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The development of effective cancer immunotherapies has been consistently hampered by several factors, including an inability to instigate long-term effective functional antitumor immunity. This is particularly true for immunotherapies that focus on the adoptive transfer of activated or genetically modified mature [CD8.sup.+] T cells. In this study, we sought to alter and enhance long-term host immunity by genetically modifying, then transplanting, mouse HSCs. We first cloned a previously identified tumor-reactive HLA-DR4-restricted [CD4.sup.+] TCR specific for the melanocyte differentiation antigen tyrosinase-related protein 1 (Tyrp1), then constructed both a high-expression lentivirus vector and a TCR-transgenic mouse expressing the genes encoding this TCR. Using these tools, we demonstrated that both mouse and human HSCs established durable, highefficiency TCR gene transfer following long-term transplantation into lethally irradiated mice transgenic for HLA-DR4. Recipients of genetically modified mouse HSCs developed spontaneous autoimmune vitiligo that was associated with the presence of a Th1-polarized memory effector [CD4.sup.+] T cell population that expressed the Tyrp1-specific TCR. Most importantly, large numbers of [CD4.sup.+] T cells expressing the Tyrp1-specific TCR were detected in secondary HLA-DR4-transgenic transplant recipients, and these mice were able to destroy subcutaneously administered melanoma cells without the aid of vaccination, immune modulation, or cytokine administration. These results demonstrate the creation of what we believe to be a novel translational model of durable lentiviral gene transfer that results in long-term effective immunity., Introduction A major obstacle in the development of effective cancer immunotherapies has been the inability to instigate long-term effective functional antitumor immunity. Recent clinical studies involving adoptive transfer of T [...]

Simple efforts are needed to enhance cord blood (CB) transplantation. We hypothesized that short-term exposure of CD34+ CB cells to 39.5°C would enhance their response to stromal-derived factor-1 (SDF-1), by increasing lipid raft aggregation and CXCR4 expression, thus leading to enhanced engraftment. Mild hyperthermia (39.5°C) significantly increased the percent of CD34+ CB that migrated toward SDF-1. This was associated with increased expression of CXCR4 on the cells. Mechanistically, mild heating increased the percent of CD34+ cells with aggregated lipid rafts and enhanced colocalization of CXCR4 within lipid raft domains. Using methyl-β-cyclodextrin (MβCD), an agent that blocks lipid raft aggregation, it was determined that this enhancement in chemotaxis was dependent upon lipid raft aggregation. Colocalization of Rac1, a GTPase crucial for cell migration and adhesion, with CXCR4 to the lipid raft was essential for the effects of heat on chemotaxis, as determined with an inhibitor of Rac1 activation, NSC23766. Application-wise, mild heat treatment significantly increased the percent chimerism as well as homing and engraftment of CD34+ CB cells in sublethally irradiated non-obese diabetic severe combined immunodeficiency IL-2 receptor gamma chain d (NSG) mice. Mild heating may be a simple and inexpensive means to enhance engraftment following CB transplantation in patients. Stem Cells S tem C ells 2015;33:1975-1984 [ABSTRACT FROM AUTHOR]

Objective: Cell-cycle checkpoints guarantee movement through the cell cycle. Mitotic arrest deficiency 2 (Mad2), a mitotic checkpoint protein, appears crucial for generating the wait anaphase signal to prevent onset of anaphase. We evaluated effects of Mad2 haploinsufficiency on hematopoietic stem (HSC) and progenitor (HPC) function in response to stress. Materials and Methods: We studied effects of Mad2+/− on in vivo recovery of bone marrow HPC from cytotoxic effects and also effects of cytostatic agents on HPC growth in vitro using Mad2+/− mice. Results: Mad2 +/− HPCs were protected from cytotoxic effects in vivo of a cell-cycle−specific agent, Ara-C, events consistent with Mad2 +/− HPCs being in a slow or noncycling state, but not from recovery of functional HPC after treatment with non-cycle−specific cyclophosphamide or sublethal irradiation. There were no differences in phenotyped HSCs in Mad2 +/− &Mad2 +/+ mice, information confirmed by no changes in short- or long-term repopulating HSC assay. To better understand Mad2 +/− HPC function, E3330, a cytostatic agent, was used to assess redox function of Ape1/Ref-1; colony growth was examined under 5% and 20% O2 tension. Mad2 +/− HPCs were less responsive to E3330 than Mad2 +/+ HPCs, and E3330 was more effective under lowered O2 tension. Mad2 +/− HPCs were not enhanced at lowered oxygen, as were Mad2 +/+ HPCs. Conclusions: Our studies have unexpectedly found that Mad2 haploinsufficiency is protective in the presence of a cycle-specific DNA synthesis agent in vivo, and Ape1/Ref-1 inhibitor in vitro. [ABSTRACT FROM AUTHOR]

Objective: Human cord blood (CB) is a potential source of hematopoietic stem cells (HSC) for gene therapy to treat patients with hematopoietic disorders. However, limited numbers of CB CD34+ cells, low transduction efficiency with lentiviral vectors (LVs), and low engraftment efficiency of nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating cells (SRC), a measure of HSC, are blocks to this procedure. To optimize culture and transduction conditions, we compared various lengths of time for prestimulation before transduction, transduction duration, and posttransduction cell culture. Materials and Methods: We used a LV to transduce human CB CD34+ cells followed by engraftment into NOD/SCID mice. We evaluated the effects of prestimulation and transduction time and optimized ex vivo cell culture duration before transplantation. Results: We were able to achieve up to 40% transduction efficiency and up to 50% engraftment efficiency of SRC in CB CD34+ cells when CB CD34+ cells were either not prestimulated or prestimulated in 1% fetal bovine serum medium for 1 hour, followed by 5 hours transduction and 3 days culture in a cocktail of growth factors after transduction. No apparent functional changes of CB CD34+ cells were noted under these conditions. Conclusion: This gene-transduction/cell-expansion protocol is the first systematic study to optimize prestimulation time, transduction time, and, very importantly, ex vivo culture time after transduction, and may be of use for LV gene transduction in a gene therapy setting. [Copyright &y& Elsevier]

Validated gene transfer and expression tracers are essential for elucidating functions of mammalian genes. Here, we have determined the suitability and unintended side effects of enhanced green fluorescent protein (EGFP) and DsRed-Express fluorescent protein as expression tracers in long-term hematopoietic stem cells (HSCs). Retrovirally transduced mouse bone marrow cells expressing either EGFP or DsRed-Express in single or mixed dual-color cell populations were clearly discerned by flow cytometry and fluorescence microscopy. The results from in vivo competitive repopulation assays demonstrated that EGFP-expressing HSCs were maintained nearly throughout the lifespan of the transplanted mice and retained long-term multilineage repopulating potential. All mice assessed at 15 months post-transplantation were EGFP positive, and, on average, 24% total peripheral white blood cells expressed EGFP. Most EGFP-expressing recipient mice lived at least 22 months. In contrast, Discosoma sp. red fluorescent protein (DsRed)-expressing donor cells dramatically declined in transplant-recipient mice over time, particularly in the competitive setting, in which mixed EGFP- and DsRed-expressing cells were cotransplanted. Moreover, under in vitro culture condition favoring preservation of HSCs, purified EGFP-expressing cells grew robustly, whereas DsRed-expressing cells did not. Therefore, EGFP has no detectable deteriorative effects on HSCs, and is nearly an ideal long-term expression tracer for hematopoietic cells; however, DsRed-Express fluorescent protein is not suitable for these cells. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Peripheral blood CD14+CD16++ monocytes (Mo) are a rare Mo subpopulation known to undergo expansion in various diseases. We show here that IL-10 in the presence of M-CSF and IL-4 triggers the generation of CD14+CD16++ cells from highly purified human cord blood (CB) and adult blood Mo. CB Mo were more sensitive to this cytokine combination than adult Mo. IL-10-induced CD14+CD16++ cells that expressed dendritic cell markers: CD80, CD86, HLA-DR, and CD83 and initiated significantly decreased allogeneic mixed lymphocyte reactions (MLRs). Blockage of CD86, but not CD80, further down-modulated MLRs induced by CD14+CD16++cells. CD14+CD16++ cells had similar features to CD14+CD16++ Mo in that they expressed increased level of CCR5, efficiently produced TNF-α, and displayed higher MLR than CD14+CD16− Mo. Together, these results demonstrate that M-CSF, IL-4, and IL-10 drive Mo into CD14+CD16++ cells similar to those identified in vivo, and CB Mo, due to their increased responsiveness, may be a useful starting cell source to study differentiation of CD14+CD16++ cells. [Copyright &y& Elsevier]

: ObjectiveUnderstanding the molecular events that contribute to survival of and drug-induced apoptosis in hematopoietic stem and progenitor cells (HSC/P) can have impact on more rational approaches to blood cancer therapeutic design, as well as on strategies to minimize toxic side effects of chemotherapeutic drugs. Here we sought to systematically evaluate the basic molecular components and main pathways that govern and mediate cellular response initiated within human CD34+ cells to etoposide-induced apoptosis.: Materials and MethodsHuman CD34+ cells were isolated from umbilical cord blood (CB) and expanded in vitro. Expression of apoptosis-related genes in the control and etoposide treated cells was determined using cDNA array and quantitative real-time RT-PCR.: ResultsWe identified a set of apoptosis-related genes expressed in highly purified normal human CB CD34+ cells and determined how the expression of these genes changed in response to etoposide treatment. In addition, TRAIL does not induce apoptosis of normal human CD34+ cells, and it has no cytotoxic effect on human CD34+ cells that are undergoing apoptosis in response to growth factor withdrawal. This may be due to upregulation of cytotoxic receptors as well as the decoy receptor for TRAIL, and c-FLIP.: Conclusionp53, c-Myc, and BAFF pathways are main pathways utilized by CD34+ cells to arrest cell-cycle progression at multiple checkpoints, to halt proliferation, and to induce apoptosis as part of their cellular response to etoposide. Multiple known pro-survival and pro-apoptotic pathways are simultaneously activated in etoposide-treated CD34+ cells. Also, TRAIL, used alone or in concert with chemotherapeutic drugs, may be of use as a safe blood cancer therapeutic with no or low toxicity for HSC/P. [Copyright &y& Elsevier]

Reports on a study that found the increased osteoclast development after estrogen loss can be changed by an antibody to interleukin-6. Method; Results; Conclusion.