Donor Markers Research Articles

Beta-cell death and dysfunction drives hyperglycaemia in organ donors.

Donor hyperglycaemia following brain death has been attributed to reversible insulin resistance. However, our islet and pancreas transplant data suggest that other mechanisms may be predominant. We aimed to determine the relationships between donor insulin use and markers of beta-cell death and beta-cell function in pancreas donors after brain death. In pancreas donors after brain death, we compared clinical and biochemical data in 'insulin-treated' and 'not insulin-treated donors' (IT vs. not-IT). We measured plasma glucose, C-peptide and levels of circulating unmethylated insulin gene promoter cell-free DNA (INS-cfDNA) and microRNA-375 (miR-375), as measures of beta-cell death. Relationships between markers of beta-cell death and islet isolation outcomes and post-transplant function were also evaluated. Of 92 pancreas donors, 40 (43%) required insulin. Glycaemic control and beta-cell function were significantly poorer in IT donors versus not-IT donors [median (IQR) peak glucose: 8 (7-11) vs. 6 (6-8) mmol/L, p = .016; C-peptide: 3280 (3159-3386) vs. 3195 (2868-3386) pmol/L, p = .046]. IT donors had significantly higher levels of INS-cfDNA [35 (18-52) vs. 30 (8-51) copies/ml, p = .035] and miR-375 [1.050 (0.19-1.95) vs. 0.73 (0.32-1.10) copies/nl, p = .05]. Circulating donor miR-375 was highly predictive of recipient islet graft failure at 3 months [adjusted receiver operator curve (SE) = 0.813 (0.149)]. In pancreas donors, hyperglycaemia requiring IT is strongly associated with beta-cell death. This provides an explanation for the relationship of donor IT with post-transplant beta-cell dysfunction in transplant recipients.

P–136 Factors predicting clinical outcomes of 511 recipients of vitrified oocyte donation from an UK-regulated egg bank

Abstract Study question Do established donor and recipient clinical markers predict recipient clinical pregnancy and live birth rates (LBRs) in a vitrified oocyte donation programme? Summary answer Recipient BMI and previous miscarriages predicted cumulative LBR. Likelihood of clinical pregnancy and LBR was higher in recipients of donors aged 23–29 than donors 18–22. What is known already The influence of age on ovarian reserve underlies the upper limit of 35 years for UK donors. However, recent evidence suggests that oocyte aneuploidy rates follow an inverse U-shaped curve in relation to a woman’s age. Conflicting evidence exists regarding the impact of other donor-related factors including BMI, AMH, oocyte yield and prior reproductive history on recipient outcomes. Moreover, the effect of recipient age, BMI, and reproductive history on oocyte donation outcome remains unclear. Study design, size, duration Retrospective cohort study of 325 altruistic oocyte donors matched to a total of 511 recipients. Only first donations taking place between January 2017 and December 2019 were included. Participants/materials, setting, methods All oocyte donors were altruistic volunteers aged 18–35 with no prior infertility diagnosis. Donor and recipient screening for suitability and safety was carried out according to the Human Fertilisation Embryology Authority guidelines. Backward stepwise logistic regression was used to identify donor, recipient and embryology parameters predictive of recipient primary outcomes defined as clinical pregnancy and live birth, either cumulative or after the first embryo transfer (ET). Main results and the role of chance A total of 705 fresh and frozen/thawed ETs were performed, of which 76% were elective single embryo transfers (eSETs) of blastocysts (96.5%), resulting in a cumulative clinical pregnancy and LBR of 83.5% and 70.5% respectively after 3 ETs. Recipient BMI and previous miscarriages were predictors of cumulative LBR (p < 0.05). The ratio of transferrable embryos per oocytes received/fertilised and the number of ETs needed to achieve the intended primary outcome were predictors of cumulative clinical pregnancy and LBR (p < 0.05). Donor age 18–22 was associated with lower incidence of recipient clinical pregnancy and live birth after the first ET, as compared to donor age 23–29 (p < 0.05). Limitations, reasons for caution The present study included only healthy oocyte donors, thus conclusions may not apply to subfertile or less healthy women. Male factors were not accounted for. Wider implications of the findings: We demonstrate the efficacy of vitrified oocyte donation treatment and identify recipient BMI, previous miscarriages and embryology parameters as predictors of cumulative LBR. Additionally, the choice of donors aged 18–22 instead of older donors is found not to be advantageous for increasing the chance of clinical pregnancy and live birth. Trial registration number Not applicable

Factors predicting clinical outcomes from 494 vitrified oocyte donation cycles at a UK-regulated egg bank

Research questionDo donor age, AMH, AFC, BMI and reproductive history predict response to ovarian stimulation? Do donor and recipient clinical markers and embryology parameters predict recipient pregnancy and live birth? DesignRetrospective cohort study of 494 altruistic oocyte donors aged 18–35 years; 340 were matched to 559 recipients. Predictors of donor total oocyte yield and total mature oocyte yield were identified. Total and mature oocyte number were compared according to stratified donor AMH and age. Donor, recipient and embryology parameters predictive of recipient primary outcomes (clinical pregnancy and live birth) were identified. ResultsDonor age and AMH predicted total oocyte yield (P = 0.030 and P < 0.001)) and total mature oocyte yield (P = 0.011 and P < 0.001). Donors aged 30–35 years with AMH 15–29.9 pmol/l had lower total oocyte yield (P = 0.004) and mature oocyte yield (P < 0.001) than donors aged 18–24 years. Up to an AMH threshold of 39.9 pmol/l, increasing AMH levels predicted higher total oocyte yield (<15 pmol/l versus 15–29.9 pmol/l, P = 0.001; 15–29.9 pmol/l versus 30–39.9 pmol/l, P < 0.001; 30–39.9pmol/l versus ≥ 40 pmol/l, P = 1.0) and mature oocyte yield (<15 pmol/l versus 15–29.9 pmol/l, P = 0.005; 15–29.9 pmol/l versus 30–39.9 pmol/l, P = 0.006; 30–39.9 pmol/l versus ≥40 pmol/l, P = 1.0). In recipients, the rate of transferrable embryos per oocytes received, fertilized and number of embryo transfers needed to achieve the primary outcome were predictors of cumulative clinical pregnancy (P = 0.011, P = 0.017 and P < 0.001) and live birth (P = 0.008, P = 0.012 and P < 0.001) rates. Recipient BMI (P = 0.024) and previous miscarriages (P = 0.045) were predictors of cumulative live birth rate. Donor age 18–22 years was associated with a lower incidence of recipient clinical pregnancy (P = 0.004) and live birth (P = 0.001) after the first embryo transfer versus donor age 23–29 years. ConclusionsDonor age and AMH are independent predictors of oocyte yield. Raised recipient BMI and history of miscarriages reduce cumulative live birth rates, which may be increased by selecting donors aged 23–29 years, instead of younger donors.

The Impact of Donor/Recipient Age Difference and HLA Matching on Graft Outcome in Pediatric Kidney Transplantation

Introduction Understanding the relationship between the factors that influence long-term kidney transplant survival in order to appropriately select the optimal initial graft remains a key priority for pediatric nephrologists. Materials and Methods Using the ANZDATA database, we conducted a retrospective cohort study of pediatric and adolescent primary kidney transplant recipients to assess the relative impact of donor/recipient age difference and HLA mismatches on long-term graft outcomes. The study population included all kidney transplant recipients less than 20 years of age who received a primary kidney transplant in Australia and New Zealand between January 1, 1990 and December 31, 2015. The primary outcome was graft survival following first kidney transplant analysed according to the Kaplan-Meier method. Secondary outcomes included delayed graft function (requiring dialysis in the first 72 hours post-transplant) and graft function at 12 months, as a surrogate marker of long term graft survival. Results and Discussion During this 26-year period, 1134 primary kidney transplants were performed in recipients less than 20 years of age. The median follow-up time after the first graft transplant was 10.2 years (IQR 4.2-16.3 years), with 30 recipients lost to follow up (2.6%). During the study period, 405 patients (35.7%) lost their graft with graft survival of 93.8% (95% CI, 92.4%-95.2%) at 1 year post-transplant, 82.5% (95%CI 80.6%-84.4) at 5-years, 65.8% (95% CI, 62.5-69.1%) at 10 years and 49.9% (95% CI, 46.9-54.1%) at 15 years. There was consistently higher graft loss of deceased donor kidneys as compared to live donor kidneys at each time point. On Cox proportional hazards regression, both increasing donor/recipient age difference (adjusted hazard ratio [aHR] 1.09 per 10 years; 95%CI, 1.01-1.18; p=0.03) and increasing HLA mismatch (aHR 1.19 per mismatch; 95%CI, 1.10-1.30; p<0.001) were associated with decreased graft survival. In addition, graft failure was associated with the following factors: Indigenous/Pacific Islander race (when compared to Caucasian race, p<0.001), glomerulonephritis as a cause of ESKD (when compared to CAKUT; p=0.01), use of T-cell or other induction agents (when compared to basiliximab; p=0.009) and increasing recipient age at kidney transplant (p<0.001), when adjusted for baseline immunosuppression. Conclusion Both donor/recipient age difference and HLA matching are important factors influencing long-term graft outcomes in pediatric kidney transplantation and HLA mismatch remains a strong predictor of graft loss. For pediatric patients without the option of a live donor, we suggest that along with age of the donor and other markers of kidney quality, the degree of HLA mismatch should not be discounted as part of the decision making process in deceased donor allocation. ANZDATA.

Application of interspecific Somatic Cell Nuclear Transfer (iSCNT) in sturgeons and an unexpectedly produced gynogenetic sterlet with homozygous quadruple haploid

Somatic cell nuclear transfer (SCNT) is a very promising cloning technique for reconstruction of endangered animals. The aim of the present research is to implement the interspecific SCNT (iSCNT) technique to sturgeon; one fish family bearing some of the most critically endangered species. We transplanted single cells enzymatically isolated from a dissociated fin-fragment of the Russian sturgeon (Acipenser gueldenstaedtii) into non-enucleated eggs of the sterlet (Acipenser ruthenus), two species bearing different ploidy (4n and 2n, respectively). Up to 12% of the transplanted eggs underwent early development, and one feeding larva (0.5%) was successfully produced. Interestingly, although this transplant displayed tetraploidism (4n) as the donor species, the microsatellite and species-specific analysis showed recipient-exclusive homozygosis without any donor markers. Namely, with regards to this viable larva, host genome duplication occurred twice to form tetraploidism during its early development, probably due to iSCNT manipulation. The importance of this first attempt is to apply iSCNT in sturgeon species, establishing the crucial first steps by adjusting the cloning-methodology in sturgeon’s biology. Future improvements in sturgeon’s cloning are necessary for providing with great hope in sturgeon’s reproduction.

Microhomology-Mediated High-Throughput Gene Inactivation Strategy for the Hyperthermophilic Crenarchaeon Sulfolobus islandicus.

Sulfolobus islandicus is rapidly emerging as a model system for studying the biology and evolution within the TACK lineage of the archaeal domain. As the tree of life grows, identifying the cellular functions of genes within this lineage will have significant impacts on our understanding of the evolution of the last archaeal eukaryote common ancestor (LEACA) and the differentiation of archaea from eukaryotes during the evolution of the modern-day cell. To increase our understanding of this key archaeal organism, we report a novel high-throughput method for targeted gene inactivation in S. islandicus through one-step microhomology-directed homologous recombination (HR). We validated the efficacy of this approach by systematically deleting 21 individual toxin-antitoxin gene pairs and its application to delete chromosomal regions as large as 50 kb. Sequence analysis of 96 ArgD+ transformants showed that S. islandicus can effectively incorporate donor markers as short segments through HR in a continuous or discontinuous manner. We determined that the minimal size of homology allowing native argD marker replacement was as few as 10 bp, whereas argD marker replacement was frequently observed when increasing the size of homology to 30 to 50 bp. The microhomology-mediated gene inactivation system developed here will greatly facilitate isolation of S. islandicus gene deletion strains, making generation of a collection of genome-wide targeted mutants feasible and providing a tool to investigate homologous recombination in this organism.IMPORTANCE Current procedures for the construction of deletion mutants of S. islandicus are still tedious and time-consuming. We developed a novel procedure based on microhomology-mediated HR, allowing for rapid and efficient removal for genetic regions as large as 50 kb. Our work will greatly facilitate functional genomic studies in this promising model organism. Additionally, we developed a quantitative genetic assay to measure HR properties in S. islandicus, providing evidence that the ability to incorporate short, mismatched donor DNA into the genome through HR was probably a common trait for members of the Sulfolobus genus that are recombinogenic.

Transplanted photoreceptor precursors transfer proteins to host photoreceptors by a mechanism of cytoplasmic fusion.

Photoreceptor transplantation is a potential future treatment for blindness caused by retinal degeneration. Photoreceptor transplantation restores visual responses in end-stage retinal degeneration, but has also been assessed in non-degenerate retinas. In the latter scenario, subretinal transplantation places donor cells beneath an intact host outer nuclear layer (ONL) containing host photoreceptors. Here we show that host cells are labelled with the donor marker through cytoplasmic transfer—94±4.1% of apparently well-integrated donor cells containing both donor and host markers. We detect the occurrence of Cre-Lox recombination between donor and host photoreceptors, and we confirm the findings through FISH analysis of X and Y chromosomes in sex-discordant transplants. We do not find evidence of nuclear fusion of donor and host cells. The artefactual appearance of integrated donor cells in host retinas following transplantation is most commonly due to material transfer from donor cells. Understanding this novel mechanism may provide alternate therapeutic strategies at earlier stages of retinal degeneration.

Identification of microsatellite markers linked to drought tolerance in rice (Oryza sativa L.) through bulked line analysis

Identification of donor parents and markers for traits conferring drought tolerance eventually hastens the breeding for drought tolerance in rice. The extent of variation for water stress indicators, physio-morphological and plant production traits were assessed by evaluating 36 rice genotypes under water stress condition. Significant variation was observed for these traits and thirteen drought tolerant and susceptible genotypes were selected and grouped as tolerant and susceptible bulks based on physio-morphological and biochemical characterization. Genomic DNA was isolated from these rice accessions and pooled, respectively into drought tolerant and susceptible bulks. Bulked line analysis was carried to identify microsatellite markers linked to drought tolerance in rice. Out of 200 SSR primers screened RM 1092; RM 129 and RM157B were polymorphic between the bulks and also co-segregated among the individual genotypes constituting the respective bulks. The genomic regions flanked by these markers have been identified to be associated with various drought tolerance traits in rice and can be useful for marker assisted selection for drought resistance improvement in rice. The genotypes identified as drought tolerant can be used us donars in drought improvement breeding programmes. Key words Rice, drought resistance, microsatellite markers, bulk line analysis

Genetic Diagnosis in Recently Transfused Patients

Analysis of recently transfused patients is usually postponed to avoid spurious results because of contamination with donor's cells. However, little is known about the extent of this influence in routine molecular diagnostic tests. To elucidate this question, we tested a mix of blood samples from 2 α-1-antitrypsin-deficient patients diagnosed as Pi*Z homozygous with 1 normal donor at 1:1, 1:10, 1:20, and 1:30 proportions. Human identification panel and Pi*Z allele detection were used to establish the detection limit of a blood mixture. Mixtures of 1:1 and 1:10 were easily detected with both techniques, whereas for 1:30, it was necessary to change the equipment settings to identify the mixture. Moreover, the heterozygous pattern observed for the mixtures on Pi*Z genotyping was weaker at this level of mixture. We further evaluated the degree of mixture detectable in 20 transfused patients who received 1 blood unit (concentrate of irradiated or nonirradiated red blood cells) using the human identification panel. Two days after the transfusion, the presence of the donor's markers was not detected, suggesting that after this time point the levels of admixture are below 1:30. The methods applied in the present study showed adequate sensitivity to identify alleles of the so-called "smaller population" of cells up to 3%, approximately. The same result was obtained in a "diagnostic situation," in which the blood mixture was submitted to a PCR-RFLP protocol to detect a mutation.

Computational molecular phenotyping of retinal sheet transplants to rats with retinal degeneration

Retinal progenitor sheet transplants have been shown to extend neuronal processes into a degenerating host retina and to restore visual responses in the brain. The aim of this study was to identify cells involved in transplant signals to retinal degenerate hosts using computational molecular phenotyping (CMP). S334ter line 3 rats received fetal retinal sheet transplants at the age of 24-40 days. Donor tissues were incubated with slow-releasing microspheres containing brain-derived neurotrophic factor or glial cell-derived neurotrophic factor. Up to 265 days after surgery, eyes of selected rats were vibratome-sectioned through the transplant area (some slices stained for donor marker human placental alkaline phosphatase), dehydrated and embedded in Eponate, sectioned into serial ultrathin datasets and probed for rhodopsin, cone opsin, CRALBP (cellular retinaldehyde binding protein), l-glutamate, l-glutamine, glutathione, glycine, taurine, γ-aminobutyric acid (GABA) and DAPI (4',6-diamidino-2-phenylindole). In large transplant areas, photoreceptor outer segments in contact with host retinal pigment epithelium revealed rod and cone opsin immunoreactivity whereas no such staining was found in the degenerate host retina. Transplant photoreceptor layers contained high taurine levels. Glutamate levels in the transplants were higher than in the host retina whereas GABA levels were similar. The transplant inner nuclear layer showed some loss of neurons, but amacrine cells and horizontal cells were not reduced. In many areas, glial hypertrophy between the host and transplant was absent and host and transplant neuropil appeared to intermingle. CMP data indicate that horizontal cells and both glycinergic and GABAergic amacrine cells are involved in a novel circuit between transplant and host, generating alternative signal pathways between transplant and degenerating host retina.

Stem Cell Therapy in Spinal Cord Injury: In Vivo and Postmortem Tracking of Bone Marrow Mononuclear or Mesenchymal Stem Cells

The aim of this study was to address the question of whether bone marrow-originated mononuclear cells (MNC) or mesenchymal stem cells (MSC) induce neural regeneration when implanted intraspinally. The study design included 4 groups of mice: Group 1, non-traumatized control group; Groups 2, 3 and 4 spinal cord traumatized mice with 1 g force Tator clips, which received intralesionally either no cellular implants (Group 2), luciferase (Luc) (+) MNC (Group 3) or MSC (Group 4) obtained from CMV-Luc or beta-actin Luc donor transgenic mice. Following the surgery until decapitation, periodical radioluminescence imaging (RLI) and Basso Mouse Scale (BMS) evaluations was performed to monitor neural activity. Postmortem immunohistochemical techniques were used to analyze the fate of donor type implanted cells. All mice of Groups 3 and 4 showed various degrees of improvement in the BMS scores, whereas there was no change in Groups 1 and 2. The functional improvement was significantly better in Group 4 compared to Group 3 (18 vs 8, p=0.002). The immunohistochemical staining demonstrated GFP(+)Luc(+) neuronal/glial cells that were also positive with one or more of these markers: nestin, myelin associated glycoprotein, microtubule associated protein or myelin oligodendrocyte specific protein, which is considered as indicator of donor type neuronal regeneration. Frequency of donor type neuronal cells; Luc + signals and median BMS scores were observed 48-64% and 68-72%; 44-80%; 8 and 18 within Groups III and IV respectively. MSCs were more effective than MNC in obtaining neuronal recovery. Substantial but incomplete functional improvement was associated with donor type in vivo imaging signals more frequently than the number of neuronal cells expressing donor markers in spinal cord sections in vitro. Our results are in favor of functional recovery arising from both donor MSC and MNCs, contributing to direct neuronal regeneration and additional indirect mechanisms.

Hematopoietic Cells Undergo Homotypic Cell Fusion: A Novel Platform to Study Genomic Instability and Leukemogenesis.

AbstractAbstract 1196Gene copy number variation (CNV) and translocation-derived gene fusion products are commonly observed in hematological malignancies. Mechanisms precipitating CNV include failed cytokinesis or spontaneous cell fusion. Heterotypic fusion of hematopoietic cells with non-hematopoeitic cells has been reported following injury in diverse tissues. Cell fusion involves blending membranes, intracellular material, and nuclear material from two parental cells to form a genetically and immunophenotypically distinct (often hyperdiploid) single daughter cell. We therefore hypothesized that if we could generate genetically chimeric, hematopoietic cells via cell-cell fusion, they might provide an instructive model for determining the role CNV plays in leukemogenesis. To generate genetically abnormal hematopoietic cells in vivo, a transplantation model with radiation induction was employed in which donor and host mice possess numerous unique cell surface and genomic markers. Fusion was determined by co-expression of donor and host markers among hematopoietic cells isolated from recipient BM, spleen, and thymus by serial fluorescence activated cell sorting. The presence of ‘bona fide’ fused cells was confirmed by performing rigorous immunophenotypic and genotypic analyses of individual cells, including immunofluoresence staining and z-stack analysis, single nucleotide polymorphism (SNP) PCR, and fluorescent in situ hybridization (FISH). Fused cell populations were detected in both mature myeloid and lymphoid lineages. Consistent with the proposed model of studying early, initiating leukemogenic events, cell fusion was also detected in the progenitor cell populations including colony forming unit (CFU-C) myeloid progenitors, and c-kit+, sca-1+, lin- (KSL) cells. Moreover, transplanted fused hematopoietic cells could reconstitute lethally irradiated secondary hosts >16 weeks after transplant. Thus, hematopoietic progenitor cells are capable of homotypic cell fusion, and subsequent to cell fusion, these cells are further able to divide, differentiate, and are competent to contribute to functional hematopoiesis. Additional assays are underway to more definitively address the functional significance of hematopoietic cell fusion as a repair mechanism, or during homeostasis. Importantly, we detected evidence of genomic instability in fused hematopoietic cells using a variety of assays. Fused cells with loss of specific donor and/or host marker genes were identified by immunofluorescence and genomic DNA analysis. DNA strand breakage, evaluated by single cell gel electrophoresis (comet assay) showed the presence of a significantly larger tail moment in fused cells suggesting a higher degree of DNA damage in these cells. Studies for evaluation of p53 status and histone H2AX activation are underway. In summary, our data not only provide novel evidence that homotypic cell fusion occurs between hematopoietic cells following injury, but also that fused cells retain their replicative potential in vitro and in vivo and exhibit greater genomic instability compared to non-fused counterparts. Because most existing murine models of leukemogenesis focus on events in the promotion of disease, we believe fusion and reductive division of hematopoietic cells shown here will serve as a novel platform to study the role of CNV as a leukemia-initiating event. Disclosures:No relevant conflicts of interest to declare.

Understanding the Impact of Inflammation on Hematopoietic Stem Cells.

AbstractAbstract 2629The overall objectives of this study are to investigate the impact of inflammatory conditions on hematopoietic stem cell (HSC) maintenance and to elucidate the underlying mechanisms. HSCs are exposed to a variety of inflammatory conditions through life. How these conditions influence the integrity of HSCs is a fundamental issue of clinical importance but it is poorly understood. Equally unknown is the molecular regulation of HSC maintenance during inflammatory. In this context, our focus is on the role of basic helix-loop-helix (bHLH) proteins, which include transcription activators such as E2A proteins and their inhibitors including Id proteins. We and others have shown that these regulators are involved in normal hematopoiesis such as stem cell function and lineage specific differentiation. Recently, we have obtained evidence to suggest that signaling through Toll-like receptors (TLRs), which is closely linked to inflammation, causes down-regulation of E2A function by stimulating Id1 expression. Therefore, we hypothesize that inflammatory conditions causes down-regulation of E protein function, which disturbs the quiescence of long-term (LT)-HSC, leading to stem cell exhaustion over time.To test this hypothesis, we induced chronic inflammation in wild type and Id1-/- mice by daily injection of 1 mg of LPS, i.p. for 30 days. Peripheral blood was collected on days 15 and 30 and levels of a panel of inflammatory cytokines were assayed using a Luminex multiplex kit. On day 15, dramatic increases were found in the levels of IL-10, IL-6, KC and TNFα but not IFN-γ, IL12-p70 and IL-1β. Interestingly, levels of IL-6 and TNFα were significantly lower in Id1-/- mice compared to wild type mice. By day 30 of LPS treatment, levels of these cytokines returned to the levels in animals without LPS injection. These results suggest that this chronic LPS treatment indeed elicited an inflammatory response that included transient elevation of inflammatory cytokines. Whether secretion of these cytokines has any direct effects on HSCs remains to be determined.To measure HSC activity in these LPS-treated mice, we performed serial bone marrow transplant assays. Lin−Sca-1+c-kit+ (LSK) stem/progenitor cells were isolated from wild type or Id1-/- mice treated with or without LPS. These cells were transplanted into lethally irradiated CD45.1+ recipients along with equal numbers of YFP-expressing LSK as competitors. Six weeks later, cohorts of mice were sacrificed and bone marrow cells were collected. Pooled whole bone marrow cells within each cohort were injected into lethally irradiated secondary recipients. Secondary recipients were sacrificed 8 and 16 weeks post transplant. For assessment of primary and secondary engraftment, bone marrow cells were examined for expression of donor and lineage specific markers. Robust engraftment was observed in primary or secondary recipients. Donor derived cells were then gated for YFP− and YFP+ cells, which separate cells originated from tester and competitor LSK, respectively. While YFP− and YFP+ cells engrafted equivalently in primary recipients transplanted with cells treated with or without LPS, LPS treatment of wild type mice caused a great disparity in secondary recipients. In contrast, HSC in Id1-/- mice did not appear to be affected by the same treatment even though HSCs in Id1 deficient mice are normally lower in numbers and activities as we previously reported. These results suggest that chronic inflammation diminishes the LT-stem cell activity and this may involve the up-regulation of Id1 expression. To investigate the underlying mechanism, we performed label retaining assays to examine the quiescence of LT-HSCs. We found that BrdU-labeling in HSCs was 2-fold lower in mice treated with LPS compared to the untreated controls, suggesting that treatment with LPS promoted the cycling of HSCs, thus impairing their stem cell function.Taken together, our study illustrates that chronic inflammation has a detrimental effect on LT-stem cell activity. Although HSCs have an enormous capability to repopulate the bone marrow by compensatory proliferation, pro-longed inflammation could eventually lead to stem cell exhaustion and seriously compromise hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

Discontinuity and Limited Linkage in the Homologous Recombination System of a Hyperthermophilic Archaeon

Genetic transformation of Sulfolobus acidocaldarius by a multiply marked pyrE gene provided a high-resolution assay of homologous recombination in a hyperthermophilic archaeon. Analysis of 100 Pyr(+) transformants revealed that this recombination system could transfer each of 23 nonselected base pair substitutions to the recipient chromosome along with the selected marker. In 30% of the recombinants, donor markers were transferred as multiple blocks. In at least 40% of the recombinants, donor markers separated by 5 or 6 bp segregated from each other, whereas similar markers separated by 2 bp did not segregate. Among intermarker intervals, the frequency of recombination tract endpoints varied 40-fold, but in contrast to other recombination systems, it did not correlate with the length of the interval. The average length of donor tracts (161 bp) and the frequent generation of multiple tracts seemed generally consistent with the genetic properties observed previously in S. acidocaldarius conjugation. The efficiency with which short intervals of diverged pyrE sequence were incorporated into the genome raises questions about the threat of ectopic recombination in Sulfolobus spp. mediated by this apparently efficient yet permissive system.

FRET for lab-on-a-chip devices — current trends and future prospects

This review focuses on the use of Förster Resonance Energy Transfer (FRET) to monitor intra- and intermolecular reactions occurring in microfluidic reactors. Microfluidic devices have recently been used for performing highly efficient and miniaturised biological assays for the analysis of biological entities such as cells, proteins and nucleic acids. Microfluidic assays are characterised by nanolitre to femtolitre reaction volumes, which necessitates the adoption of a sensitive optical detection scheme. FRET serves as a strong 'spectroscopic ruler' for elucidating the tertiary structure of biomolecules, as the efficiency of the non-radiative energy transfer is extremely sensitive to nanoscale changes in the separation between donor and acceptor markers attached to the biomolecule of interest. In this review, we will review the implementation of various microfluidic assays which employ FRET for diverse applications in the biomedical field, along with the advantages and disadvantages of the various approaches. The future prospects for development of microfluidic devices incorporating FRET detection will be discussed.

Nuclear Transplants from Adult Somatic Cells Generated by a Novel Method Using Diploidized Eggs as Recipients in Medaka Fish (Oryzias latipes)

We previously reported the generation of fertile diploid adult fish with a donor marker by transfer of adult somatic cell nuclei to recipient diploidized eggs without enucleation in medaka (Oryzias latipes). Although transplants appeared similar to clones of donor fish, the possibility existed that they were chimeras of cells originating from both the donor and recipient nuclei. To clarify the nuclear origin of transplants, the green fluorescent protein gene (GFP) was used as the recipient marker and the DMY/dmrt1bY gene, which directs male differentiation in medaka, was used as the donor marker. The marker genes were examined in the transplants by fluorescence microscopy, polymerase chain reaction assays, and transmission to the progeny. Of the seven adult fish obtained from 974 nuclear transfer procedures, six were analyzed in detail. Three of these exhibited the donor phenotype but did not have the recipient marker, suggesting that they were donor clones. The other three showed GFP expression, with one exhibiting an apparent chimerism in both donor and recipient genetic markers and the other two considered to be parthenogenic. Elucidation of a mechanism capable of eliminating recipient nuclei from nuclear transplants is considered to be key to the establishment of cloning techniques in fish.

Clonal T Cell Large Granular Lymphocyte Leukaemia of Recipient Origin after Related Mismatched Allogeneic Haematopoietic Stem Cell Transplantation for Acute Myeloid Leukaemia as a Possible Consequence of Anti-Thymocyte Globulin in Conditioning Regimen

Polyclonal or oligoclonal expansion of T cell large granular lymphocyte (T-LGL) after allogeneic haematopoietic stem cell transplantation (HSCT) has been described. A few cases of clonal T-LGL leukaemia of donor origin has been reported as a post transplantation lymphoproliferative disorder (PTLD) and described after allogeneic HSCT (Chang, Am J Clin Pathol, 2005). We report here a patient with a clonal T-LGL of recipient origin occurring after allogeneic HSCT for acute myeloid leukaemia (AML). A 56 year old male patient with an AML received an HLA mismatched (disparity on locus A) related HSCT from his sister in first complete remission. Conditioning regimen was myeloablative and combined Busilvex (12.8 mg/kg) and Cyclophosphomide (120 mg/kg) (BuCy2). Anti-Thymocyte Globulin (ATG Sangstat) was added because of the HLA disparity between donor and recipient at a dose of 15 mg/kg. Prophylaxis of Graft versus Host Disease (GVHD) combined cyclosporine and methotrexate. Complete engraftment was observed with full haematopoietic recovery and 100% donor type chimerism detected by real time quantitative PCR (RQ-PCR). A grade II acute GVHD resolved under steroid and CMV infection was treated with valganciclovir. Ten months post HSCT while all immunosuppressive treatment was discontinued, a severe neutropenia (poly morpho nuclear <0.2 × 109/l) and an hyperlymphocytosis up to 10×109/l occurred with a thrombocytopenia of 100×109/l. Relapse of AML and viral infection (CMV, HHV6, EBV, HCV, HBV, HIV, HTLV1, parvovirus B19) were eliminated. Seach for autoantibodies was negative. No chronic GVHD was diagnosed. The predominant cells in blood films were typical LGLs. Flow cytometric analysis showed a population CD3+/CD8+/CD57+/CD56− consistent with T-LGL. Bone marrow biopsy confirmed agranulocytosis and T-LGL infiltration CD3+/CD8+/CD57+/CD56−. Secondarily agranulocytosis and thrombocytopenia spontaneously resolved within one month, but hyperlymphocytosis persisted with the same T-LGL profile. Chimerism by RQ-PCR on CD3+ and CD3− peripheral blood mononuclear cells (PBMNC) population at time of profound agranulocytosis revealed that donor cells were only 8.2 % and 1.2% on CD3+ and CD3− fractions respectively. Chimerism done when cytopenia resolved became mixed with 47% and 39% donor markers on CD3+ and CD3− fractions respectively. Chimerism was then performed on selected subpopulations; donor markers were 98% on CD14+/15+ myeloid cells, 100% on CD56+ natural killer (NK) cells and 78 % on CD19+ B cells. Cell sorting isolated two T-LGL subpopulations according to CD57 expression: CD57+bright and CD57+weak. Donor markers were 0.02% on CD3+/CD8+/CD57+bright T-LGL cells and 1.2% on CD3+/CD8+/CD57+weak T-LGL cells. These results were in favor of a recipient origin of the T-LGL population without loss of the graft. The diagnosis of clonal T-LGL population was confirmed by TCRg gene rearrangement performed on the selected CD3+/CD8+/CD57+bright and CD3+/CD8+/CD57+weak population. In addition a polyclonal background was observed only in the CD3+/CD8+/CD57+weak population. These data show that T-LGL leukaemia of recipient origin can occur after allogeneic HSCT for AML and can be included as a PTLD. The role of ATG as an intensive immunosuppressive treatment cannot be excluded in the occurrence of this PTLD. In this patient no treatment of T-LGL leukaemia and a wait and watch attitude were undertaken. Eighteen months post HSCT the patient is alive and well with the same stable PTLD profile and no relapse of AML The cytolytic activity of the recipient T-LGL clone could explained the absence of AML relapse and a transient toxicity against donor haematopoiesis. In vitro cytolytic assay will be done to confirm the cytotoxic activity of T-LGL against leukaemic cells and haematopoietic stem cells.

Hepatocyte Nuclear Factor-1 as Marker of Epithelial Phenotype Reveals Marrow-Derived Hepatocytes, but Not Duct Cells, After Liver Injury in Mice

The potential bone marrow origin of hepatocytes, cholangiocytes, and ductal progenitor cells in the liver was examined in female mice after transplantation of bone marrow cells from male green fluorescent protein (GFP) transgenic donors. Following stable hematopoietic engraftment, the livers of the recipients were injured with carbon tetrachloride (CCl(4), with or without local irradiation of the liver) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC, with or without local irradiation of the liver). The presence of numerous marrow-derived, GFP-positive inflammatory cells had the potential to lead to erroneous interpretation of marrow-derived hepatocytes, cholangiocytes, and ductal progenitor cells. Identification of marrow-derived ductal progenitor or cholangiocyte phenotype using colocalization of GFP or Y chromosome with pancytokeratin staining also failed to distinguish epithelial cells from closely apposed inflammatory cells. To address this inadequacy, we developed a rigorous new immunofluorescence protocol to identify marrow-derived epithelial cells in the liver using Y chromosome (donor marker) and hepatocyte nuclear factor-1 (HNF1, a nuclear marker of liver epithelial, nonhematopoietic phenotype). Using the Y/HNF1 method, rare (approximately one in 20,000) hepatocytes in female mice transplanted with male bone marrow contained a donor-derived Y chromosome. On the other hand, no Y chromosomes were found in cholangiocytes or ductal progenitor cells in mice with liver injury due to DDC or CCl(4). The use of a nuclear marker of mature hepatocytes or cholangiocytes, such as HNF1, improves discrimination of marrow-derived epithelial cells in tissue sections.

Bone Marrow–Derived Hepatic Oval Cells Differentiate Into Hepatocytes in 2-Acetylaminofluorene/Partial Hepatectomy–Induced Liver Regeneration

The ability of the bone marrow cells to differentiate into liver, pancreas, and other tissues led to the speculation that these cells might be the source of adult stem cells found in these organs. The present study analyzed whether the bone marrow cells are a source of hepatic oval cells involved in rat liver regeneration induced by 2-acetylaminofluorene (2-AAF) and 70% partial hepatectomy (PHx). Three groups of mutant F344 dipeptidyl peptidase IV-deficient (DPPIV(-)) rats were required for the study. Groups A and B received the mitotic inhibitor monocrotaline, followed by male F344 (DPPIV(+)) bone marrow transplantation. Next, group A received PHx only, while group B received the 2-AAF/PHx required for the oval cell activation. The last group C was used to analyze the effects of monocrotaline on transplanted bone marrow cells. These rats underwent transplantation with bone marrow cells and were then treated with monocrotaline. Subsequently, the animals were treated with 2-AAF/PHx. In group A, DPPIV(+) hepatocytes were found in the liver. Group B showed that approximately 20% of the oval cell population expressed both donor marker (DPPIV) and alpha-fetoprotein, and some differentiated into hepatocytes. In contrast, animals in group C failed to significantly induce oval cells with the donor DPPIV antigen. In addition, X/Y-chromosome analysis revealed that fusion was not contributing to differentiation of donor-derived oval cells. Our results suggest that under certain physiologic conditions, a portion of hepatic stem cells might arise from the bone marrow and can differentiate into hepatocytes.

Hematopoietic Stem Cells Contribute to Lymphatic Endothelium.

The lymphatic system plays an important physiological role in vascular and immune homeostasis. Lymphatic vessel function is implicated in a number of pathological conditions including tumor metastasis and impaired wound healing. The identity and origin of lymphatic endothelial precursors is poorly understood. Previously we have shown that adult bone marrow-derived, hematopoietic stem cells (HSCs, c-kit+, Sca-1+, lineage−) can differentiate into functional blood vascular endothelial cells. Given the close relationship between the blood and lymphatic vascular systems, we have investigated whether HSCs also give rise to lymphatic endothelial cells (LEC). GFP+ HSCs were transplanted into lethally irradiated (1200 cGy) recipient mice. Donor-derived LEC expressing lymphatic endothelial markers including LYVE-1 and VEGFR3 were clearly distinguished from hematopoietic cells by the absence of CD45 and F4/80 expression. Deconvolution microscopy confirmed the co-localization of donor and LEC marker expression in individual cells. Transplanted HSCs gave rise to LEC in the liver, gut, gastric and kidney. Donor-derived LEC were detected in 2.4% of liver lymphatic vessels at 4 weeks and persisted for at least 12 months (mean of 3.4%). The self-renewal capacity of HSC-derived lymphatic progenitor cells was demonstrated by serial transplantation. The contribution of these progenitors to tumor lymphatics was evaluated. Transplantation of HSCs into young Min−/− mice resulted in the incorporation of donor-derived LEC into the lymphatics of intestinal adenomas that spontaneously develop in these mice. In addition, CD45+F4/80+ leukocytes were detected in the vessel lumens indicating that these are functional tumor lymphatic vessels. Finally, to determine if LEC progenitors contribute to lymphatic vessels in the absence of radiation injury or tumorigenesis, a parabiosis model was evaluated. Donor-derived LEC were detected in parabiotic mice at a frequency similar to that observed for donor-derived blood vascular endothelial cells. This finding suggests that circulating progenitor cells contribute to lymphangiogenesis during steady-state conditions. Our results indicate that hematopoietic stem cells have the potential to contribute to lymphatic endothelium and therefore HSC-derived progenitors may be potential therapeutic targets for hematopoietic and lymphatic disease.