Kidney Marrow Research Articles

New tools for 'ZEBRA-FISHING'.

Zebrafish has been established as a classical model for developmental studies, yet in the past years, with the explosion of novel technological methods, the use of zebrafish as a model has expanded. One of the prominent fields that took advantage of zebrafish as a model organism early on is hematopoiesis, the process of blood cell generation from hematopoietic stem and progenitor cells (HSPCs). In zebrafish, HSPCs are born early during development in the aorta-gonad-mesonephros region and then translocate to the caudal hematopoietic tissue, where they expand and finally take residence in the kidney marrow. This journey is tightly regulated at multiple levels from extracellular signals to chromatin. In order to delineate the mechanistic underpinnings of this process, next-generation sequencing techniques could be an important ally. Here, we describe genome-wide approaches that have been undertaken to delineate zebrafish hematopoiesis.

Gadolinium retention within multiple rat organs after intra-articular administration of gadolinium-based contrast agents

ObjectiveTo characterize the extent of retention and biodistribution of gadolinium (Gd) following intra-articular (IA) injection of linear and macrocyclic gadolinium-based contrast agents (GBCAs) into the knee joint of a rat model.Materials and methodsFifteen Wistar rats were divided into five groups and underwent fluoroscopically-guided injections of both knee joints of (1) clinical 1:200 dilution (low dose, LD) gadodiamide (linear GBCA), (2) LD gadobutrol (macrocyclic GBCA), (3) undiluted (high dose, HD) gadodiamide, (4) HD gadobutrol, and (5) saline. Gd concentrations were quantified by inductively coupled plasma mass spectrometry in (1) blood and urine samples obtained over a 72 h period and (2) knee joint tissues, brain, kidney, and bone marrow at 3 days post-injection.ResultsBoth HD and LD gadodiamide and gadobutrol were rapidly absorbed from the joint with peak serum and urine concentration at 1 h post-injection, with relatively faster clearance of gadobutrol. All GBCA-exposed groups had detectable levels of Gd in the joint tissues, bone marrow, and/or kidneys (median tissue gadolinium range: 0.1–71 μg Gd/g tissue), with higher amounts observed with gadodiamide versus gadobutrol. Retention within brain tissues was only detected following HD gadodiamide administration but not LD gadodiamide nor HD or LD gadobutrol.ConclusionThere was rapid systemic absorption, redistribution, and widespread multi-organ retention of Gd following IA injection of both linear and macrocyclic GBCAs, despite substantial amounts of urinary excretion. Higher concentrations of Gd were observed with administration of gadodiamide compared to gadobutrol in most tissues and biofluids.

Expansion of CD45RA−FOXP3++ regulatory T cells is associated with immune tolerance in patients with combined kidney and bone marrow transplantation

ObjectivesSimultaneous transplantation of a solid organ and bone marrow from the same donor is a possible means of achieving transplant tolerance. Here, we attempted to identify biomarkers that indicate transplant tolerance for discontinuation of immunosuppressants in combined kidney and bone marrow transplantation (CKBMT).MethodsConventional kidney transplant (KT) recipients (n = 20) and CKBMT recipients (n = 6) were included in this study. We examined various immunological parameters by flow cytometry using peripheral blood mononuclear cells (PBMCs), including the frequency and phenotype of regulatory T (Treg) cell subpopulations. We also examined the suppressive activity of the Treg cell population in the setting of mixed lymphocyte reaction (MLR) with or without Treg cell depletion.ResultsAmong six CKBMT recipients, three successfully discontinued immunosuppressants (tolerant group) and three could not (non‐tolerant group). The CD45RA−FOXP3++ Treg cell subpopulation was expanded in CKBMT recipients compared to conventional kidney transplant patients, and this was more obvious in the tolerant group than the non‐tolerant group. In addition, high suppressive activity of the Treg cell population was observed in the tolerant group. The ratio of CD45RA−FOXP3++ Treg cells to CD45RA−FOXP3+ cells indicated good discrimination between the tolerant and non‐tolerant groups.ConclusionThus, our findings propose a biomarker that can distinguish CKBMT patients who achieve transplant tolerance and are eligible for discontinuation of immunosuppressants and may provide insight into tolerance mechanisms in CKBMT.

Idiosyncratic Reaction to Single 7.5 mg Dose of Methotrexate

Methotrexate (MTX) is one of the most common systemic immunosuppressive agents prescribed in dermatology. MTX toxicity targets skin, gastrointestinal mucosa, kidney, liver, and bone marrow. Toxic effects of MTX presenting as hepatic, renal, pulmonary, and bone marrow disorders occur less frequently than the minor effects but may be life-threatening. Characteristic signs and symptoms of acute MTX toxicity are based on extent and severity of organ involvement. Here, we report a 55-year-old married male, known case of chronic plaque-type psoriasis presenting with reaction to single dose of tablet MTX 7.5 mg as advised by the dermatologist for his disease. On history and clinical examination, diagnosis of idiosyncratic reaction to single dose of tablet MTX 7.5 mg was made.

Toxicological Study of an Exhumed Body Using Alternative Matrices for the Detection of Psychoactive Drugs

In forensic toxicology, systematic toxicological analyses (STA) and interpretation of results are difficult when routine matrices such as blood (typically from a central and peripheral source), urine, bile, vitreous humor, stomach or gastric contents are not available. Furthermore, in instances of limited remains, only muscle, hair, or a skeleton may be available for toxicological examination to unambiguously identify as many toxicologically relevant compounds as possible to assist in determining the cause of death. This article describes a case of the exhumed corpse of a 53-year-old male known to have been addicted to several types of drugs. The STA was carried out on alternative biological matrices, including brain, kidney and bone marrow, using gas chromatography–mass spectrometry (GC-MS) and high-performance liquid chromatography– mass spectrometry (HPLC-MS) and subsequently the identified substances were quantified by GC-MS and ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS). The mixture found included multiple agents such as barbiturates, benzodiazepines (BZD) and anaesthetic agent. We demonstrate the possibility of detection of several drugs in an exhumed body 2 years after interment. Despite the problematic of interpreting the quantitative toxicological results, the data could provide complementary information about aetiology of death.

15483 Low adherence to skin cancer screening recommendations in organ transplant recipients

Skin cancer is the most common malignancy in organ transplant recipients (OTRs). Despite an increased incidence, surveillance in OTRs is underprovided. This study aimed to identify the dermatology follow-up rate of OTRs at a hospital with transplant service as well as demographic characteristics influencing follow-up rates. This study was a retrospective chart review completed at a tertiary care center from 1/2008 to 1/2019. ICD codes for kidney, heart, liver, and bone marrow transplants were used to identify patients. 1153 charts were reviewed; age, sex, race, insurance, and dermatology visits were recorded. 118 (10%) of patients were seen by dermatology. Of those 118 patients, 40 were diagnosed with skin cancer (33.9%). Clinic notes identified an additional 41 patients who were seen by an outside dermatology provider. Overall, only 14% of OTRs were seen by any dermatologist after their transplant. Factors that significantly increased visits to dermatology were male sex (P < .01) and white race (P < .0001). Kidney transplant patients had the lowest rate of dermatology follow-up (11% vs 19.6% in all others; P < .0001). Insurance status did not affect follow-up rates. Only 6.9% of all OTRs saw dermatology without a skin cancer diagnosis, highlighting missed opportunities for screening and education prior to cancer development. OTRs not seen by dermatologists have decreased skin cancer awareness and compliance with photo-protective measures. Our study demonstrates a need for quality improvement measures to improve dermatology follow-up. We encourage other programs to monitor their transplant populations, in hopes of decreasing morbidity and mortality in this population.

Hemophagocytic lymphohistiocytosis and miliary tuberculosis in a previously healthy individual: a case report

BackgroundHemophagocytic lymphohistiocytosis (HLH) is a rare heterogenous genetic or acquired hyperinflammatory syndrome associated with a high degree of morbidity and mortality. HLH has clinical manifestations related to abnormal prolonged activation of T lymphocytes and macrophages with an excess of proinflammatory cytokines. The main causes of secondary HLH are malignancies and infectious diseases.Case presentationThe patient was a 54-year-old man, originally from Eastern Africa, who had lived in Northern Europe for 30 years. Here we describe the clinical features, laboratory parameters, diagnostic workup, management and outcome data of a previously healthy 54-year-old man diagnosed with HLH secondary to tuberculosis. The patient was initially treated for a community-acquired pneumonia. He developed multiorgan failure with acute respiratory distress syndrome, hypertransaminasemia, and kidney and bone marrow dysfunction. The clinical course together with a simultaneous increase in serum ferritin raised the suspicion of HLH. The patient fulfilled seven out of eight diagnostic criteria for HLH. A thorough diagnostic workup with respect to HLH and a potential underlying disease was initiated. Cultivation of bronchoalveolar lavage fluid, stool and urine, and polymerase chain reaction of epithelioid cell granulomas in the bone marrow were all positive for Mycobacterium tuberculosis. He was treated for both HLH and tuberculosis, and he survived without any sequelae.ConclusionsWe present one of few published cases of a patient who survived HLH triggered by miliary tuberculosis. The current case illustrates the need for awareness of these two diagnoses, and the timely initiation of specific and supportive treatment to reduce mortality.

Cellular Barcoding of HSCs during Development Reveals Long-Term Persistence of T Cell Progenitor Clones in the Thymus

Developmental hematopoietic stem and progenitor cells (HSPCs) have been thought to be largely replaced by adult hematopoietic stem cells (HSCs) after birth. Here, we performed simultaneous genetic lineage tracing by cellular barcoding and transcriptional profiling of zebrafish hematopoiesis to investigate HSPC clonal dynamics and gain insight into the developmental origin of adult blood cells. We took advantage of a recently developed dynamic lineage tracing system, scGESTALT, a CRISPR-based approach that allows for inducible cellular barcoding at two different time points during zebrafish embryonic development. We induced the first stage of lineage recording in the early embryo via microinjection at the one-cell stage and selected 28 hours post-fertilization, just before the birth of definitive blood stem cells, for additional recording activity. In this way, we were able to barcode HSCs, which will pass their unique barcodes on to their progeny, thereby enabling lineage tracing. To study the clonal dynamics and developmental origins of adult blood cells, we dissected paired adult kidney marrow and thymi tissues from barcoded fish between 3 and 7 months of age. We then performed single-cell RNA sequencing (scRNA-seq) in combination with lineage tracing by specifically amplifying the GESTALT barcode and sequencing both transcriptional and GESTALT barcode libraries. In total, we recovered transcriptional profiles from 71,109 cells across 9 fish. By optimizing promoter choice, we improved barcode recovery by almost 3-fold (17.7% vs. 6.5%) for detection in cells from the zebrafish kidney marrow. In two of these more efficient lineage recording fish, we detected 61 and 72 unique HSPC clones that contributed to adult hematopoiesis. The majority of these HSPC clones did not exhibit significant lineage biases; however, we demonstrated that 6/61 and 4/72 clones from the two fish, respectively, were significantly enriched in the myeloid lineage and 8/61 and 8/72 clones were significantly enriched in the lymphoid lineage (p&amp;lt; 0.05). These findings demonstrate that myeloid and lymphoid biased clones arise during normal development. From our paired thymus and kidney marrow sample, we identified 51 unique kidney marrow clones, 10 shared, and 2 unique thymus clones. This latter finding of unique thymus clones was surprising and suggests that long-lived embryonic T cell progenitors persist and contribute to adult T cell production in the zebrafish. Taken together, we have demonstrated how scGESTALT can uncover complex lineage relationships in blood, mapping the origins and contributions of HSCs and embryonic progenitors to the adult hematopoietic system. Disclosures Zon: Amagma Therapeutics: Current equity holder in private company, Other: Founder; Celularity: Consultancy; Cellarity: Consultancy; CAMP4 Therapeutics: Current equity holder in private company, Other: Founder; Fate Therapeutics: Current equity holder in publicly-traded company, Other: Founder; Scholar Rock: Current equity holder in publicly-traded company, Other: Founder.

Cxcl8/PKC Signaling in the Endothelial Cell Niche Regulates Long-Term Hematopoietic Stem Cell Fate

The sinusoidal endothelial cell niche is a component of the hematopoietic microenvironment with an important role in regulating hematopoietic stem cell (HSC) fate. Mechanisms by which the niche control the long-term functional and phylogenetic diversity of the hematopoietic system are poorly understood. We performed gene expression profiling on FACS-purified endothelial cells from the zebrafish caudal hematopoietic territory (CHT), the sinusoidal endothelial niche equivalent to the mammalian fetal liver. Kdrl:GFP+lyve1b:DsRed+ CHT endothelial cells were compared to kdrl:GFP+lyve1b:DsRed- non-CHT endothelial cells by bulk RNA-seq and 106 genes were downregulated in the CHT with q&amp;lt;0.05. Enforced expression of candidate genes in the CHT was achieved using a zebrafish E-selectin (sele) enhancer/promoter element. Microinjection of a sele:prkcda-2A-mCherry (protein kinase C (PKC), delta a) construct into Runx1+23:GFP HSPC reporter zebrafish embryos resulted in a significant increase in the number of HSPCs compared to empty vector control (5.4 ± 1.0 vs 2.5 ± 0.4, p = 0.007). The same construct was microinjected into hemizygous GESTALT zebrafish with barcoding sgRNAs and Cas9. Barcoded embryos were grown to adulthood, peripheral blood was sampled at 3, 6, 9 and 12 months post-fertilization (mpf), and phylogenetic diversity of the hematopoietic system was measured in a longitudinal manner by enumerating unique GESTALT clones. Transgenic animals microinjected with the sele:prkcda-2A-mCherry construct consistently showed greater HSC clonal diversity compared to empty vector control (9.5 ± 0.9 vs 6.4 ± 0.7 clones at 12 mpf, p = 0.024). Single cell RNA sequencing (scRNA-seq) of the marrow of these animals revealed expanded populations of macrophages and erythrocyte precursors in those with dysregulated expression of prkcda. PKC family members are signaling intermediates for cxcr1, the receptor for the chemokine cxcl8, and a factor we have previously shown to positively regulate CHT colonization. Treatment of human umbilical vein endothelial cells in vitro with recombinant cxcl8 showed no change in prkcd transcript levels by bulk RNAseq or quantitative RT-PCR, but did show enhanced accumulation of PKC-d protein after 2 hours consistent with induction of this pathway at the post-transcriptional level. To understand how cxcl8/PKC signaling might alter the niche in vivo, transgenic zebrafish with enforced expression of cxcl8 were generated by microinjecting embryos with a Runx1+23:cxcl8 expression construct. These were raised to 12 mpf and kidney marrow was harvested and subjected to scRNA-seq, demonstrating over 2-fold expansion of stromal and endothelial elements in Runx1:cxcl8 animals compared to controls. Hematopoietic cells were identified, a developmental trajectory was plotted using pseudotime analysis, and this was anchored using expression of genes characteristic of early (meis1b, tal1) and late (lyz, mpx) myeloid development. Runx1:cxcl8 mosaic transgenics had a relative expansion of early myeloid cells compared to control animals (61% vs 38% early myeloid cells, p = 0.0002). Our data support the following model: HSPCs or other myeloid cells may actively remodel the niche via cxcl8 expression, signaling through cxcr1 and PKC, increasing niche capacity and thereby expanding the number of HSCs that can contribute to hematopoiesis. The presence of reserve capacity within the niche may be relevant to conditions where loss of HSC clonal diversity carries a risk of myeloid neoplasia. Disclosures Zon: Amagma Therapeutics: Current equity holder in private company, Other: Founder; CAMP4 Therapeutics: Current equity holder in private company, Other: Founder; Celularity: Consultancy; Scholar Rock: Current equity holder in publicly-traded company, Other: Founder; Cellarity: Consultancy; Fate Therapeutics: Current equity holder in publicly-traded company, Other: Founder.

Ultrastructure of the Adult Hematopoietic Stem Cell Niche Defined Using Correlative Light and Electron Microscopy

Hematopoietic stem and progenitor cells (HSPCs) that originate from the hemogenic endothelium in the dorsal aorta finally home and engraft within the adult niche, the fetal bone marrow. Within the bone marrow, HSPCs are retained in a complex microenvironment surrounded by niche cells such as megakaryocytes, peripheral nerves, endothelial and mesenchymal stromal cells. While it is known that niche cells secrete hematopoietic factors that are essential for HSPCs to self-renew and repopulate the blood lineages, the ultrastructure of this niche is not well defined as current imaging technology does not allow direct visualization of the fetal bone marrow niche. Zebrafish share a similar hematopoietic ontogeny to mammals, and because the embryos are transparent, intrinsic HSPC interactions with the niche can be directly visualized. To precisely locate rare HSPCs within the large dense kidney marrow, the presumptive adult niche, we genetically tagged endogenous HSPCs to track them live using lightsheet microscopy, followed by high-resolution serial block-face scanning electron microscopy (SBEM) (XY = 10.8 nm/pixel, Z = 70 nm/pixel). Using this technique, we could visually track single mCherry+ HSPCs, then confirm their exact location in the SBEM dataset with high contrast APEX2 peroxidase label. We found HSPC clusters within vessel lumens, as well as single HSPC in a novel perivascular niche. In this perivascular site, a single HSPC simultaneously contacted one mesenchymal stromal cell, multiple endothelial cells, a glial-like cell, and other hematopoietic cells. After extensive tracing of the glial-like cells within the 3D SBEM data, we identified that these cells extended as a long chain and formed contact with HSPCs. Our search for candidate structures in the kidney marrow region suggested that the glial-like cells could be dopamine beta-hydroxylase positive (dbh+) cells. Through fluorescence imaging, we observed cytoplasmic projections extending from dbh+ cells into Runx+ HSPC clusters. To confirm the identity of these glial-like cells, we performed fluorescence imaging of dbh+ transgenic larvae followed by SBEM. By correlating the two datasets, we identified dbh+ glial-like cell in contact with a single HSPC in the niche. Furthermore, treatment with a neurotoxin, 6-hydroxydopamine, led to a significant reduction in HSPC numbers within the niche highlighting the biological significance of dbh+ cells. In summary, through this approach, we could identify a previously unknown cell type within the zebrafish adult hematopoietic niche that is pivotal in maintaining the HSPC pool. Our finding validates the importance of having multiple niche cells that concurrently regulate HSPCs within their niche. Importantly, our approach can be applied to characterize the ultrastructure of rare cells, such as other stem cells, and their niche by using multiple imaging modalities and can also lead to the identification of previously uncharacterized cell types. Further, we can now identify novel intercellular structures that form between an unperturbed HSPC in its endogenous perivascular niche. Disclosures No relevant conflicts of interest to declare.

A novel method to purify neutrophils enables functional analysis of zebrafish hematopoiesis.

Zebrafish is a useful model to study vertebrate hematopoiesis, but lack of antibodies to zebrafish proteins has limited purification of hematopoietic cells. Here, we purified neutrophils from larval and adult zebrafish using the lectin Phaseolus vulgaris erythroagglutinin (PHA-E) and DRAQ5, a DNA-staining fluorescent dye. In adult kidney marrow, we purified neutrophil-like PHA-E4low DRAQ5low cells, which neutrophil-type granules. Specifically, at 96-hr post-fertilization, we sorted large-sized cells from larvae using forward scatter and found that they consisted of PHA-Elow DRAQ5low populations. These cells had myeloperoxidase activity, were Sudan Black B-positive and expressed high levels of neutrophil-specific (csf3r and mpx) mRNAs, all neutrophil characteristics. Using this method, we conducted functional analysis suggesting that zyxin (Zyx) plays a role in neutrophil generation in zebrafish larvae. Overall, PHA-E and DRAQ5-based flow cytometry serves as a tool to purify zebrafish neutrophils.

Protective role of chlorogenic acid on DNA damage caused by ochratoxin A exposure

Chlorogenic acid (ChlA) has shown short-term protective effects against the cyto-genotoxic effects of ochratoxin A (OTA). The present study evaluated the effect of oral administration of ChlA in male Wistar rats exposed to OTA. OTA (0.4 mg/kg bw/day), ChlA (5 mg/kg bw/day), or the combination of both, were administered orally to animals during 28 days. No deaths, decrease in feed consumption or change in the body weight of animals were observed in any group. In the OTA-treated group a decrease in locomotion as well as increased DNA damage in blood, kidney and bone marrow cells were observed. ChlA alone was not genotoxic for animals. The combination of OTA+ChlA decreased the DNA damage by 37% in blood cells, by 55% in kidney cells and by 80% in bone marrow cells compared to OTA-treated group. In conclusion, oral treatment with ChlA showed a good protective effect on genotoxicity produced by OTA in rats during 28 days exposure.

Evaluation of the pharmacokinetic drug-drug interaction potential of iohexol, a renal filtration marker.

Carboplatin dose is calculated based on kidney function, commonly estimated with imperfect creatinine-based formulae. Iohexol is used to measure glomerular filtration rate (GFR) and allows calculation of a more appropriate carboplatin dose. To address potential concerns that iohexol administered during a course of chemotherapy impacts that therapy, we performed in vitro and in vivo pharmacokinetic drug-drug interaction evaluations of iohexol. Carboplatin was administered IV to female mice at 60mg/kg with or without iohexol at 300mg/kg. Plasma ultrafiltrate, kidney and bone marrow platinum was quantitated by atomic absorption spectrophotometry. Paclitaxel microsomal and gemcitabine cytosolic metabolism as well as metabolism of CYP and UGT probes was assessed with and without iohexol at 300µg/mL by LC-MS/MS. In vivo carboplatin exposure was not significantly affected by iohexol co-administration (platinum AUC combination vs alone: plasma ultrafiltrate 1,791 vs 1920µg/mL min; kidney 8367 vs 9757µg/g min; bone marrow 12.7 vs 12.7µg/mg-protein min). Paclitaxel microsomal metabolism was not impacted (combination vs alone: 6-α-OH-paclitaxel 38.3 versus 39.4ng/mL/60min; 3-p-OH-paclitaxel 26.2 versus 27.7ng/mL/60min). Gemcitabine human cytosolic elimination was not impacted (AUC combination vs gemcitabine alone: dFdU 24.1 versus 23.7µg/mL/30min). Iohexol displayed no relevant inhibition of the CYP and UGT enzymes in human liver microsomes. Iohexol is unlikely to affect the clinical pharmacokinetics of carboplatin, paclitaxel, gemcitabine, or other agents used in combination with carboplatin treatment. Measuring GFR with iohexol to better dose carboplatin is unlikely to alter the safety or efficacy of chemotherapy through pharmacokinetic drug-drug interactions.

Postmortem Findings in Italian Patients With COVID-19: A Descriptive Full Autopsy Study of Cases With and Without Comorbidities.

BackgroundDescriptions of the pathological features of coronavirus disease-2019 (COVID-19) caused by the novel zoonotic pathogen severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emanate from tissue biopsies, case reports, and small postmortem studies restricted to the lung and specific organs. Whole-body autopsy studies of COVID-19 patients have been sparse.MethodsTo further define the pathology caused by SARS-CoV-2 across all body organs, we performed autopsies on 22 patients with COVID-19 (18 with comorbidities and 4 without comorbidities) who died at the National Institute for Infectious Diseases Lazzaro Spallanzani-IRCCS Hospital, Rome, Italy. Tissues from the lung, heart, liver, kidney, spleen, and bone marrow (but not the brain) were examined. Only lung tissues were subject to transmission electron microscopy.ResultsCOVID-19 caused multisystem pathology. Pulmonary and cardiovascular involvement were dominant pathological features. Extrapulmonary manifestations included hepatic, kidney, splenic, and bone marrow involvement, and microvascular injury and thrombosis were also detected. These findings were similar in patients with or without preexisting medical comorbidities.ConclusionsSARS-CoV-2 infection causes multisystem disease and significant pathology in most organs in patients with and without comorbidities.

THE REQUIREMENT OF THYMIC IRRADIATION FOR INDUCTION OF MIXED CHIMERISM AND RENAL ALLOGRAFT TOLERANCE IN NON-HUMAN PRIMATES

Introduction: Thymic irradiation (TI) has been required for consistent induction of allograft tolerance through a mixed chimerism approach in both murine and nonhuman primate (NHP) models. In this study, we investigated the mechanisms of TI requirement in combined kidney (KTx) and bone marrow transplantation (BMT) in NHPs. Materials and Methods: In Group A, 5 recipients received BMT (combined KTx was evaluated in three) from MHC mismatched donors with a conditioning regimen that included total body irradiation (TBI, 1.5Gy), a Bcl-2 inhibitor (venetoclax), ATG and TI (7Gy). After transplant, the recipients were treated with a short course of anti-CD154 monoclonal antibody and a one-month course of cyclosporine. No immunosuppression was given after one month. In Group B, 3 recipients received combined KTx and BMT with the same regimen but without TI. Results: All recipients in Group A (TI) developed significantly higher chimerism than Group B (p < 0.02). Three recipients in Group A with KTx achieved long-term renal allograft survival without rejection (survival days 313, >646,>982 days) while all three Group B (No TI) recipients rejected renal allografts shortly after discontinuation of cyclosporine (97, 100, 163 days; p < 0.025 Groups A vs. B in Kaplan-Meier; Figure 1).Although recent thymic emigrants (RTE; CD4+CD31+CD45RA+) and naïve T cells of Group A were completely depleted after the preconditioning, those of Group B were substantially observed and significantly higher (Figure 2). On the other hand, no difference was observed in memory T cell subsets or regulatory T cells between two groups. Conclusion: Peripheral T cell deletion by the conditioning regimen may result in enhanced thymopoiesis with increased post-transplant RTE and naïve cells if TI is not administered. Higher post-transplant naïve T cells potentially inhibited chimerism and tolerance induction in recipients without TI.

3047 – THE ADULT HEMATOPOIETIC STEM CELL NICHE IN ZEBRAFISH DEFINED USING CORRELATIVE LIGHT AND ELECTRON MICROSCOPY

Hematopoietic stem and progenitor cells (HSPCs) that originate from the hemogenic endothelium in the dorsal aorta to finally home and engraft within the adult niche, the fetal bone marrow (BM), become surrounded by niche cells in a complex microenvironment. The ultrastructure of this niche is not well defined as current imaging technology does not allow direct visualization of the fetal BM niche. Zebrafish have a similar hematopoietic ontogeny to mammals, and because the embryos are transparent, intrinsic HSPC interactions with the niche can be directly visualized. To track HSPCs during colonization of the presumptive adult niche, the kidney marrow (KM), we used our previously validated HSPC-specific transgenic reporter lines. In 5 days post fertilization fixed larvae, we detected ∼100 HSPCs/larva. To precisely locate these rare HSPCs within the larger dense KM, we genetically tagged endogenous HSPCs to track them live using lightsheet microscopy, followed by high resolution serial block-face scanning electron microscopy (SBEM). Using this technique, we could visually track single mCherry+ HSPCs, then confirm their exact location in a SBEM dataset with high contrast APEX2 peroxidase label. We found HSPC clusters within vessel lumens, as well as single HSPC in a novel perivascular niche. In this perivascular site, a single HSPC was seen to simultaneously contact one mesenchymal stromal cell, multiple endothelial cells, a glial-like cell and other hematopoietic cells. The glial-like cells were dopamine beta hydroxylase positive (dbh+) cells and treatment with small molecules inhibitor of dopamine led to significantly reduced HSPC numbers within the niche. Our approach can be used to identify the ultrastructure of single rare cells within dense tissues by using multiple imaging modalities and can also lead to the identification of previously uncharacterized cell types.

3071 – IMPROVED HOMING IN A CONDITIONING-FREE ZEBRAFISH TRANSPLANTATION MODEL

Hematopoietic stem cell (HSC) transplantation is a treatment used to replace the defective blood system of patients with malignant and non-malignant blood disorders. However, the biology behind donor-cell engraftment and cell-niche interaction is incompletely understood. Zebrafish is an ideal model to visualize engraftment and cell-niche interaction due to its optical transparency, which allow for whole-organism imaging. We have established a novel conditioning-free transplantation model using an HSC-deficient (runx1 W84X) mutant zebrafish. The lack of definitive hematopoiesis in these mutants provides empty HSC niche and no adaptive immune system thus diminishing the risk of graft failure or rejection. Transplantation of donor marrow cells that ubiquitously express green fluorescent protein (ubi:GFP) into these HSC-deficient mutants at 2 days-post fertilization resulted in significantly higher GFP+ donor cell engraftment than in HSC-competent animals. Transplanted runx1 mutants showed improved survival by more than 3-fold with sustained long-term, serial-repopulating, multi-lineage engraftment in adults. To decipher when the differences in engraftment occurs, we employed serial imaging to visualize hematopoietic cell homing throughout zebrafish larvae. From 4-7 days post fertilization, we observed seeding of GFP+ cells into all hematopoietic tissues including the thymus and kidney marrow (equivalent to mammalian bone marrow) with HSC-deficient runx1 mutants showing better donor cell seeding compared to HSC-competent (wild type and runx1 heterozygote) animals as early as 3 days post-transplant. This finding demonstrates the importance of the availability of recipient HSC niche in the success of engraftment, and indicate the utility of the model to provide novel insight into the earliest stages of engraftment improving our understanding of HSC transplantation outcome.

PDGFRα+ Cell-Derived SCF Regulates Hematopoiesis of Adult Mice

To investigate the effect of PDGFRα+ stromal cells derived SCF on hematopoiesis of adult mice. Pdgfrα-CreER; R26-tdTomato mice model was constructed, and the proportion and distribution of PDGFRα+ cells in the liver, spleen, lung, kidney and bone marrow were analyzed by flow cytometry and confocal microscopy. Then the Pdgfrα-CreER; Scf flox/flox mice model was further constructed, the Scf in PDGFRα+ was knocked out specifically, the effect of Scf-knocked out in PDGFRα+ stromal cells in the propitiation of HSPCs in the bone marrow was analyzed by flow cytometry. The effect of SCF on the proportion on number of peripheral blood cells in mice was analyzed by whole blood analyzer. After Scf was knocked out in PDGFRα+ stromal cells, the propitiation and number of LKS- cell, LKS+ cell, HSC, MPP1, MKP, PreGM, PreMegE, and CFU-E in the bone marrow of mice was decreased, as well as in the number of red blood cells and hemoglobin concentration of peripheral blood. However, Scf knocked out from PDGFRα+ cells showed no effect on the hematopoiesis in spleen. specific knocked out of Scf in PDGFRα+ stromal cells in adult mice can decrease the proportion of HSPCs in the bone marrow and the number of red blood cells in peripheral blood, and finally lead to anemia in mice.

3115 – GATA2A DETERMINES THE SURVIVAL AND HOMEOSTATIC LINEAGE OUTPUT OF HAEMATOPOIETIC STEM AND PROGENITOR CELLS

The transcription factor Gata2 is key regulator of haematopoietic stem and progenitor cells (HSPCs) during embryonic development of vertebrates. We took advantage of a partial genome duplication in zebrafish that lead to the appearance of two Gata2 genes, Gata2a and Gata2b, to address how Gata2 programmes the haemogenic endothelium (HE) towards the HSPC cell fate. Through deletion of a conserved intronic enhancer (i4 enhancer) in the gata2a locus, we show that reducing gata2a levels in the endothelium leads to decreased expression of gata2b and runx1, thus demonstrating that Gata2a activity is required for HE programming in embryos. By contrast, loss of gata2b impaired cmyb expression in the HSPC population but did not affect HE programming, indicating that Gata2a programmes HE first and Gata2b modulates HSPC programming later. Single cell transcriptional profiling of the adult kidney marrow revealed that gata2a and gata2b are expressed in HSPCs, but in different subsets. Strikingly, homozygous Gata2a mutants are viable and fertile, but show hypocellularity in the marrow niche and neutropenia, resembling GATA2 deficiency syndrome in humans. Gata2a enhancer mutants show fewer HSPCs and a lineage skewing towards the erythroid and B cell fates at the expense of the neutrophilic and monocytic lineages. These differences are already detectable in HSPCs. In addition, myeloid lineages in gata2a enhancer mutants ectopically express B- and erythroid lineage markers and fail to express lineage-specific genes at wild type levels, suggesting that Gata2a activity in HSPCs initiates and maintains lineage commitment in haematopoietic cells.

GMP production of [68Ga]Ga-BOT5035 for imaging of liver fibrosis in microdosing phase 0 study

IntroductionEarly detection of liver fibrosis and monitoring response to treatment crucial for the management of patients are currently not feasible in clinical practice. Platelet derived growth factor receptor β (PDGFR-β) expression is regarded as a potential biomarker to determine the stages of fibrotic diseases including liver fibrosis. [68Ga]Ga-BOT5035 comprising a bicyclic peptide was developed for specific targeting of PDGFR-β overexpressed in pathological fibrosis. The realization of microdosing phase 0 study using [68Ga]Ga-BOT5035 positron emission tomography required automated good manufacturing practice (GMP) compliant production of [68Ga]Ga-BOT5035 presented herein. Moreover, the investigation of radiation dosimetry was conducted to ensure possibility of multiple annual examinations for disease monitoring in clinical setup. MethodsThe active pharmaceutical ingredient starting material BOT5035 (GMP grade) was provided by BiOrion Technologies BV. The 68Ga-labelling process was developed and automated using synthesis platform (Modular-Lab PharmTrace, Eckert & Ziegler), disposable cassettes for 68Ga-labelling, and pharmaceutical grade 68Ge/68Ga generator (GalliaPharm®) purchased from Eckert & Ziegler. Radiolysis sensitive BOT5035 required development and systematic optimization of the labelling synthesis parameters such as time, temperature, precursor concentration, radical scavenger, buffer concentration and pH. The validation process was conducted with regard to the product quality and quantity, as well as production reproducibility. Human organ equivalent doses and total body effective doses were calculated using Organ Level Internal Dose Assessment Code software (OLINDA/EXM 1.1), based on ex vivo organ distribution in Sprague-Dawley rats. ResultsThe GMP compliant automated production of [68Ga]Ga-BOT5035 with on-line documentation demonstrated high reproducibility. The time for the labelling synthesis and quality control was approximately 60 min. The non-decay corrected radiochemical yield and radiochemical purity of the radiopharmaceutical were 43.7 ± 7.6% (n = 3, process validation) and 97.7 ± 0.4% (n = 3, process validation), respectively. Predefined acceptance criteria were met for the sterility, endotoxins level, radionuclidic purity and residual solvent content. The stability at ambient temperature was controlled for 120 min with approved results. Ex vivo organ distribution data revealed fast blood clearance and washout from most of the organs. The dose-limiting organs were kidney and bone marrow. The total effective dose as limiting parameter would allow for up to 3–4 PET scans per annum. ConclusionThe fully automated and GMP compliant production of [68Ga]Ga-BOT5035 was developed and thoroughly validated. The radiopharmaceutical was approved by Swedish Medicinal Products Agency and the Ethical Review Authority for the Phase 0 clinical study of the quantitative imaging of liver fibrosis. Human dosimetry calculations extrapolated from animal experiment indicated possibility of 3–4 PET examinations per year.