Academic literature on the topic 'Fetal; Donor; Tumour'

While hematopoietic stem cells from adult and fetal stages of murine development are capable of long term reconstitution of all mature blood lineages in vivo, embryonic hematopoietic stem cell repopulation in vivo has proved difficult. It is thought that there are many fewer hematopoietic stem cells in the embryo than in the fetal/adult stages of mouse development and that these cells possess a different developmental potential. One source of such cells are embryonic stem (ES) cells which can differentiate into most mature blood lineages in vitro. We have therefore used transplantation of differentiated ES cells to assess the hematopoietic potential of embryonic hematopoietic cells in vivo. We demonstrate here that precursors obtained from in vitro cultures of normal ES cells can contribute only to restricted and limited hematopoiesis in a mouse without leading to tumour formation. Repopulation occurs for greater than 6.5 months at levels ranging from 0.1% to 6% in B and T cell lineages in peripheral blood. In contrast to in vitro colony data demonstrating the myeloid lineage developmental potential of ES cells, no donor-derived myeloid repopulation was observed in CFU-S assays and no macrophage and mast cells were found in long term repopulated recipients. Thus, the hematopoietic potential of ES cells in vivo is limited to low levels of repopulation and is restricted to the lymphoid lineage.

Abstract The SSX family includes five functionally active and highly homologous members which are only expressed in the thyroid and testis in normal tissues. However these genes are expressed in several cancers including melanoma. Of note SSX1, SSX2, or SSX4 may be fused to the SYT gene as a result of the t(X;18) translocation in synovial sarcoma. Previous investigators used yeast two-hybrid systems to elucidate the SSX2 interacting protein (SSX2IP) through its association with SSX2. SSX2IP has been shown to be ubiquitously expressed in many normal tissues. We found SSX2IP through the immunoscreening of a testis cDNA library with pooled presentation M4 and M5 acute myeloid leukaemia (AML) sera. SSX2IP was found to be preferentially recognized by 62% of acute myeloid leukaemia (AML) sera (n=22) compared to 21% of normal donor sera (n=20). By RT-PCR SSX2IP was found to be expressed by four of 12 (30%) AML patient samples at presentation but none of eight normal donor haematopoietic samples (bone marrow and peripheral blood). By immunocytochemistry (ICC) we found SSX2IP protein expression in three of six myeloid leukaemia cell lines (K562, P39 and HL60), three of nine AML samples at presentation but none of three normal donor haematopoietic samples (peripheral blood). By confocal microscopy we noticed SSX2IP was predominantly expressed on the surface of K562 cells in early mitosis. We then synchronized K562 cells (as demonstrated by flow cytometry) using either serum starvation (0.1% fetal calf serum for 5 days) in the G0 phase or using 0.3mM hydroxyurea for 3 days at the G1/S interface of the cell cycle. On release back into the cell cycle (as shown by cell counts and flow cytometry) we observed a 3hr period, post-synchronisation, at 24–26 hours for serum starvation or 14–17 hours for hydroxyurea treatment during which time SSX2IP expression peaked, as detected by ICC and confocal microscopy. We transfected K562 cells with the cell surface expressed costimulatory molecule CD80 and demonstrated that SSX2IP was almost entirely restricted in its expression to the cell surface, mimicing the expression pattern of CD80. SSX2IP has a murine counterpart with 87% amino acid sequence similarity called ADIP. ADIP is a novel Afadin- and alpha-actinin-binding protein which localises at cell-cell adherens junctions. ADIP is at least partly involved in the physical association of nectins and cadherins and has more recently been implied to play a role in vesicle trafficking from the Golgi to the endoplasmic reticulum and through the Golgi complex. The mouse protein ADIP has been shown to interact with AF6, the human equivalent of which is involved in the human MLL-AF6 translocation in AML and acute lymphocytic leukaemia. However AF6 was not found to colocalise with SSX2IP in K562 cells as determined by confocal microscopy in our studies although both were expressed. P39 cells only expressed detectable levels of SSX2IP and not AF6. We have demonstrated a novel expression of the tumour antigen SSX2IP on the surface of AML cells in early mitosis. As such SSX2IP may provide a novel antibody target for the depletion of the AML cells from harvests prior to autologous transplant, when no suitable allogeneic donor can be found.

Interspecies somatic cell nuclear transfer (iSCNT) is an invaluable tool for studying nucleous-cytoplasm interactions, and may provide an alternative for cloning endangered animals, whose oocytes are difficult to obtain. Using readily available oocytes from domestic/farm animals as recipients for iSCNT would greatly benefit ongoing research on somatic cell reprogramming. However, little information is available concerning the development of canine iSCNT embryos reconstructed with bovine oocyte cytoplasm. In the first experiment, we investigated the influence of donor cell type on the development of canine iSCNT embryos reconstructed with enucleated bovine oocytes. Canine mammary gland tumour (MGT) cells and cumulus cells were used as donor cell. The bovine oocytes matured for 22 h were enucleated by the micromanipulator, and the donor cells were transferred into the perivitelline space adjacent to the plasma membrane of the oocyte. The couples were fused and activated simultaneously with a single DC pulse of 2.3 kV cm–1 for 30 μs, using an electro cell fusion generator. The reconstructed embryos were cultured for 72 h in the mSOF medium supplemented with 0.4% BSA. After 72 h of culture, only cleaved embryos were further co-cultured with bovine cumulus cells in mSOF supplemented with 5% fetal bovine serum (FBS) for an additional 5 days. In the second experiment, we examined the effects of serum type on the development of canine iSCNT embryos. The embryos reconstructed with canine cumulus cells were co-cultured with canine cumulus cells in mSOF supplemented with 5% FBS, and canine oestrous and diestrous serum for 5 days after 72 h of culture with 0.4% BSA. Data were analysed by chi-square analysis with a Yates’ correction. More than 75% of the canine somatic cells successfully were fused with bovine enucleated oocytes following electrofusion, irrespective of the types of the donor cells. There were no significant differences in the cleavage rates of iSCNT embryos between the cumulus cell and MGT cell (66.2% v. 62.6%). Although none of the embryos reconstructed with MGT cells (n = 123) developed to the 16-cell stage, 6% of embryos with cumulus cells (n = 133) reached at least the 16-cell stage. There were no significant differences in the cleavage rates of iSCNT embryos among the types of serum. The iSCNT embryos could not develop to the blastocyst stage, irrespective of the type of donor cell and serum. In conclusion, our results indicate that the bovine oocytes partly supported the remodelling and reprogramming of the canine somatic cell nuclei, but they were unable to support the development to the blastocyst stage of canine iSCNT embryos. Moreover, the development to the late embryonic stage of iSCNT embryos may be influenced by the type of donor cell but not serum.

Abstract Background Most metastatic melanoma patients treated with Programed cell Death (PD)-1 blockers fail to achieve a durable response. The gut microbiota profoundly affects host immunity, and fecal microbiota transplantations (FMT) have been shown to enhance anti-PD-1 effectiveness in murine models. We report initial safety and efficacy results from the first patients treated in a Phase I study of FMT and re-induction anti-PD-1 therapy in anti-PD-1 refractory metastatic melanoma. Methods FMT donors were two metastatic melanoma patients who achieved a durable complete response to treatment. FMT recipients were metastatic melanoma patients who failed at least one anti-PD-1 line of treatment. FMT was conducted by both colonoscopic and oral administration, followed by anti-PD-1 re-treatment. Each recipient underwent pre- and post-treatment stool sampling, tissue biopsy of both gut and tumor, and total body imaging. Results Five patients with treatment-resistant metastatic melanoma were recruited. No FMT-related or immunotherapy-related adverse events were observed. To assess engraftment of the new microbiota, recipients were paired with their respective donors and stool 16S rDNA gene sequence analysis was performed. Sequencing results demonstrated post-FMT compositional dissimilarity (Unweighted UniFrac, P = 0.04, FDR q = 0.22) between the two recipient–donor groups. Specific taxonomic dynamics included post-FMT increased abundance of Paraprevotellaceae, previously associated in descriptive studies with responsiveness to treatment, and significant reductions in abundance of β-proteobacteria, previously associated with reduced response to treatment. Immunohistochemical stains of biopsies demonstrated an increased post-FMT infiltration of antigen presenting cells (CD68+) in the gut (paired T-test, P = 0.008) and in the tumor (P = 0.0076). Post-treatment intra-tumoral CD8+ T-cell infiltration was also increased. Three patients had a partial or complete response to treatment post-FMT. Conclusion FMT in metastatic melanoma patients seems to be safe and may alter recipient gut microbiota to resemble that of a responder donor. This alteration may result in intra-tumoral T-cell activity, and conferred clinical and radiological benefit in several recipients previously unresponsive to treatment. Disclosures All Authors: No reported Disclosures.

Abstract [Objective] Our previously work has demonstrated that allogeneic bone marrow transplantation (allo-BMT) combined with thymus transplantation (TT) was effective in restoring donor-derived T cell function and was beneficial for enhancing graft versus tumor (GVT) effects. However, since the thymic cell functions differ with age, the most effective age of thymus should be explored. In the present study, we examined the effects of allo-BMT plus thymus transplantation (TT) from various ages (fetal, newborn, adult) to determine it’s anti tumor effects. [Methods] BALB/c mice (H-2d ) bearing Meth-A sarcoma (H-2d )were treated with allo-BMT combined with or without TT from various age B6 mice(H-2b), the tumor size and survival period of the recipient BALB/c mice were examined, histological studies were performed in the liver, intestine, and the engrafted thymus from the recipients 4 weeks after the BMT. Surface markers on lymphocytes from the spleen were analyzed by 3-color fluorescence staining using a FACScan system to determine chimerism. Cytokine production was examined for monitoring lymphocyte function. [Results]. All mice treated with BMT with or without TT showed fully donor-derived chimerism. The tumor size were significantly smaller in the mice treated with BMT plus TT than those treated with BMT alone. Interestingly, the mice treated with BMT plus newborn or fetal thymus showed the greatest degree of tumor regression. The survival rate in mice treated with BMT plus newborn thymus was significantly prolonged compared with those treated with BMT plus adult thymus or BMT plus fetal thymus. Histologically, both the cortex and medullar areas were clearly shown in each group. Normal T-cell differentiation was also observed in the engrafted thymus. The number of CD4+ T cells significantly increased in the mice treated with BMT plus TT compared with those treated BMT alone. The numbers were highest in the mice treated with BMT plus newborn thymus or BMT plus fetal thymus. Microscopic founding of small intestine and liver indicated no evidence of GVHD in all mice treated with BMT combined with or without TT. The production of IL-2 and IFN-γ was significantly elevated in the mice treated with BMT plus TT compared with those treated with BMT alone. However, the production of IL-2 has no significantly difference in all various age thymus transplantation groups. In contrast, the production of IFN-γ was the highest in the mice treated with BMT plus newborn thymus transplantation. [Conclusion]. The present study indicated that allo-BMT combined with TT induces high thymopoiesis, elicit strong GVT effects, and is effective for the host with cancer. And the combination of allo-BMT with newborn thymus is the most effect. We thus found that donor-derived T cells play an important role in the treatment of leukemia. As human thymus tissue can be obtained from patient with congenital heart disease or from aborted fetuses, so the results of the present study suggest this strategy will become a new way for the treatment of malignant tumors in human. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 953 Recently developed culture conditions for expanding and activating natural killer (NK) cells may improve immunotherapeutic options for patients with multiple myeloma. We have previously shown that co-incubation of NK cells from multiple myeloma patients and healthy donors with K562 cells genetically modified to express membrane-bound interleukin 15 and the co-stimulatory molecule 41BBL (K562-mb15-41BBL) leads to a dramatic increase in both NK cell number and anti-myeloma activity in vitro. In this study, we tested the anti-myeloma activity of these expanded NK cells in vivo using a murine model, which supports the growth of myeloma cells in a microenvironment that reproduces that of the human bone marrow. We first implanted human fetal bones subcutaneously into NOD/SCID/IL2Rγ null mice and allowed them to engraft. Luciferase transfected OPM2 myeloma cells were then injected into the human bone fragment. Tumor burden was followed by bio-imaging and ELISA for human Ig. NK cells were expanded from healthy donor PBMC by co-culture with irradiated K562-mb15-41BBL cells in the presence of 300U/ml IL2 for 10-12 days. Myeloma tumor bearing mice were dosed 6-7 days after OPM2 injection (bio-imaging confirmed myeloma engraftment) with expanded NK cells via tail vein injection followed by subcutaneous IL2 to prolong NK cell survival. Flow cytometry was used to track the human NK cells in blood. Histology was performed by H&E staining of formalin-fixed, paraffin embedded tissues harvested at the end of the study. Sufficient NK cells (3×109) for dosing mice were obtained from one unit of healthy donor blood and their ability to kill OPM2 myeloma was confirmed in vitro by chromium release assays. In experiment 1, OPM2-bearing mice received two tail vein injections, 48h apart, comprising PBS, 4 ×107, or 16 ×107 (total dose) expanded NK cells with 100U IL2 given twice per week. We observed significant myeloma growth inhibition in the cohort given 16 ×107 NK cells (p<0.04) and circulating human NK cells could be detected up to day 21 post-administration. Measurement of tumor burden by bio-imaging and ELISA were highly concordant (correlation coefficient = 0.995). Histologic analyses confirmed a dramatic reduction in tumor burden in the mice treated with expanded NK cells and revealed that bone loss was more pronounced in the implanted fetal bones of the control cohort not receiving NK cells. We observed in a subsequent experiment that increasing the IL2 dose to 1000U daily led to in vivo expansion of CSFE-labeled NK cells of up to 10 generations by day 6 post-infusion, which was associated with an enhanced anti-tumor effect in the 16 ×107 dose cohort. Furthermore, we found that multiple injections of NK cells (four injections of 4 ×107 NK cells versus two injections of 8 ×107) were better tolerated in the highest dose cohort (3/3 surviving versus 6/12 surviving). In conclusion, we observed a significant anti-myeloma effect against the aggressive plasma cell leukemia cell line OPM2 with expanded NK cells and low dose IL2. Adjusting the IL2 dose to 1000U daily led to in vivo expansion of NK cells, longer term NK cell persistence, and an increased anti-myeloma effect. Importantly, this IL2 dose is comparable to the 3×106 U daily dose well tolerated in a previous trial with non-expanded NK cells in humans. Single doses of NK cells >8×107 led to high morbidity in this model while multiple doses of 4×107 were well tolerated. Experiments are in progress testing the in vivo effect of expanded NK cells on primary myeloma cell tumors in this model. Our findings support the planned clinical application of ex vivo expanded NK cells. Disclosures: No relevant conflicts of interest to declare.

The extracellular matrix protein tenascin (TN) is expressed with precise temporo-spatial patterns during embryonic and fetal development and is induced in healing wounds, inflammatory lesions and solid tumors. These tissue patterns suggest that TN synthesis may be modulated by soluble factors present in developing tissues or released from injured, inflammatory or neoplastic cells. To characterize the extrinsic control of human TN we examined the effects of several signalling molecules on cultured neural, melanocytic and fibroblastic cells. Results obtained with alpha TN antibodies in enzyme-linked immunosorbent and immunoprecipitation assays indicate that TN expression is tightly regulated in a cell type-specific manner: (1) Primitive neuroectodermal tumor (PNET) cells grown in chemically defined, serum-free media show up to &gt; 100-fold TN induction in response to fibroblast growth factors (aFGF, bFGF, K-FGF) and phorbol ester, independent of changes in cell proliferation or total protein synthesis; no induction is seen in PNET cultures stimulated with serum or other growth and differentiation factors. (2) Normal melanocytes, which require FGF and phorbol ester for survival in vitro, fail to express TN; however, they produce TN following oncogenic transformation. (3) Fibroblasts derived from disparate tissues differ up to 100-fold in basal TN production; for example, fetal lung fibroblasts are TNhigh, but conjunctival fibroblasts derived from the same donors and fetal leptomeningeal cells are TNlow. (4) TNlow fibroblasts treated with interleukin-1, tumor necrosis factor-alpha, and interleukin-4 show up to &gt; 100-fold increased TN secretion and TN incorporation into their extracellular matrix. Transforming growth factor-beta, which acts as an inducer of fibronectin, collagen, and integrin-type matrix receptors, has variable effects on fibroblast TN, ranging from increased deposition in the extracellular matrix of fetal conjunctival fibroblasts to reduced secretion in newborn foreskin fibroblasts. In contrast, FGFs (which are potent fibroblast mitogens), phorbol ester, bone morphogenetic proteins, and several other factors tested produced no discernible effects on fibroblast TN expression. These findings suggest that discrete sets of extrinsic signals modify TN expression in specific cell types, with the effects of a given ligand/receptor system determined by cell type-specific signalling pathways that may be linked to unique cis-regulatory elements of the TN gene. As a result, a limited set of regulatory peptides may produce highly diversified TN distribution patterns in developing and lesional tissues.

Abstract Differential gene expression profiling of pediatric common acute lymphoblastic leukemia (cALL) versus non-malignant tissues enables identification of aberrantly expressed genes in malignant cells, facilitating discrimination of leukemic from normal cells and possibly revealing specific disease mechanisms. Expression patterns of 29 pediatric cALL samples were analyzed by use of high-density DNA microarrays HG-U133A. Leukemic patients’ bone marrow samples were compared to sorted B cells from cord blood of healthy donors expressing CD19 and CD10 surface antigens. Principal component analysis clearly distinguished leukemia samples from normal controls. Significance analysis of microarrays revealed 723 genes significantly up-regulated, and 617 down-regulated genes in leukemic cells. Independent validation of deregulated genes by RT-PCR was chosen to address enrichment limitations of controls. A comparison to previous publications investigating genetically defined subsets of cALL revealed only 5 – 22% match with our differentially expressed genes. Furthermore, class prediction with only 14 differentially expressed genes correctly classified tumors and controls not included in the training set and hitherto not investigated samples including 3 leukemic tumor lines (NALM-6, CALL-2, 697) as cALL. Interestingly, terminal deoxynucleotidyl-transferase (DNTT) as well as in the context of cALL unknown genes, were found to be the strongest predictive genes for the malignant phenotype signifying the diagnostic value of our approach.

Somatic cell nuclear transfer (SCNT) has been utilized to study various genetic and epigenetic contributions of specific biomedical diseases and developmental events by using various donor cell types such as mature lymphocytes, brain tumor cells, and other unique genotypes. Previously, we produced cloned fetuses and offspring derived from SCNT of adult ear skin fibroblasts obtained from a sub-fertile cow harboring an X-autosome translocation as a model to study X-inactivation and chromosome dynamics during female meiosis. The aim of this study was to assess the cloning efficiency of the fibroblasts derived from a cloned calf with the X-autosome translocation t(Xp+;23q-) compared to the original adult fibroblast donor containing the same chromosome translocation. Primary cultures of cells were established in DMEM +15% fetal calf serum (FCS). To serve as nuclear donors, cells at 5-7 passages were cultured for 5 days until confluent. Oocytes matured for 18 h in TCM-199 with hormones were removed of their chromatin, and reconstructed by transfer of donor cells and fusion with two DC pulses (1.2 kV/cm, 15 �s), delivered by a BTX 2000 Electro Cell Minupulator (BTX, Inc., San Diego, CA, USA), in 0.28 M mannitol containing 0.01 mM MgCl2. After 1 h of fusion, the eggs were activated with 5.5 �M ionomycin for 5 min, followed by 11 �g/mL cyclohexamide for 5 h. The eggs were cultured for 9 days in L-SOF at 39�C in a humidified atmosphere of 5% CO2, 5% O2, 90% N2. Chi-square analysis revealed no significant (P > 0.05) differences in the rates of cleavage, blastocyst frequencies, and cell numbers between the 1st and 2nd generation cloned embryos. Cleavage rates were 87.4% and 85.4% for 1st and 2nd generation cloned embryos, respectively. The frequencies of blastocyst development and hatched blastocyst formation on Day 9 were 41.4% (91/220) and 38.7% (92/238), and 26.4% (58/220) and 22.7% (54/238) for the 1st and 2nd generation cloned embryos, respectively. The numbers of total cells and inner cell mass (ICM) cells of Day 9 blastocysts were 183 and 52, respectively, in the 1st generation embryos and 167 and 51 cells in the 2nd-generation cloned embryos. In summary, 2nd generation cloned embryos derived from fibroblasts of a cloned calf with an X-autosome translocated chromosome showed embryo development and cell numbers similar to those of the 1st generation clones. These results demonstrate that serial nuclear transfer does not improve the blastocyst development rate of cloned embryos containing the X-autosome translocation t(Xp+;23q-). This work was funded by OCAG, OMAF, and CRC.

Dendritic cells (DCs) are a heterogeneous population of antigen-presenting cells derived from hematopoietic progenitors that bridge the transition between the innate and adaptive immune responses, while maintaining self-tolerance and Th1/Th2 homeostasis, by priming other cells in either an immunogenic or tolerogenic direction. Through their role in both innate and adaptive immunity, DCs play a major part in transplant engraftment and rejection and in graft-versus-host disease (GvHD). Preferentially tolerogenic or immunogenic DC subtypes offer targets for immunotherapy, to optimize transplant success rates and prolong disease-free and overall survival. Cord blood DCs are immature and preferentially tolerogenic, due to maternal-fetal tolerance, leading to better graft acceptance and immune reconstitution and explaining the lower incidence and severity of GvHD in CB transplantation, despite donor-host mismatching. Manipulation of DC maturation and cell loading with tumor-antigens can direct antitumor immunity and target minimal residual disease, as demonstrated for acute myeloid leukemia, optimizing the graft-versus-leukemia effect.