6, 13.3%±1.9 vs 8.1%±2.1, p=0.76, και 14%±2.2, p=0.68). 4) significantly decreases their response rate to allo-stimulus in MLC (n=8, 45%±0.5 vs65%±0.2, p=0.006 και 64%±0.2, p=0.008). The p-T cells regained their alloproliferative capacity after FACS-sorting removal of CD4+/CD25high Tregs. Conclusions Our results show that ex vivo priming of T cells with low dose of IL-2 reduces their in vitro alloproliferative capacity. This reduction is not due to late differentiation or immune – exhaustion of T cells but to selective induction of Foxp3+ cells with immunomodulatory properties in the culture. It remains to be seen whether IL2-primed DLI is safe and effective in transplant patients. Disclosures: No relevant conflicts of interest to declare.

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Interferon-Gamma Impairs Expansion and Hematopoietic Support of Bone Marrow Mesenchymal Stromal Cells

Blood ◽

10.1182/blood.v128.22.3884.3884 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3884-3884

Author(s):

Marieke Goedhart ◽

Anne Cornelissen ◽

Carlijn Kuijk ◽

Sulima Geerman ◽

Fernanda Pascutti ◽

...

Keyword(s):

Stromal Cells ◽

Stromal Compartment ◽

Self Renewal ◽

Hematopoietic Stem ◽

Ifn Γ ◽

Immunomodulatory Properties ◽

Pre Treatment ◽

The Impact

Abstract Maintenance of hematopoietic stem cells (HSCs) and regulation of their quiescence and self-renewal is critical for maintaining a lifelong supply of blood cells. The ability of HSCs to stay quiescent is thought to depend on their specific niche in the bone marrow (BM). Mesenchymal stromal cells (MSC) in the BM are multipotent stem cells that form part of the vascular HSC niche and provide micro-environmental support to HSCs both in vivo and upon expansion ex vivo. Culture-expanded MSCs also exhibit immunomodulatory properties that can be enhanced by pre-treatment with interferon-gamma (IFN-γ). BM MSC are thus attractive candidates for cellular therapy after hematopoietic stem cell transplantation, for promoting rapid hematopoietic recovery and reducing the incidence or severity of graft versus host disease. Although IFN-γ pre-treatment can improve the immunomodulatory properties of MSCs, elevated IFN-γ levels have also been associated with anemia and BM failure in multiple chronic inflammatory diseases. While the impact of IFN-γ on HSC has been elucidated in recent years, it remains largely unknown whether IFN-γ can also influence hematopoietic support by BM stromal cells. In this study, we aim to elucidate the impact of IFN-γ on hematopoietic support of BM MSC. We show that in vitro expansion of primary BM MSC cultures from healthy donors was significantly reduced in the presence of IFN-γ, and this effect could be reproduced in the BM stromal cell line MS-5. Concurrently, IFN-γ diminished the clonal capacity of BM MSC, as measured by CFU-F assays. In addition, BM MSC that were pre-stimulated with IFN-γ produced significantly lower levels of CXCL12, suggesting a loss of hematopoietic support potential. Indeed, support of CD34+ hematopoietic stem and progenitor cells (HSPC) in a co-culture assay was greatly reduced in when MSC were pre-treated with IFN-γ. To determine the impact of IFN-γ on BM MSC in vivo, we investigated the BM stromal compartment of IFN-γ AU-rich element deleted (ARE-Del) mice, which constitutively express IFN-γ in steady state conditions. FACS analysis revealed a remodeling of the BM stromal compartment in ARE-Del mice compared to littermate controls, with significantly fewer MSCs, identified as CD45-Ter119-CD31-CD51+PDGFRa+ cells. Numbers of other stromal cell subsets, such as osteoblasts and fibroblasts, were not altered. The reduction of BM MSC in ARE-Del mice coincided with a loss of quiescence in HSCs; only 35% of long term HSC (LT-HSC) in ARE-Del mice were quiescent, compared to 70% in WT mice, as determined by Ki-67 staining. Loss of quiescence in LT-HSC did not lead to increased self-renewal, but rather induced increased differentiation towards short-term HSC and multi-potent progenitors. We then sorted LT-HSC from WT and ARE-Del mice and performed in vitro HSC culture assays in the absence of IFN-γ. Absolute numbers of LT-HSC were rapidly decreased in ARE-Del compared to WT cultures after 3 and 7 days of HSC culture, while numbers of more differentiated progenitors were increased. These data indicate that an IFN-γ-mediated loss of BM MSC in ARE-Del mice leads to loss of quiescent LT-HSCs and induces a tendency towards HSC differentiation over self-renewal. In conclusion, we have shown that IFN-γ has a negative impact on expansion and hematopoietic support of BM MSC in vitro and in vivo across species. Although IFN-γ treatment enhances the immunomodulatory function of MSCs in a clinical setting, it is obvious from our data that IFN-γ impairs their HSC supporting function. These data also provide more insight in the underlying mechanism by which IFN-γ contributes to the pathogenesis of anemia and BM failure. Disclosures No relevant conflicts of interest to declare.

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