Improving approaches for hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) mobilization is clinically important because increased numbers of these cells are needed for enhanced transplantation. Chemokine stromal cell derived factor-1 (also known as CXCL12) is believed to be involved in retention of HSCs and HPCs in bone marrow. AMD3100, a selective antagonist of CXCL12 that binds to its receptor, CXCR4, was evaluated in murine and human systems for mobilizing capacity, alone and in combination with granulocyte colony-stimulating factor (G-CSF). AMD3100 induced rapid mobilization of mouse and human HPCs and synergistically augmented G-CSF–induced mobilization of HPCs. AMD3100 also mobilized murine long-term repopulating (LTR) cells that engrafted primary and secondary lethally-irradiated mice, and human CD34+ cells that can repopulate nonobese diabetic-severe combined immunodeficiency (SCID) mice. AMD3100 synergized with G-CSF to mobilize murine LTR cells and human SCID repopulating cells (SRCs). Human CD34+ cells isolated after treatment with G-CSF plus AMD3100 expressed a phenotype that was characteristic of highly engrafting mouse HSCs. Synergy of AMD3100 and G-CSF in mobilization was due to enhanced numbers and perhaps other characteristics of the mobilized cells. These results support the hypothesis that the CXCL12-CXCR4 axis is involved in marrow retention of HSCs and HPCs, and demonstrate the clinical potential of AMD3100 for HSC mobilization.
IL-31R is involved in positive regulation of absolute numbers and cycling status of immature subsets of HPC in vivo. While IL-31 in vitro does not modulate proliferation of HPC, it does enhance their survival, which may contribute to effects on cycling and numbers of HPC in vivo. Under steady-state conditions, loss of IL-31R on HPC does not appear to influence the activity of competitive repopulating HSC. These results with HPC may be of future utility for manipulation of hematopoiesis in a preclinical setting.
Arsenical keratosis and skin cancer are among the most common health effects associated with acute and chronic exposures to arsenic. This study examines the acute and chronic dose-responses of arsenic in established human cell lines using keratinocytes (HaCaT), melanocytes (CRL1675) and dendritic cells (THP-1 + A23187). Chronic conditions were established by treating the three cell lines with at least 8 passages in 0.2 microg/mL arsenic trioxide. Cytotoxicity was assessed using the fluorescein diacetate assay after 72 hrs of exposure. Single cell gel electrophoresis (Comet assay) was used to measure DNA damage. Acute exposure to arsenic had LD10 and LD25 values of 0.38 microg/mL and 3.0 microg/mL for keratinocytes; 0.19 microg/mL and 0.38 microg/mL for melanocytes; and 0.38 microg/mL and 0.75 microg/mL for dendritic cells. Cytotoxicity assays for chronically exposed cells resulted in LD10, and LD25 values of 0.4 microg/mL and 0.8 microg/mL for keratinocytes; 0.10 microg/mL and 0.20 microg/mL for melanocytes; and 0.10 microg/mL and 1.0 microg/mL for dendritic cells. The Comet assay showed that arsenic was highly genotoxic to the three cell lines. No significant differences (p > 0.05) in DNA cleavage were observed between acute and chronic exposures. In acute exposure arsenic genotoxicity was more severe with dendritic cells while melanocytes were more sensitive to arsenic cytotoxicity. Similarly, chronically exposed dendritic cells showed the maximum genotoxic damage while melanocytes were more sensitive to arsenic cytotoxicity. In conclusion, this research shows that arsenic is dermatotoxic, showing a high degree of genotoxicity and cytotoxicity to skin cells.
Inorganic arsenic is a known environmental toxicant and carcinogen of global public health concern. Arsenic is genotoxic and cytotoxic to human keratinocytes. However, the biological pathways perturbed in keratinocytes by low chronic dose inorganic arsenic are not completely understood. The objective of the investigation was to discover the mechanism of arsenic carcinogenicity in human epidermal keratinocytes. We hypothesize that a combined strategy of DNA microarray, qRT-PCR and gene function annotation will identify aberrantly expressed genes in HaCaT keratinocyte cell line after chronic treatment with arsenic trioxide. Microarray data analysis identified 14 up-regulated genes and 21 down-regulated genes in response to arsenic trioxide. The expression of 4 up-regulated genes and 1 down-regulated gene were confirmed by qRT-PCR. The up-regulated genes were AKR1C3 (Aldo-Keto Reductase family 1, member C3), IGFL1 (Insulin Growth Factor-Like family member 1), IL1R2 (Interleukin 1 Receptor, type 2), and TNFSF18 (Tumor Necrosis Factor [ligand] SuperFamily, member 18) and down-regulated gene was RGS2 (Regulator of G-protein Signaling 2). The observed over expression of TNFSF18 (167 fold) coupled with moderate expression of IGFL1 (3.1 fold), IL1R2 (5.9 fold) and AKR1C3 (9.2 fold) with a decreased RGS2 (2.0 fold) suggests that chronic arsenic exposure could produce sustained levels of TNF with modulation by an IL-1 analogue resulting in chronic immunologic insult. A concomitant decrease in growth inhibiting gene (RGS2) and increase in AKR1C3 may contribute to chronic inflammation leading to metaplasia, which may eventually lead to carcinogenicity in the skin keratinocytes. Also, increased expression of IGFL1 may trigger cancer development and progression in HaCaT keratinocytes.
Based on the hypothesis that arsenic exposure results in toxicity and mitogenecity, this study examined the dose-response of arsenic in established human cell lines of keratinocytes (HaCaT), melanocytes (1675), dendritic cells (THP-1/A23187), dermal fibroblasts (CRL1904), microvascular endothelial cells (HMEC), monocytes (THP-1), and T cells (Jurkat). Cytotoxicity was determined by incubating THP-1, THP-1+ A23187 and JKT cells in RPMI 1640, 1675 in Vitacell, HMEC in EBM, and dermal fibroblasts and HaCaT in DMEM with 10% fetal bovine serum, 1% streptomycin and penicillin for 72 hrs in 96-well microtiter plates, at 37 o C in a 5% CO 2 incubator with different concentrations of arsenic using fluorescein diacetate (FDA). Cell proliferation in 96-well plates was determined in cultured cells starved by prior incubation for 24 hrs in 1% FBS and exposed for 72 hours, using the 96 cell titer proliferation solution (Promega) assay. Cytotoxicity assays yielded LD 50 s of 9 µg/mL for HaCaT, 1.5 µg/mL for CRL 1675, 1.5 µg/mL for dendritic cells, 37 µg/mL for dermal fibroblasts, 0.48 µg/mL for HMEC, 50 µg/mL for THP-1 cells and 50 µg/mL for JKT-T cells. The peak proliferation was observed at 6 µg/mL for HaCaT and THP-1 cells, 0.19 µg/mL for CRL 1675, dendritic cells, and HMEC, and 1.5 µg/mL for dermal fibroblasts and Jurkat T cells. These results show that arsenic is toxic at high doses to keratinocytes, fibroblasts, monocytes and T cells, and toxic at lower doses to melanocytes, microvascular endothelial cells and dendritic cells. Proliferation studies showed sub-lethal doses of arsenic to be mitogenic.
Tolerogenic dendritic cells (DCs) may be valuable in transplantation for silencing immune reaction. Macrophage colony-stimulating factor (M-CSF)/IL-4 induces differentiation of cord blood (CB) monocytes into DCs (M-DCs) with tolerogenic phenotype/function. We assessed whether factors produced by tolerogenic DCs could modulate hematopoiesis. TGF-β1 added to CB M-DC cultures induced bona fide DC morphology (TGF-M-DCs), similar to that of DCs generated with TGF-β and granulocyte-macrophage colony-stimulating factor (GM-CSF)/IL-4 (TGF-GM-DCs). Of conditioned media (CM) produced from TGF-M-DCs, TGF-GM-DCs, M-DCs, and GM-DCs, TGF-M-DC CM was the only one that enhanced SCF, Flt3 ligand, and TPO expansion of myeloid progenitor cells ex vivo. This effect was blocked by neutralizing anti–M-CSF Ab, but protein analysis of CM suggested that M-CSF alone was not manifesting enhanced expansion of myeloid progenitors. LPS-stimulated TGF-M-DCs induced T-cell tolerance/anergy as effectively as M-DCs. TGF-M-DCs secreted significantly lower concentrations of progenitor cell inhibitory cytokines and were less potent in activating T cells than TGF-GM-DCs. Functional differences between TGF-M-DCs and TGF-GM-DCs included enhanced responses to LPS-induced ERK, JNK, and P38 activation in TGF-M-DCs and their immune suppressive–skewed cytokine release profiles. TGF-M-DCs appear unique among culture-generated DCs in their capability for silencing immunity while promoting expansion of myeloid progenitors, events that may be of therapeutic value.
Arsenic is a toxic metalloid that causes skin cancer and binds to cysteine residues—a property that could be used to infer arsenic responsiveness of a target protein. Non-synonymous Single Nucleotide Polymorphisms (nsSNPs) result in amino acid substitutions and may alter arsenic binding with cysteine residues. Thus, the objective of this investigation was to identify and analyze nsSNPs that lead to substitutions to or from cysteine residues as an indication of increased or decreased arsenic responsiveness. We hypothesize that integration of data on molecular impacts of nsSNPs and arsenic-gene relationships will identify nsSNPs that could serve as arsenic responsiveness markers. We have analyzed functional and structural impacts data for 5,811 nsSNPs linked to 1,224 arsenic-annotated genes. In addition to the identified candidate nsSNPs for increased or reduced arsenic responsiveness, we observed i) a nsSNP that results in the breakage of a disulfide bond, as candidate marker for reduced arsenic responsiveness of KLK7, a secreted serine protease participate in normal shedding of the skin; and ii) 6 pairs of vicinal cysteines in KLK7 protein that could be binding sites for arsenic. In summary, our analysis identified non-synonymous SNPs that could be used to evaluate responsiveness of a protein target to arsenic. In particular, an epidermal expressed serine protease with crucial function in normal skin physiology was prioritized on the basis of abundance of vicinal cysteines for further research on arsenic-induced keratinocyte carcinogenesis.
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