Good Engraftment Research Articles

Dihydrolipoamide dehydrogenase deficiency in five siblings with variable phenotypes, including fulminant fatal liver failure despite good engraftment of transplanted liver.

Dihydrolipoamide dehydrogenase (DLD) deficiency can, in one of its forms, be a rare cause of acute liver failure. Clinical presentation is nonspecific. Biochemical findings can reflect metabolic block, but vary depending on patient and his condition. Consensus treatment guidelines do not exist. We present a family with five members suffering from DLD deficiency. Patient 1 presented with emesis, mental deterioration, and fulminant hepatic failure, which required high-urgency liver transplantation. His younger brother, patient 2, experienced unexplained hypoglycemia and metabolic acidosis on the second day after cardiac surgery. Three affected younger siblings were asymptomatic. In patients with acute liver failure of unknown etiology urgent metabolic work-up should be done, and whole exome sequencing considered. Liver transplantation remains life-saving treatment option, but its outcome may be dependent on etiology-specific supportive treatment.

Immature platelet fraction levels predict the development of prolonged thrombocytopenia after haematopoietic stem cell transplantation

Prolonged thrombocytopenia (PT) is a serious complication after haematopoietic stem cell transplantation (HSCT). PT has been suggested to be associated with an increased platelet transfusion requirement and poor outcomes after transplantation. Due to the complex mechanism of PT development, it is difficult to diagnose in the early post-transplant period. Our study aimed to identify an early predictive marker for PT after HSCT. Previous studies showed that the clinical utility of immature platelet fraction (IPF) predicts platelet recovery after chemotherapy and successful engraftment. However, the relationship between IPF and PT after HSCT remains unclear. Fifty-two patients with malignant haematological diseases who underwent HSCT were included in the study. We observed the kinetics of recovery of haematological parameters after transplantation and performed receiver operating characteristics (ROC) curve analysis using data from the 52 HSCT patients. The days to rise and peak of IPF, absolute IPF count (A-IPF) and highly fluorescent IPF (H-IPF) were almost synchronised in all patients, at day 10 and day 15, respectively. The begin to rise levels of IPF, H-IPF and A-IPF were all significantly lower in the PT group than in the good engraftment (GE) group (p=0.0016, p=0.0094, p=0.0086, respectively). The peak levels of IPF were significantly lower in the PT group than the GE group (p=0.0036). However, the peaks of H-IPF and A-IPF were not statistically significant between the two groups (p=0.3383, p=0.0887, respectively). The area under the ROC curve (AUC) of IPF rise was 0.739 (95% CI 0.583-0.896; p<0.05) and the cut-off value was 3.5%, while the AUC of IPF peak was 0.800 (95% CI 0.637-0.962; p<0.01) and the cut-off value was 8.0%. In conclusion, early low levels of IPF predict the development of PT after HSCT. These findings may help improve the management and treatment strategies for PT after HSCT.

Good engraftment after reduced intensity targeted busulfan-based conditioning and matched related donor hematopoietic cell transplantation in hemoglobinopathies.

Hematopoietic cell transplantation (HCT) is an established curative therapy for transfusion-dependent thalassemia (TDT) and sickle cell disease (SCD). The latest American Society of Hematology guidelines recommend myeloablative preparative regimen in patients under 18years of age. The objective was to demonstrate safety and efficacy of a reduced intensity conditioning (RIC) regimen including high-dose fludarabine, anti-thymocyte globulin, and targeted busulfan as a single alkylator to sub-myeloablative exposures. Between 2012 and 2021, 11 patients with SCD and five patients with TDT and matched related donor (MRD) HCT were included. The median age at transplantation was 8.3years (range: 3.7-18.8years). The median administered busulfan AUC was 67.4mg/L×h (range: 60.7-80mg/L×h). Overall survival was 93.8% and event-free survival 87.5% with one engrafted SCD patient with pre-existing moyamoya disease succumbing after drainage of a subdural hematoma. One SCD patient developed a secondary graft failure and was treated with a second HCT. Myeloid chimerism was full in all other patients with a median follow-up time of 4.1years (range: 2.0-11.1years), whereas T-cell donor chimerism was frequently mixed. This RIC conditioning followed by MRD HCT is sufficiently myeloablative to cure pediatric patients with hemoglobinopathies without the need for additional total body irradiation or thiotepa.

Specificities of Management of Perineal Burns.

There is a paucity of articles addressing the management strategy for perineal burns. Pelvic floor burns present a multitude of problems. These relate to the management of stool, urine, areas of maceration, significant strain, and the risk of infection. The objective of this study was to analyze a consecutive series of perineal burns by studying their characteristics, treatment, and management of urine and feces. This was a retrospective study including 100 patients between January 2018 and December 2022. The patients had all been hospitalized after suffering burns to the perineum. In 28% of the patients, the perineal burn was complicated by infection. In 61% of cases, a cutaneous infection, in 32% of cases, a urinary tract infection, in 25% of cases, a respiratory tract infection, and in 7% of cases, an infection of the digestive tract. Superinfection is associated with an increased risk of poor engraftment. A total of 100% of the patients with poor or average engraftment had an infection in the aftermath of the burn, compared with 20% of the patients with excellent engraftment and 61% of the patients with good engraftment. In our series, the analysis revealed that, in 95% of cases, no specific procedures were implemented for stool management. Perianal involvement was associated with a risk of sub-excellent engraftment. The results were statistically significant, with P = .005 and an OR = 8.72 after multivariate analysis. We favor the least invasive approach to stool management. Indications for a rectal catheter arise in patients with the following characteristics: the patient must be sedated, the burns must be deep and close to the anal opening, and the stools must be watery and abundant. In 95% of cases, we do not install a stool management device. Colostomies should remain exceptional.

Allogeneic hematopoietic stem cell transplantation for aplastic anemia

The addition of the thrombopoietin receptor agonist eltrombopag to immunosuppressive therapy (IST) with horse antithymocyte globulin and cyclosporin A has improved response to initial therapy for aplastic anemia, but about one-third of patients still require allogeneic hematopoietic stem cell transplantation (HSCT) due to resistance or relapse. Allogeneic HSCT as initial therapy is indicated when the patient is younger than 40 years of age (especially <20 years) and has an HLA-matched sibling donor. On the other hand, fulminant aplastic anemia, in which the neutrophil count is 0 even with G-CSF administration, requires transplantation regardless of donor type. When an HLA-matched donor is not available and an early transplant is needed, cord blood transplantation and haploidentical transplantation are options. In the past few years, haploidentical transplantation with post-transplantation cyclophosphamide (PTCY) as GVHD prophylaxis has been shown to produce favorable outcomes in patients with aplastic anemia. A retrospective study in Japan also showed a good engraftment rate after haploidentical transplantation with PTCY. This review discusses transplant indications for aplastic anemia and selection of donor type and conditioning regimen.

Assessment of Transduction of CD34+ Human Hematopoietic Stem Cells from Patients with Severe Hemophilia-Α with Lentiviral Vector Carrying a High Expression FVIII Transgene (CD68-ET3-LV)

Background: Alternative strategies are needed for the large number of people with hemophilia who are ineligible for AAV based gene therapy due to age or high levels of anti-AAV neutralizing antibodies. High inter-individual variability of expression and ill sustained factor levels are also challenges with AAV based gene therapy for hemophilia A, in particular. We have developed a third-generation lentiviral vector mediated hematopoietic stem cell-based gene therapy for hemophilia A. (Doering et al Human Gene Therapy 2018; 29: 1183-1201) This vector (CD68-ET3-LV) has a high expression FVIII transgene with a CD68 promoter targeting expression in monocytic cells predominantly. We report here successful transduction of severe hemophilia A patient derived human hematopoietic stem cells with this vector. Methods: Mobilized peripheral blood stem cells were collected by apheresis from three patients with severe hemophilia A without inhibitors who also received prophylactic clotting factor replacement therapy during this period. CD34+ hematopoietic stem cells (HSCs) were enriched on the CliniMACS Plus® system (Miltenyi Biotec, Bergish Gladbach, Germany) using the CliniMACS CD34® reagent system. Purified HSC were then transduced with this CD68-ET3-LV as follows - HSCs cultured on retronectin coated surfaces in xenofree media with cytokines were exposed to clinical grade CD68-ET3-LV vector in two ways - a double transduction protocol without any enhancer and a single transduction with an enhancer. After completion of this step, the product was assessed for viability and vector copy number (VCN) by trypan blue labelling and Q-PCR performed on genomic DNA from CFU cells, respectively. Transduced HSCs were also assessed for their engraftment potential. CD68-ET3-LV vector transduced HSC (1x10 6) were transplanted into NBSGW mice via tail vein injection. Engraftment was assessed at 16 weeks after transplantation. This protocol was approved by the Institutional Review Board of the Christian Medical College, Vellore, India. Results: G-CSF (10 ug/kg/day) based peripheral blood stem cell mobilization was well tolerated by all participants. The apheresis procedures which were done with plasma FVIII levels in the normal range after prophylactic replacement therapy were unremarkable. The total collection of mobilized CD34+ cells from the three donors was 268±80.5x10 6 CD34+ cells. An aliquot of 2x10 6 CD34+ cells/ml was used in the transduction experiments. The data on viability and vector copy number post transduction shown in the tables 1 and 2 confirm that viability was not affected by the manipulation of these HSCs in both double and single transduction methods. The vector copy number (VCN) in the genomic DNA obtained from CFU cells collected from CD34+ HSCs cultured in MethoCult H4434™ (Stem Cell Technologies™, Vancouver, Canada) varied from 0.62 to 1.21 in double transduction (n=3) and nearly doubled in the single transduction method with an enhancer to VCNs of 1.5 and 2.4 in two samples tested. Transplantation studies in NBSGW mice showed successful engraftment of human CD34+ HSCs with a mean engraftment of 80.99 ± 4.2% of CD45+ multilineage hematopoietic cells in the bone marrow 16 weeks after transplantation. Conclusion: To the best of our knowledge, this is the first report of transduction of mobilized peripheral blood CD34+ HSCs from patients with severe hemophilia A using a clinical grade lentiviral vector with a high expression FVIII transgene. These data establish feasibility and safety of the procedure. The transduction protocol showed good efficiency with very high viability, significant VCN and good engraftment of these gene modified human HSCs in a mouse model. These protocols are currently being evaluated in a first in human phase 1 clinical trial of gene therapy for severe hemophilia A without inhibitors.

Кератопротезы в современной офтальмохирургии

The shortage of donor corneas for keratoplasty in patients with severe disorders (persistent opacity, corneal leucoma of IV–V categories, including leucoma with vascularization, after burns, multiple transplant failures, with extensive symblepharon, Stevens – Johnson syndrome, Lyell's syndrome, ocular cicatricial pemphigoid, etc) is currently compensated by keratoprostheses, which partly or completely consist of synthetic materials, show good engraftment and high vision restoration efficacy. The article is a review of main scientific papers on keratoprostheses. The authors state characteristics of different types of keratoprosthesis, indications and outcomes of their use, complications, efficacy rate and postoperative visual acuity. Each of the presented types of keratoprosthesis has its own benefits and drawbacks, so their improvement to achieve better surgical outcomes remains relevant and needs further study. Key words: ophthalmic surgery, corneal opacity, keratoplasty, multiple transplant failures, keratoprostheses

CD51 labels periosteal injury-responsive osteoprogenitors.

The periosteum is a critical source of skeletal stem and progenitor cells (SSPCs) that form callus tissue in response to injury. There is yet to be a consensus on how to identify SSPCs in the adult periosteum. The aim of this study was to understand how potential murine periosteal SSPC populations behave in vivo and in response to injury. We evaluated the in vivo differentiation potential of Sca1-CD51+ and Sca1+CD51+ cells following transplantation. In vitro, the Sca1+CD51+ population appears to be more primitive multipotent cells, but after transplantation, Sca1-CD51+ cells showed superior engraftment, expansion, and differentiation into chondrocytes and osteoblasts. Despite representing a clear population with flow cytometry, we identified very few Sca1+CD51+ cells histologically. Using a periosteal scratch injury model, we successfully mimicked the endochondral-like healing process seen in unstable fractures, including the expansion and osteochondral differentiation of αSMA+ cells following injury. CD51+ cells were present in the cambium layer of resting periosteum and expanded following injury. Sca1+CD51- cells were mainly localized in the outer periosteal layer. We found that injury increased colony-forming unit fibroblast (CFU-F) formation in the periosteum and led to rapid expansion of CD90+ cells. Several other populations, including Sca1-CD51+ and CD34+ cells, were expanded by day 7. Mice with enhanced fracture healing due to elevated Notch signaling mediated by NICD1 overexpression showed significant expansion of CD51+ and CD34hi cells in the early stages of healing, suggesting these populations contribute to more rapid healing. In conclusion, we demonstrate that periosteal injury leads to the expansion of various SSPC populations, but further studies are required to confirm their lineage hierarchy in the adult skeletal system. Our data indicate that CD51+ skeletal progenitor cells are injury-responsive and show good engraftment and differentiation potential upon transplantation.

Therapeutic effect of a hydrogel-based neural stem cell delivery sheet for mild traumatic brain injury

Objective: There are no effective clinically applicable treatments for neuronal dysfunction after mild traumatic brain injury (TBI). Here, we evaluated the therapeutic effect of a new delivery method of mouse neural stem cell (mNSC) spheroids using a hydrogel, in terms of improvement in damaged cortical lesions and cognitive impairment after mild TBI. Methods: mNSCs were isolated from the subventricular zone and subgranular zone by a hydrogel-based culture system. GFP-transduced mNSCs were generated into spheroids and wrapped into a sheet for transplantation. Male C57BL/6J mice were randomly divided into four groups: sham operation, TBI, TBI with mNSC spheroids, and TBI with mNSC spheroid sheet transplantation covering the damaged cortex. Histopathological and immunohistochemical features and cognitive function were evaluated 7, 14, and 28 days after transplantation following TBI. Results: Hydrogel-based culture systems and mNSC isolation were successfully established from the adult mice. Essential transcription factors for NSCs, such as SOX2, PAX6, Olig2, nestin, and doublecortin (DCX), were highly expressed in the mNSCs. A transplanted hydrogel-based mNSC spheroid sheet showed good engraftment and survival ability, differentiated into TUJ1-positive neurons, promoted angiogenesis, and reduced neuronal degeneration. Also, TBI mice treated with mNSC spheroid sheet transplantation exhibited a significantly increased preference for a new object, suggesting improved cognitive function compared to the mNSC spheroids or no treatment groups. Conclusion: Transplantation with a hydrogel-based mNSC spheroid sheet showed engraftment, migration, and stability of delivered cells in a hostile microenvironment after TBI, resulting in improved cognitive function via reconstruction of the damaged cortex. Statement of Significance: This study presents the therapeutic effect of a new delivery method of mouse neural stem cells spheroids using a hydrogel, in terms of improvement in damaged cortical lesions and cognitive impairment after traumatic brain injury. Collagen/fibrin hydrogel allowed long-term survival and migratory ability of NSCs spheroids. Furthermore, transplanted hydrogel-based mNSCs spheroids sheet showed good engraftment, migration, and stability of delivered cells in a hostile microenvironment, resulting in reconstruction of the damaged cortex and improved cognitive function after TBI. Therefore, we suggest that a hydrogel-based mNSCs spheroids sheet could help to improve cognitive impairment after TBI.

Lichen Transplantation on Polyporales Fungi. An Attempt to Verify the Compatibility after Translocation from a Hill Forest to Anthropized City Parks at Livorno (Tuscany, Italy)

The transplantation of genus Cladonia terricolous lichens on Polyporales parasitic xylophagous fungi was attempted. Three species of Cladonia (C. pyxidata, C. rangiformis, C. foliacea) have been moved (translocated) from Livorno hill woods (Tuscany, Italy) to some public Livorno parks which suffer by an excessive anthropization. After a short period (a week) of adaptation to the new environment we transplanted thirteen specimens of Cladonia lichens on four different species of Polyporales fungi (Fomitopis betulina, Phellinus torulosus, Stereum hirsutum and Ganoderma applanatum) identified on the trees of the parks. The transplants obtained good engraftment and every periodic qualitative control showed a persistent photosynthetic activity even eight months after the start. Only one transplant (on Ganoderma applanatum) failed due by the toxicity of a secretion after incision. These observations lead us to consider those experiments as a demonstration of the compatibility of the implied tissues on transplantation and also allow us to introduce the difference of the term transplantation (the insert of an organism to another) from the often-confused translocation (relocation of an organism in different habitat). Future checks will indicate how and for how long this artificial association complex might persist.

PLX-R18 (Avoplacel), a Placental Expanded Mesenchymal-like Stromal Cell Therapy Shows Favorable Safety and Appears to Improve Incomplete Hematopoietic Recovery after Hematopoietic Cell Transplantation (HCT)

Introduction: Persistent cytopenia in the post-HCT setting remains a challenge in some recipients despite good engraftment of donor cells. PLX-R18 is a placental-derived mesenchymal-like cell product, expanded ex vivo in a 3-dimentional environment according to Good Manufacturing Practices. PLX-R18 cells secrete a large array of hematopoietic factors including stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), interleukin-6 and interleukin-11, which promote regeneration, maturation, and differentiation of hematopoietic cells in bone marrow and stimulate their migration to peripheral blood. Reported herein are the final encouraging safety and efficacy results from a phase 1 study (NCT03002519). Methods: 21 patients > 18 years who experienced incomplete hematopoietic recovery at least 3 months post-HCT, were enrolled into the study. Eligibility criteria were defined as meeting at least one of the following: neutrophil count (ANC) < 1000/μL, platelet (PLT) < 50,000/μL or hemoglobin (HGB) < 8 g/dL. Patients had no evidence of other causes for cytopenia such as an active infection or malignancy or severe acute or chronic Graft vs. Host Disease. For allogeneic (allo) HCT, complete, stable donor chimerism was required. Safety of PLX-R18 therapy was the primary objective. PLX-R18 was administered via multiple IM injection in 2 treatment sessions one week apart. Patients were treated in 3 cohorts: 1 million cells/kg (n=3); 2 million cells/kg (n=6); and 4 million cells/kg (n=12). Safety: 19 of 21 treated patients (90%) received an allo-HCT; for 5 patients (24%) this was a repeat HCT. The median time from HCT to PLX-R18 administration was ~8 months (236 days). Acute lymphocytic leukemia (ALL) was the most common underlying malignancy (33%) followed by acute myeloid leukemia (14%) and myelofibrosis (14%). The most common adverse events (AEs) were local injection-site reactions (pain), observed in 85.7% of patients, and were transient and of mild to moderate severity. No clinically meaningful changes were observed in vital signs, ECG and blood counts, liver and kidney function or coagulation laboratory results. A transient and reversible elevation in inflammatory markers, high sensitivity C-reactive protein (hsCRP) and complement factor 5a (C5a), were observed on the day following PLX-R18 administration and returned to normal levels thereafter. One patient developed severe febrile neutropenia two days after the second infusion (PLX-R18 4 million cells/kg) which was considered related to PLX-R18 and resolved 2 days later. Four AEs were associated with a fatal outcome (1 sudden death, 2 events of sepsis and 1 event of ALL recurrence); none were considered by the Investigator, or the Sponsor, as related to PLX-R18. One relapse was reported, an ALL patient who relapsed 296 days following study treatment and passed away 2 weeks later. No other events of relapse or recurrence were reported. Efficacy: For all PLX-R18 doses, a positive mean change from baseline in PLT and HGB was observed starting as early as 1 month after PLX-R18 administration and lasting up to 12 months following treatment (p<0.05). Positive mean change from baseline in ANC was observed in all doses as early as one month, peaking at 6 months for the 4 million cells/kg dose (p=0.062). Among patients who received blood transfusions at any time during follow up, a meaningful reduction in the mean number of transfused units per month, for both PLT and RBC transfusions, was observed as early as 3 months following PLX-R18 treatment and lasted throughout the 12-month follow up period. For PLT, the mean monthly number of transfused units dropped from 5.09 at baseline to 0.55 at 12 months (p<0.05); for RBC, from 2.91 at baseline to 0.00 at 12 months (p=0.0005). Conclusion: PLX-R18 therapy appears to be safe and well tolerated and shows promise in improving incomplete hematopoietic recovery after HCT. Further clinical studies are warranted to optimize the efficacy of PLX-R18 in treatment of cytopenia in the post-HCT setting and expand the potential to other marrow failure disorders as well as cytopenia associated with certain treatment modalities such as CAR-T therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Comparable Outcomes Following Non-First-Degree and First-Degree Related Donor Haploidentical Hematopoietic Cell Transplantation for Acute Leukemia Patients in Complete Remission: A Study from the Global Committee and the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation

Background Non-first-degree (NFD) related donors can be considered as alternatives for first-degree (FD) related donors in haploidentical hematopoietic cell transplantation (haplo-HCT), but the efficacy of these transplants remains uncertain. We therefore compared the outcomes of NFD and FD related haplo-HCTs for acute leukemia patients in complete remission (CR). Patients and Methods We retrospectively analyzed 2703 acute myeloid leukemia (AML: n=2047) and acute lymphoblastic leukemia (ALL: n=656) patients in CR who received a haplo-HCT from 2010-2021 in 177 EBMT centers participating in the study. Exact matching and propensity score (PS) matching was used for pair-matching. The following variables were used for exact matching: diagnosis, status at transplant, patient age per 5 years, GVHD prophylaxis. The propensity score was based on patient sex and conditioning intensity. The NFD-to-FD ratio was 1:3, but patients with only 1 or 2 controls were included in the analysis. Results One hundred and twenty-three NFD were matched with 324 FD. The patients were well matched with standardized mean difference estimates of less than 5% for all matched parameters. . Myeloablative conditioning was used in 65% and 69% of FD and NFD patients, respectively. The source of stem cells was peripheral blood (PB) alone in 46% and 68%, respectively. Both cohorts reached good engraftment rates (FD: 95.6% vs. NFD: 96.7%, p= 0.78). The 180-day cumulative incidence (CI) of grade II-IV acute graft-versus-host disease (GVHD) (FD: 29.1% vs. NFD: 24%, p= 0.31), and the 2-year CI of extensive chronic GVHD (FD: 9.9% vs. NFD: 15.3%, p= 0.2) were comparable between the two cohorts. No differences were observed between the two cohorts in 2-year CIs of relapse (FD: 22.6% vs. NFD: 22.1%, p=0.84) and non-relapse mortality (NRM) (FD: 17.7% vs. NFD: 13.2%, p=0.33). The 2-year probabilities of overall survival (OS) (FD: 68.3% vs. NFD: 71.8%, p=0.56), leukemia-free survival (LFS) (FD: 59.7% vs. NFD: 65.7%, p=0.6), and GVHD-free, relapse-free survival (GRFS) (FD: 47.8% vs. NFD: 50.9%, p=0.69) were similar between the two cohorts (Table 1 and Figure 1). The three major causes of death in FD and NFD were relapse (41% and 46%), GVHD (13% and 29%), and infection (30% and 15%), respectively. Conclusion No significant differences were observed in outcomes following haplo-HCT using NFD or FD donors in this retrospective matched-pair analysis. Our findings support the consideration of NFD donors for haplo-HCT when there is no available FD donor. If both donor types are available, an additional question may be to identify other important factors such as donor age and cytomegalovirus (CMV) status where the NFD donor might be a better choice than a FD donor. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Procurement and Evaluation of Hepatocytes for Transplantation From Neonatal Donors After Circulatory Death

Hepatocyte transplantation is a promising treatment for liver failure and inborn metabolic liver diseases, but progress has been hampered by a scarcity of available organs. Here, hepatocytes isolated from livers procured for a neonatal hepatocyte donation program within a research setting were assessed for metabolic function and suitability for transplantation. Organ donation was considered for infants who died in neonatal intensive care in the Stockholm region during 2015–2021. Inclusion was assessed when a decision to discontinue life-sustaining treatment had been made and hepatectomy performed after declaration of death. Hepatocyte isolation was performed by three-step collagenase perfusion. Hepatocyte viability, yield, and function were assessed using fresh and cryopreserved cells. Engraftment and maturation of cryopreserved neonatal hepatocytes were assessed by transplantation into an immunodeficient mouse model and analysis of the gene expression of phase I, phase II, and liver-specific enzymes and proteins. Twelve livers were procured. Median warm ischemia time (WIT) was 190 [interquartile range (IQR): 80–210] minutes. Median viability was 86% (IQR: 71%–91%). Median yield was 6.9 (IQR: 3.4–12.8) x106 viable hepatocytes/g. Transplantation into immunodeficient mice resulted in good engraftment and maturation of hepatocyte-specific proteins and enzymes. A neonatal organ donation program including preterm born infants was found to be feasible. Hepatocytes isolated from neonatal donors had good viability, function, and engraftment despite prolonged WIT. Therefore, neonatal livers should be considered as a donor source for clinical hepatocyte transplantation, even in cases with extended WIT.

Učestalost slabosti kalema nakon alogene transplantacije matičnih ćelija hematopoeze

Introduction: Poor engraftment represents one of the possible complications after allogeneic hematopoietic stem cell transplantation (allo-HSCT). It presents as pancytopenia or bicytopenia, with or without complete donor chimerism. There are three entities of poor engraftment: poor graft function (PGF), graft failure (GF), and graft rejection (GR). Aim: This study aims to show the frequency of poor engraftment, as well as the frequency of all of its entities individually, among the patients of the Clinic of Hematology of the University Clinical Center of Serbia (UCCS), who underwent allo-HSCT between December 20, 2017 and December 25, 2020, for the purpose of achieving improved management and understanding of this serious complication. Materials and methods: This retrospective cohort study included 58 patients. Diagnosis of poor engraftment was confirmed by pancytopenia (cut off values: hemoglobin &lt; 70g/L; platelet count &lt; 20 x 109 /L; absolute neutrophil count (ANC) &lt; 0.5 x 109 /L), for three consecutive days, as of day D+28, with the exclusion of severe graft versus host disease (GvHD) and relapse, with complete donor chimerism in PGF and with incomplete donor chimerism in GF. GR presented as acute rejection of the graft by the recipient with bone marrow aplasia or pancytopenia. Results: Poor engraftment was confirmed in 13 of 58 patients (22.4%). Patients with PGF were the majority, with 12.1% (seven patients), while patients with GF and those with GR had the same incidence of 5.2% (three patients). Overall survival for patients with poor engraftment after allo-HSCT was five months, which is significantly less than the overall survival of the patients who had good engraftment after allo-HSCT (57 months). Conclusion: The three types of poor engraftment must be precisely discriminated and diagnosed in relation to donor chimerism in order to decrease morbidity and mortality in patients, post allo-HSCT.

Comprehensive treatment of gunshot wounds using vacuum therapy and technomolecular silver preparations

Introduction. Burn wounds from the moment of their occurrence are primarily infected, and are accompanied by a violation of microcirculation, which contributes to the formation of massive necrosis of soft tissues, and leads to the delayed healing of such wounds. The speed of wound healing depends on a number of factors, not least of which is local treatment. An effective solution to this problem is the use of vacuum therapy (Vacuum Assisted Closure), which in combination with technomolecular silver preparations improves the course of all phases of the wound process, promotes active drainage of the wound, which reduces the level of microbial contamination of the wound, reduces local swelling of tissues, activates local blood circulation, which promotes faster healing of wounds. The aim of the study. To improve the results of treatment of patients with gunshot wounds of soft tissues, using vacuum therapy and technomolecular silver preparations. Materials and methods. In 2022, there were 35 patients with gunshot wounds of soft tissues of various degrees of complexity undergoing inpatient treatment in the surgical and trauma departments of the Uzhhorod City Multidisciplinary Hospital. Patients were divided into two groups. The first group, the control group, included 18 patients who were treated with VAC therapy and water-based antiseptics in the complex treatment of wounds after dismantling the VAC therapy device. In the second, main group of patients, the complex of wound healing measures included VAC therapy and the use of technomolecular silver preparations in the form of a spray, which was applied to the wound once a day, after dismantling the VAC therapy device. Research results . The use of VAC-therapy accelerates the time of wound cleaning, and the use of technomolecular silver preparations promotes the active growth of granulations in significant wound defects, and active marginal epithelization on the surface where the granulations filled the defect. After dismantling the VAC-therapy device, the bacterial landscape of wounds decreased in almost all patients compared to the beginning of treatment. Also, preparations of technomolecular silver were applied for a long time to wounds in which autodermoplasty was performed, which contributed to good engraftment of transplanted skin flaps and faster healing of wounds. The duration of inpatient treatment of patients in the control group was 23.5±2.3 days, and in the main one – 19.4±2.1 days, which is 1.2 times faster than in the control group of patients. Conclusion. The use of the vacuum therapy method in the complex treatment of gunshot wounds, in combination with technomolecular silver preparations, helps to quickly clean the wound from necrotic tissues, activates the growth of granulations, and promotes good engraftment of transplanted skin flaps, which in general contributes to faster healing of wounds, and accordingly shortens the duration of an inpatient treatment.

In vitro recovery of FIX clotting activity as a marker of highly functional hepatocytes in a hemophilia B iPSC model.

Background and AimsPluripotent stem cell–derived hepatocytes differentiated in monolayer culture are known to have more fetal than adult hepatocyte characteristics. If numerous studies tend to show that this immature phenotype might not necessarily be an obstacle to their use in transplantation, other applications such as drug screening, toxicological studies, or bioartificial livers are reliant on hepatocyte functionality and require full differentiation of hepatocytes. New technologies have been used to improve the differentiation process in recent years, usually evaluated by measuring the albumin production and CYP450 activity. Here we used the complex production and most importantly the activity of the coagulation factor IX (FIX) produced by mature hepatocytes to assess the differentiation of hemophilia B (HB) patient’s induced pluripotent stem cells (iPSCs) in both monolayer culture and organoids.Approach and ResultsIndeed, HB is an X‐linked monogenic disease due to an impaired activity of FIX synthesized by hepatocytes in the liver. We have developed an in vitro model of HB hepatocytes using iPSCs generated from fibroblasts of a severe HB patient. We used CRISPR/Cas9 technology to target the genomic insertion of a coagulation factor 9 minigene bearing the Padua mutation to enhance FIX activity. Noncorrected and corrected iPSCs were differentiated into hepatocytes under both two‐dimensional and three‐dimensional differentiation protocols and deciphered the production of active FIX in vitro. Finally, we assessed the therapeutic efficacy of this approach in vivo using a mouse model of HB.ConclusionsFunctional FIX, whose post‐translational modifications only occur in fully mature hepatocytes, was only produced in corrected iPSCs differentiated in organoids. Immunohistochemistry analyses of mouse livers indicated a good cell engraftment, and the FIX activity detected in the plasma of transplanted animals confirmed rescue of the bleeding phenotype.

Hemoglobinuria in the Early Poststem-Cell-Transplant Period: Risk Factors and Association with Outcomes.

Information on risk factors of hemoglobinuria after hematopoietic stem-cell transplant (HSCT) and its association with AKI, mortality, and engraftment is limited. We conducted a retrospective cohort study on all consecutive adults that underwent HSCT from January 6, 1999, to November 6, 2017. The study included 6039 patients that underwent bone marrow transplantation (BMT), umbilical cord blood, and peripheral blood stem-cell transplantation (PBSCT). Early post-HSCT, AKI occurred in 393 (7%) patients, and 52 (0.9%) patients had post-HSCT hemoglobinuria. Post-HSCT hemoglobinuria was associated with graft type (BMT+Cord), underlying disease (lymphoma, acute leukemia), and fludarabine-based conditioning regimen. Post-HSCT hemoglobinuria was associated with early (48-72 hours) post-HSCT AKI. Graft type (BMT+Cord) was associated with AKI among patients with hemoglobinuria. AKI in patients with hemoglobinuria was associated with delayed platelet engraftment and delayed WBC engraftment but not 100-day mortality. Close monitoring is recommended in this patient group to facilitate a good engraftment outcome.

Good Engraftment but Quality and Donor Concerns for Cryopreserved Hemopoietic Progenitor Cell Products Collected During the COVID-19 Pandemic

Changes to donor availability, collection center capacity, and travel restrictions during the early phase of the COVID-19 pandemic led to routine cryopreservation of most unrelated donor products for hematopoietic transplantation prior to the recipient commencing the conditioning regimen. We investigated the effect of this change on unrelated donor product quality and clinical outcomes. Product information was requested from transplantation centers in Australia and New Zealand and clinical outcome data from the Australasian Bone Marrow Transplant Recipient Registry (ABMTRR). In total, 191 products were collected between April 1, 2021, and September 30, 2021, and most (74%) were from international collection centers. Median post-thaw CD34 recovery was 78% (range 25% to 176%) and median post-thaw CD34 viability was 87% (range 34% to 112%). Median time to neutrophil recovery was 17 days (interquartile range 10 to 24 days), and graft failure occurred in 6 patients (4%). These clinical outcomes were similar to those of “fresh” unrelated donor transplants reported to the ABMTRR in 2019. However, recipient transplantation centers reported problems with 29% of products in the form of damage during transit, low cell dose, inadequate labeling, missing representative samples, or missing documentation. These problems were critical in 7 cases (4%). At last follow-up, 22 products (12%) had not been infused. Routine cryopreservation of unrelated donor hemopoietic progenitor cell products has enabled safe continuation of allogeneic transplant services during the COVID-19 pandemic. However, practices for product tracing, documentation, and transportation can be optimized, and measures to reduce the incidence of unused unrelated donor product are required.

Serially Transplantable Patient-Derived Xenograft Model of Chronic Myelomonocytic Leukemia (CMML)

Introduction: The establishment of preclinical mouse models for MDS or MDS/MPN overlap syndromes from patient cells often fails or results in low engraftment. If successful, engrafting cells in primary mouse recipients usually do not engraft secondary or later recipients, thus precluding genetic manipulation of the cells for functional studies. We hypothesized that oncogenic complementation of patient cells would a) allow engraftment and serial transplantation, b) enable genetic modification of the cells for functional evaluation and c) preserve the disease-associated biology of the cell. To study these hypotheses we chose the MN1 oncogene for complementation of cells from a patient with chronic myelomonocytic leukemia (CMML). Importantly, healthy cord blood cells, when transduced with MN1, induce myeloproliferation in vivo, but cannot be serially transplanted and do not induce acute myeloid leukemia (Imren et al. Blood 2014).Aim: To establish a serially transplantable CMML patient-derived xenograft (PDX) model and perform a targeted in vivo shRNA screening in this model.Patient and Methods: Bone marrow (BM) cells were harvested from a CMML patient with 15% blasts in BM (CMML-2). BM cells were depleted for T cells and transduced with a lentiviral vector encoding MN1 and GFP. One million MN1-transduced or untransduced cells were injected intravenously in sublethally irradiated NOD/SCID IL-2Rγ-/- mice transgenic for SCF, IL3 and GM-CSF (NSGS). BM and spleen cells from engrafted mice were immunophenotyped and retransplanted in NSGS recipients up to 6 times. Mutations were evaluated by whole exome sequencing and confirmed by Sanger sequencing. A targeted shRNA library was generated and lentivirally transduced in serially transplantable CMML cells. Representation of transduced cells was monitored by next-generation sequencing of individual shRNAs.Results: MN1 transduction of human CMML cells resulted in mean engraftment of human CD45+ cells of 9% in peripheral blood (PB) and 41% in BM at week 12 after first transplantation. In contrast, untransduced CMML cells were hardly detected in PB and BM even 22 weeks after transplantation (mean engraftment 1.1% and 1.7%, respectively). Transplantation of BM cells from primary into secondary recipients led to stable engraftment of MN1-transduced CMML cells in PB (11%), spleen (4%) and BM (26%). In contrast, transplantation from primary to secondary recipients of untransduced CMML cells showed no engraftment in PB over a period of 40 weeks. Importantly, MN1+CMML cells have been serially transplanted up to the sixth recipient with good engraftment levels in PB, BM and spleen and could be re-derived from frozen cells. Overall, MN1+CMML cells could be expanded in vivo to allow engraftment in 33 mice so far.MN1+CMML cells in spleen and BM showed a myeloid phenotype expressing CD33, CD38 and CD14, but lacked expression of lymphoid markers CD3 and CD19. The stem and progenitor phenotype CD34+CD38- was found in 2.5% of these cells. By morphologic evaluation, monocytic cells prevailed in BM up to the third transplantation, while blast cells increased in later transplantations. Molecular analysis of primary patient cells identified mutations in BCOR, DNMT3A, U2AF1, NOTCH1 and NRAS . All mutations were still present in human cells isolated from recipient mice after the 4th and 6th transplantation, confirming the genetic stability of the model.A shRNA pool directed against the above mentioned mutated genes, MN1, and luciferase was lentivirally transduced into cells from the fifth recipient and transplanted into the sixth recipient. After 8 weeks, shRNAs against luciferase and BCOR were well preserved in vivo in MN1+CMML cells, while shRNAs against U2AF1 and MN1 were depleted. Thus, MN1+CMML cells remained dependent on at least one additional gene besides MN1, thus validating our hypothesis that the disease biology is preserved in our induced PDX model.Conclusion: We present a novel approach to establish serially transplantable MDS/MPN-PDX models by complementation with the MN1 oncogene. Our CMML model preserves the genetic and morphologic characteristics of the disease and allows dissection of the functional architecture of CMML and preclinical drug studies in this underserved disease. DisclosuresNo relevant conflicts of interest to declare.

Wide-Detector Computed Tomography for Evaluating the Effectiveness of Microsurgical Autotransplantation of Tissue Complexes in the Urogenital Region

Objective : to demonstrate the possibilities and advantages of wide-detector computed tomography for performing dynamic studies and evaluating the effectiveness of microsurgical autotransplantation of tissue complexes in the urogenital region, by using a perfusion computed tomography and dynamic voiding computed cystourethrography. Subjects and methods. This investigation enrolled 36 patients with different urogenital diseases, who were examined using a wide-detector computed tomographic scanner and the procedures of dynamic perfusion computed tomography and dynamic voiding computed cystourethrography. Results. Perfusion examination revealed that none of the patients had hypoperfused areas of autografts, which suggests their good engraftment. Dynamic voiding computed cystourethrography estimated the width of the urethral lumen in all segments and identified the regions of its greatest narrowing and their extension. Computed uroflowmetry was scheduled for all patients. Conclusion. The advantages of wide-detector systems with their unique properties made it possible to apply them in the new area – in planning and evaluating the effectiveness of the microsurgical reconstruction of the urogenital region, by obtaining additional, previously unavailable diagnostic information.