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• Allogeneic stem cell transplant (SCT) is curative for patients with severe thalassemia. It can be safely performed with a sequence of a pretransplant immunosuppressive therapy (PTIS) of pulse fludarabine and dexamethasone, followed by a reduced-intensity regimen of fludarabine and i.v. busulfan, as well as peripheral blood progenitor transplant with post-transplant cyclophosphamide-based immunosuppression. • Patients with very high titers of anti-donor-specific antibodies (1:3000) further benefited from the addition of bortezomib and Rituxan to the PTIS phase to alleviate antibody-mediated graft rejection. • With this approach, SCT now be safely carried out using both matched and mismatched (aka haploidentical) donors, with a 3-year event-free and overall survival of 95%, regardless of donor type, and without having any increased risk for mortality in patients who are older than 7 years and having hepatomegaly (i.e., the Lucarelli group III high-risk subset). • This demonstrates that there is no reason to take the risk(s) of total body irradiation to secure engraftment in this group of patients who are at high risk for graft failure. Patients with severe thalassemia commonly have a survival that is significantly shorter than that of the general population. Allogeneic hematopoietic stem cell transplantation (allo-SCT) is the only established treatment that is potentially curative, but it is limited by the availability of donors and the medical condition of the patient. To expand the donor pool to include haploidentical related donors, we introduced a program consisting of a pharmacologic pretransplant immune suppression phase (PTIS) and 2 courses of dexamethasone and fludarabine, followed by pretransplant conditioning with fludarabine-i.v. busulfan and post-transplant graft-versus-host disease (GVHD) prophylaxis with cyclophosphamide, tacrolimus, and mycophenolate mofetil. We transplanted 83 consecutive transfusion-dependent patients with thalassemia (median age, 12 years; range, 1 to 28 years) with a minimum follow-up of 6 months (median, 15 months; range, 7 to 53 months); the 3-year projected overall and event-free survival is over 96%, and there have been no secondary graft failures. Of the first 31 patients, we had 2 graft failures, both of them occurring in patients with extremely high titers of anti-donor-specific HLA antibodies (anti-DSAs), but after adjusting the PTIS to include bortezomib and rituximab for patients with high titers of anti-DSAs and using pharmacologic dose guidance for busulfan, we had no graft failures in the last 52 patients. Six (7%) of 83 patients developed severe GVHD. We conclude that this is a safe and efficacious approach to allogeneic SCT in thalassemia, yielding results comparable to those available for patients with fully matched donors. [ABSTRACT FROM AUTHOR]

Immune reconstitution of T cells has been proven to be a protective factor against cytomegalovirus (CMV) reactivation in patients post-hematopoietic stem cell transplant. Recently, more evidence has suggested that natural killer (NK) cells also play role in the protection against CMV reactivation in these patients. CMV-specific T cells and CMV-reactive NK cells from pediatric patients undergoing hematopoietic stem cell transplant were examined by flow cytometry. These cells were defined as cells producing interferon gamma (IFNγ) upon stimulation with CMV antigens. This study demonstrated that NK cells reactive to CMV do exist in pediatric patients after stem cell transplant. These cells vigorously responded to stimulation with CMV peptides (pp65 and IE1) and to a lesser extent to CMV whole lysate by secretion of IFNγ. Patients with CMV reactivation tended to have less CMV-reactive NK cells than those without. Reconstitution of CMV-reactive NK cells, together with CMV-specific T cells, may play a role in the control of CMV infections in patients after stem cell transplant. • NK cells reactive to CMV reconstituted after pediatric HSCT • CMV reactive NK cells responded vigorously to CMV peptides • CMV reactive NK cells tended to be higher in patients without CMV reactivation [ABSTRACT FROM AUTHOR]

Viral hemorrhagic cystitis ( HC) after hematopoietic stem cell transplantation ( HSCT) can be devastating. Standard treatment modalities have not been well established, but immune reconstitution may be necessary for sustained viral clearance. We studied five pediatric patients who developed viral HC after haplo-identical HSCT. All patients developed virus-specific CD4- and CD8-positive T cells, and the emergence of these viral-specific T cells was temporally associated with successful viral clearance. [ABSTRACT FROM AUTHOR]

Purpose: A multimodality approach is generally considered for pediatric low-grade gliomas (LGG); however, the optimal management remains uncertain. The objective of the study was to evaluate treatment outcomes of pediatric LGG, focusing on long-term survival and factors related to outcomes. Methods: A retrospective review of 77 pediatric LGG cases treated at Ramathibodi Hospital, Thailand between 2000 and 2018 was performed. The inclusion criteria were all pediatric LGG cases aged ≤ 15 years. Diffuse intrinsic pontine gliomas and spinal cord tumors were excluded. Results: The median follow-up time was 8.2 years (range, 0.6–19.7). The median age at diagnosis was 6.2 years (interquartile range, 3.6–11.4). Treatments modality included tumor surgery (93%), chemotherapy (40%), and radiation therapy (14%). The 10-year overall survival (OS) and 10-year progression-free survival were 94% and 59%, respectively, for the entire cohort. The 10-year OS was 100% in three subgroups of patients: pilocytic subtype, WHO grade 1 tumors, and recipient of gross total resection. After multivariable analysis, no tumor surgery had a significantly unfavorable influence on overall survival. Conclusions: With a multimodality approach, pediatric LGGs had excellent outcome. Gross total resection is the standard primary treatment. Chemotherapy is the alternative standard treatment in incomplete resection cases, unresectable patients, or patients with progressive disease. Radiation therapy should be reserved as a salvage treatment option because of late complications that usually affect patients' quality of life. [ABSTRACT FROM AUTHOR]

Allergic contact dermatitis (ACD) is a type IV hypersensitivity mainly mediated by Th1/Th17 immune response. Topical corticosteroid is currently the first-line treatment for allergic contact dermatitis (ACD) and systemic administration of immunosuppressive drugs are used in patients with severe disseminated cases. However, increased risk of adverse effects has limited their use. Thus, the development of a novel immunosuppressant for ACD with low toxicity is a challenging issue. In this study, we began our study by using a murine contact hypersensitivity (CHS) model of ACD to examine the immunosuppressive effects of DYRK1B inhibition. We found that mice treated with a selective DYRK1B inhibitor show reduced ear inflammation. In addition, a significant reduction of Th1 and Th17 cells in the regional lymph node upon DYRK1B inhibition was observed by FACS analysis. Studies in vitro further revealed that DYRK1B inhibitor does not only suppressed Th1 and Th17 differentiation, but also promotes regulatory T cells (Treg) differentiation. Mechanistically, FOXO1 signaling was enhanced due to the suppression of FOXO1Ser329 phosphorylation in the presence of DYRK1B inhibitor. Therefore, these findings suggest that DYRK1B regulates CD4 T cell differentiation through FOXO1 phosphorylation and DYRK1B inhibitor has a potential as a novel agent for treatment of ACD. [ABSTRACT FROM AUTHOR]

Copolymers of (D,L-lactide-random-ε-caprolactone)-block-poly(ethylene glycol)-block-(D,L-lactide-random-ε-caprolactone) (PLECs) were synthesized with varied D,L-lactide (LA) content and different molecular weights (20 and 50 kDa). Polymer ratios, particularly the content of LA, had significant effect on the release of trypan blue. A lower trypan blue release rate was observed from monolithic rods composed of PLECs with higher CL/LA ratio. High LA content in polymer rod led to increase of the hydrophilicity of the polymer rod and the decrease of CL content resulted in the increase of the hydrophilicity of PLECs and hydrolysis rate. Spin-coating technique was employed to coat PCL-b-PEG-b-PCL membrane on PLEC rod with controlled thickness. Results showed that membrane encased rods can produce different release pattern including delayed release, zero-ordered release and burst release depending on the types of inner rod and membrane. Results for this study indicated that PLEC rods could provide the zero-order release profile of highly water-soluble molecules. Moreover, the longer lag phase was observed at higher thickness. These results suggest that these polymer rods were potential drug delivery systems that can provide controlled drug release profiles. [ABSTRACT FROM AUTHOR]

This work describes the preparation and characterization of anticancer-loaded injectable polymeric depots that consisted of d, l-lactide (LA), ε-caprolactone (CL), and poly(ethylene glycol) (PEG) or [poly(ε-caprolactone)-random-poly( d, l-lactide)]-block-poly(ethylene glycol)-block-[poly(ε-caprolactone)-random-poly( d, l-lactide)] (PLEC) copolymers for malignant gliomas treatment. PLECs were polymerized with different percentages of LA to deliver 7-ethyl-10-hydroxycamptothecin (SN-38), a highly potent anticancer drug. SN-38-loaded depots could form directly in phosphate buffer saline with more than 98% encapsulation efficiency. The release rate of SN-38 from depots was found to depend on the amount of LA in PLECs, loading content of SN-38 in the depots, and depot weight. Encapsulation of SN-38 inside depots could enhance the stability of SN-38 where all of SN-38 released after 60 days was in an active form. Depots without SN-38 were evaluated as noncytotoxic against U-87MG, whereas SN-38-loaded depots showed cytotoxic effect as a function of concentration. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:3708-3717, 2012 [ABSTRACT FROM AUTHOR]

Copolymers of (D,L-lactide-random-ε-caprolactone)-block-poy(ethylene glycol)-block-(D,L-lactide-random-ε-caprolactone) or PLEC were explored as materials for injectable drug delivery system. A series of six PLECs were successfully synthesized with varied D,L-lactide (LA) content (0, 10 and 20%) and molecular weight (20 and 50 kDa). All polymers were able to form depots with more than 90% encapsulation efficiency of trypan blue leading to the loading density as high as 27% w/w. The variation of trypan blue loading, LA content and molecular weight were found to have profound effects on trypan blue release profiles. Even though, GPC and SEM confirmed the higher degradation of PLEC chains, trypan blue release rate and burst release was greater as the content of hydrophilic moiety, i.e. LA, was decreased. This was primarily due to the smooth and dense surface and cross-section of PLEC depots. The results from this study suggest a possible application of these depots as injectable, self-solidifying drug delivery systems. [ABSTRACT FROM AUTHOR]

Abstract: Recently published reports indicate that the outcome of unrelated donor transplantations in patients with leukemia is currently comparable to that of transplantation from identical family donors. We investigated the possibly favorable outcomes of related and unrelated transplantation in children with severe thalassemia. We reviewed transplantation outcome in 49 consecutive children with severe thalassemia who underwent allogeneic stem cell transplantation with related-donor (n = 28) and unrelated-donor (n = 21) stem cells between September 1992 and May 2005 at the Faculty of Medicine, Ramathibodi Hospital, Mahidol University (Bangkok, Thailand). Analysis of engraftment, frequency of procedure-related complications, and thalassemia-free survival showed no advantage from use of related-donor stem cells. The 2-year thalassemia-free survival estimate for recipients of related-donor stem cells was 82% compared with 71% in the unrelated-donor stem cell group (P = .42). The present study provides evidence to support the view that it is quite reasonable to consider unrelated-donor stem cell transplantation an acceptable therapeutic approach in severe thalassemia, at least for patients who are not fully compliant with conventional treatment and do not yet show irreversible severe complications of iron overload. [Copyright &y& Elsevier]

Gene therapy using lentiviral vectors (LVs) that harbor a functional β-globin gene provides a curative treatment for hemoglobinopathies including beta-thalassemia and sickle cell disease. Accurate quantification of the vector copy number (VCN) and/or the proportion of transduced cells is critical to evaluate the efficacy of transduction and stability of the transgene during treatment. Moreover, commonly used techniques for LV quantification, including real-time quantitative polymerase chain reaction (PCR) or fluorescence-activated cell sorting, require either a standard curve or expression of a reporter protein for the detection of transduced cells. In the present study, we describe a digital droplet PCR (ddPCR) technique to measure the lentiviral VCN in transduced hematopoietic stem and progenitor cells (HSPCs). After HSPCs were transduced with an LV encoding the therapeutic β-globin (βA-T87Q) gene, the integrated lentiviral sequence in the host genome was amplified with primers that targeted a sequence within the vector and the human RPP30 gene. The dynamic range of ddPCR was between 5 × 10−3 ng and 5 × 10−6 ng of target copy per reaction. We found that the ddPCR-based approach was able to estimate VCN with high sensitivity and a low standard deviation. Furthermore, ddPCR-mediated quantitation of lentiviral copy numbers in differentiated erythroblasts correlated with the level of βA-T87Q protein detected by reverse-phase high-performance liquid chromatography. Taken together, the ddPCR technique has the potential to precisely detect LV copy numbers in the host genome, which can be used for VCN estimation, calculation of infectious titer and multiplicity of infection for HSPC transduction in a clinical setting. [ABSTRACT FROM AUTHOR]

More than 58 million individuals worldwide are inflicted with chronic HCV. The disease carries a high risk of end stage liver disease, i.e., cirrhosis and hepatocellular carcinoma. Although direct-acting antiviral agents (DAAs) have revolutionized therapy, the emergence of drug-resistant strains has become a growing concern. Conventional cellular models, Huh7 and its derivatives were very permissive to only HCVcc (JFH-1), but not HCV clinical isolates. The lack of suitable host cells had hindered comprehensive research on patient-derived HCV. Here, we established a novel hepatocyte model for HCV culture to host clinically pan-genotype HCV strains. The immortalized hepatocyte-like cell line (imHC) derived from human mesenchymal stem cell carries HCV receptors and essential host factors. The imHC outperformed Huh7 as a host for HCV (JFH-1) and sustained the entire HCV life cycle of pan-genotypic clinical isolates. We analyzed the alteration of host markers (i.e., hepatic markers, cellular innate immune response, and cell apoptosis) in response to HCV infection. The imHC model uncovered the underlying mechanisms governing the action of IFN-α and the activation of sofosbuvir. The insights from HCV-cell culture model hold promise for understanding disease pathogenesis and novel anti-HCV development. [ABSTRACT FROM AUTHOR]

Hematopoietic stem-cell (HSC) transplantation using a donor with a homozygous mutation in the HIV co-receptor CCR5 (CCR5Δ32/Δ32) holds great promise as a cure for HIV-1. Previously, there were three patients that had been reported to be completely cured from HIV infection by this approach. However, finding a naturally suitable Human Leukocyte Antigen (HLA)-matched homozygous CCR5Δ32 donor is very difficult. The prevalence of this allele is only 1% in the Caucasian population. Therefore, additional sources of CCR5Δ32/Δ32 HSCs are required. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system is one method to mediate CCR5 knockout in HSCs that has been successfully employed as a gene editing tool in clinical trials. Additional anti-HIV-1 strategies are still required for broad-spectrum inhibition of HIV-1 replication. Here in this study, we combined an additional anti-HIV-1 therapy, which is C46, a cell membrane-anchored HIV-1 fusion inhibitor with the CRISPR/Cas9 mediated knockout CCR5. The combined HIV-1 therapeutic genes were investigated for the potential prevention of both CCR5 (R5)- and CXCR4 (X4)-tropic HIV-1 infections in the MT4CCR5 cell line. The combinatorial CRISPR/Cas9 therapies were superior compared to single method therapy for achieving the HIV-1 cure strategy and shows potential for future applications. [ABSTRACT FROM AUTHOR]

Simple Summary: Liquid biopsy is a technique that uses minimally invasive or noninvasive methods to detect disease biomarkers in biofluids, such as blood and urine. For childhood cancers, this approach is promising, especially when tissue biopsies are challenging. By analyzing tiny particles called extracellular vesicles, the small particles in biofluids which carry genetic and molecular information from cancer cells, this liquid biopsy technique can aid early diagnosis, treatment monitoring, and outcome prediction, potentially improving the outcomes of childhood cancers. This review article introduces the concept of extracellular vesicle liquid biopsy, summarizes the progress that is being made in the diagnosis of multiple types of pediatric malignancies, and provides prospects for using this extracellular vesicle method to guide the development of novel cancer therapeutics. Liquid biopsy involves the utilization of minimally invasive or noninvasive techniques to detect biomarkers in biofluids for disease diagnosis, monitoring, or guiding treatments. This approach is promising for the early diagnosis of childhood cancer, especially for brain tumors, where tissue biopsies are more challenging and cause late detection. Extracellular vesicles offer several characteristics that make them ideal resources for childhood cancer liquid biopsy. Extracellular vesicles are nanosized particles, primarily secreted by all cell types into body fluids such as blood and urine, and contain molecular cargos, i.e., lipids, proteins, and nucleic acids of original cells. Notably, the lipid bilayer-enclosed structure of extracellular vesicles protects their cargos from enzymatic degradation in the extracellular milieu. Proteins and nucleic acids of extracellular vesicles represent genetic alterations and molecular profiles of childhood cancer, thus serving as promising resources for precision medicine in cancer diagnosis, treatment monitoring, and prognosis prediction. This review evaluates the recent progress of extracellular vesicles as a liquid biopsy platform for various types of childhood cancer, discusses the mechanistic roles of molecular cargos in carcinogenesis and metastasis, and provides perspectives on extracellular vesicle-guided therapeutic intervention. Extracellular vesicle-based liquid biopsy for childhood cancer may ultimately contribute to improving patient outcomes. [ABSTRACT FROM AUTHOR]

Reviews the management of hematologic complications particularly transient myeloid dysplasia associated with the use of valproic acid. Presentation of patient's clinical report; Serial complete blood count results; Review of adverse reactions to valproic acid.

Looks at the results of bone-marrow transplantation from matched-sibling donors as being superior to grafts from HLA-matched but unrelated donors. The assessment in light of donor selection and post-transplant supportive care improvements; The methods; The findings; The interpretation.

• Graft-versus-host disease may mimic eosinophilic gastrointestinal disorders in a pediatric patient who underwent bone marrow transplantation. • Clues for graft-versus-host disease include persistent hypereosinophilia and gastrointestinal symptoms that were unresponsive to conventional treatment of eosinophilic gastrointestinal disorders. • Ruxolitinib might be a treatment of choice for patients with a steroid-refractory disease. Eosinophilic gastrointestinal disorders (EGIDs) are well-documented entities in pediatric solid organ transplantation. However, the diseases are rare after bone marrow transplantation (BMT). We present an adolescent male with hemoglobin E-β-thalassemia who underwent BMT and developed chronic graft-versus-host disease (GVHD) mimicking EGIDs. Initially, the patient presented with a presumed diagnosis of eosinophilic gastroenteritis (subserosal type) and received corticosteroids for 12 weeks. Six months after corticosteroids cessation, he again developed abdominal pain, treated with corticosteroids, azathioprine, and a six-food elimination diet. Still, he later had similar symptoms with persistent hypereosinophilia. The patient was subsequently diagnosed with chronic GVHD after excluding various potential causes. Ruxolitinib also led to significant clinical improvement and the disappearance of eosinophilia. The differential diagnosis of chronic GVHD should be a concern in BMT recipients with persistent gastrointestinal symptoms and eosinophilia. Ruxolitinib may be a treatment option in children with a steroid-refractory disease. [ABSTRACT FROM AUTHOR]

Discusses a study which demonstrated that donor lymphocyte infusion can eliminate mixed chimerism in nonmyeloablative stem cell transplantation for correction of hyper-immunoglobulin M (HIM) syndrome. Recommended treatment for HIM; Study subjects and methods; Indication of the study findings.

Introduction Methods Results Conclusion Immune reconstitution (IR) kinetics of paediatric patients underwent haploidentical haematopoietic stem cell transplantation (HSCT) with post‐transplant cyclophosphamide (PTCy) have not been extensively studied. We compared IR patterns of children receiving HSCT from haploidentical (n = 92) and HLA‐matched donors (n = 36), and analysed risk factors for viral infection in these patients.We prospectively measured lymphocyte subset numbers before HSCT and at 1, 3, 6 and 12 months after HSCT. Blood cytomegalovirus (CMV), Epstein–Barr virus, adenovirus, BK virus (BKV) and urine adenovirus and BKV viral loads were measured at designated time points.The median numbers of total T and T helper cells at 1 month were significantly lower in the haploidentical group compared with the HLA‐matched group. Haploidentical HSCT recipients had significantly lower median numbers of several T cell subsets and B cells for 1 year after HSCT. The median NK cell count of the haploidentical group was lower at 1 month. BKV haemorrhagic cystitis, blood CMV and urine adenovirus reactivation were more frequently found in the haploidentical group. Post‐haploidentical HSCT patients receiving anti‐T lymphocyte globulin (ATG) had significantly lower median numbers of total T cells (at 1 month) and T helper cells (at 6 and 12 months) and higher rate of blood BKV reactivation compared with those without ATG.Paediatric patients who undergo haploidentical HSCT with PTCy are likely to have delayed IR and an increased risk of viral reactivation/infection compared with HLA‐matched HSCT. The addition of ATG to PTCy delayed T cell recovery and increased risk of BKV reactivation. [ABSTRACT FROM AUTHOR]

Gaucher disease (GD) is a lysosomal storage disorder caused by a mutation in the GBA1 gene, responsible for encoding the enzyme Glucocerebrosidase (GCase). Although neuronal death and neuroinflammation have been observed in the brains of individuals with neuronopathic Gaucher disease (nGD), the exact mechanism underlying neurodegeneration in nGD remains unclear. In this study, we used two induced pluripotent stem cells (iPSCs)-derived neuronal cell lines acquired from two type-3 GD patients (GD3-1 and GD3-2) to investigate the mechanisms underlying nGD by biochemical analyses. These iPSCs-derived neuronal cells from GD3-1 and GD3-2 exhibit an impairment in endoplasmic reticulum (ER) calcium homeostasis and an increase in unfolded protein response markers (BiP and CHOP), indicating the presence of ER stress in nGD. A significant increase in the BAX/BCL-2 ratio and an increase in Annexin V-positive cells demonstrate a notable increase in apoptotic cell death in GD iPSCs-derived neurons, suggesting downstream signaling after an increase in the unfolded protein response. Our study involves the establishment of iPSCs-derived neuronal models for GD and proposes a possible mechanism underlying nGD. This mechanism involves the activation of ER stress and the unfolded protein response, ultimately leading to apoptotic cell death in neurons. [ABSTRACT FROM AUTHOR]

Background: The diarylheptanoid ASPP 049 has improved the quality of adult hematopoietic stem cell (HSC) expansion ex vivo through long-term reconstitution in animal models. However, its effect on hematopoietic regeneration from human induced pluripotent stem cells (hiPSCs) is unknown. Method: We utilized a defined cocktail of cytokines without serum or feeder followed by the supplementation of ASPP 049 to produce hematopoietic stem/progenitor cells (HSPCs). Flow cytometry and trypan blue exclusion analysis were used to identify nonadherent and adherent cells. Nonadherent cells were harvested to investigate the effect of ASPP 049 on multipotency using LTC-IC and CFU assays. Subsequently, the mechanism of action was explored through transcriptomic profiles, which were validated by qRT-PCR, immunoblotting, and immunofluorescence analysis. Result: The supplementation of ASPP 049 increased the number of phenotypically defined primitive HSPCs (CD34+CD45+CD90+) two-fold relative to seeded hiPSC colonies, indicating enhanced HSC derivation from hiPSCs. Under ASPP 049-supplemented conditions, we observed elevated HSPC niches, including CD144+CD73− hemogenic- and CD144+CD73+ vascular-endothelial progenitors, during HSC differentiation. Moreover, harvested ASPP 049-treated cells exhibited improved self-renewal and a significantly larger proportion of different blood cell colonies with unbiased lineages, indicating enhanced HSC stemness properties. Transcriptomics and KEGG analysis of sorted CD34+CD45+ cells-related mRNA profiles revealed that the Hippo signaling pathway is the most significant in responding to WWTR1/TAZ, which correlates with the validation of the protein expression. Interestingly, ASPP 049-supplemented HSPCs upregulated 11 genes similarly to umbilical cord blood-derived HSPCs. Conclusion: These findings suggest that ASPP 049 can improve HSC-generating protocols with proliferative potentials, self-renewal ability, unbiased differentiation, and a definable mechanism of action for the clinical perspective of hematopoietic regenerative medicine. [ABSTRACT FROM AUTHOR]

Establishing a drug-screening platform is critical for the discovery of potential antiviral agents against SARS-CoV-2. In this study, we developed a platform based on human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to investigate SARS-CoV-2 infectivity, with the aim of evaluating potential antiviral agents for anti-SARS-CoV-2 activity and cardiotoxicity. Cultured myocytes of iPSC-CMs and immortalized human cardiomyocyte cell line (AC-16) were primarily characterized for the expression of cardiac markers and host receptors of SARS-CoV-2. An infectivity model for the wild-type SARS-CoV-2 strain was then established. Infection modeling involved inoculating cells with SARS-CoV-2 at varying multiplicities of infection (MOIs) and then quantifying infection using immunofluorescence and plaque assays. Only iPSC-CMs, not AC16 cells, expressed angiotensin-converting enzyme 2 (ACE-2), and quantitative assays confirmed the dose-dependent infection of iPSC-CMs by SARS-CoV-2, unlike the uninfectable AC16 cells lacking the expression of ACE2. Cytotoxicity was evaluated using MTT assays across a concentration range. An assessment of the plant-derived compound panduratin A (panA) showed cytotoxicity at higher doses (50% cytotoxic concentration (CC50) 10.09 μM) but promising antiviral activity against SARS-CoV-2 (50% inhibition concentration (IC50) 0.8–1.6 μM), suppressing infection at concentrations 10 times lower than its CC50. Plaque assays also showed decreased viral production following panA treatment. Overall, by modeling cardiac-specific infectivity, this iPSC-cardiomyocyte platform enables the reliable quantitative screening of compound cytotoxicity alongside antiviral efficacy. By combining disease pathogenesis and pharmacology, this system can facilitate the evaluation of potential novel therapeutics, such as panA, for drug discovery applications. [ABSTRACT FROM AUTHOR]

Objectives: Multiple myeloma (MM) accounts for 10% of hematologic malignancies. However, most of the patients suffered from relapsed/refractory disease. We would like to expand CAR T cell therapy to treat MM using our current platform. Methods: BCMA CAR T lymphocytes were generated for volunteers or MM patients. The transduction efficiency was detected by the ddPCR technique. Immunophenotyping and exhaustion markers were monitored by flow cytometry. The efficacy of BCMA CAR T cells was tested using coculturing with BCMA CAR or mock, and the positive and negative targets, K562/hBCMA‐ECTM and K562, respectively. Results: BCMA CAR T cells were generated from consented volunteers or MM patients and could be detected CAR BCMA expression at a mean of 4.07 ± 1.95 or 4.65 ± 1.21 copies/cell, respectively. Those modified T cells were primarily effector memory T cells. Our BCMA CAR T cells could explicitly eradicate the K562/hBCMA‐ECTM cell line while the K562 cell line survived. Interestingly, the BCMA CAR, mock T cells, and peripheral blood mononuclear cells from MM patients expressed similar levels of the exhaustion makers, TIM‐3, LAG‐3, and PD1. Conclusions: Our BCMA CAR T cells, mainly effector/effector memory, could eliminate BCMA‐expressing cells in vitro and had similar levels of exhaustion markers among different populations. [ABSTRACT FROM AUTHOR]

Cytomegalovirus (CMV) infection is a major opportunistic infection after liver transplantation (LT) that necessitates monitoring. Because of the lack of studies in children, we aimed to investigate CMV-specific T cell immune reconstitution among pediatric LT recipients. The recipients were monitored for CMV infection and CMV-specific T cells from the start of immunosuppressive therapy until 48 weeks after LT. Clinically significant CMV viremia (csCMV) requiring preemptive therapy was defined as a CMV load of >2000 IU/mL. Peripheral blood CMV-specific T cells were analyzed by flow cytometry based on IFNγ secretion upon stimulation with CMV antigens including immediate early protein 1 (IE1) Ag, phosphoprotein 65 (pp65) Ag, and whole CMV lysate (wCMV). Of the 41 patients who underwent LT, 20 (48.8%) had csCMV. Most (17/20 patients) were asymptomatic and characterized as experiencing CMV reactivation. The onset of csCMV occurred approximately 7 weeks after LT (interquartile range: 4–12.9); csCMV rarely recurred after preemptive therapy. Lower pp65-specific CD8+ T cell response was associated with the occurrence of csCMV (p = 0.01) and correlated with increased viral load at the time of csCMV diagnosis (ρ = −0.553, p = 0.02). Moreover, those with csCMV had lower percentages of IE1-specific CD4+ and wCMV-reactive CD4+ T cells at 12 weeks after LT (p = 0.03 and p = 0.01, respectively). Despite intense immunosuppressive therapy, CMV-specific T cell immune reconstitution occurred in pediatric patients post-LT, which could confer protection against CMV reactivation. [ABSTRACT FROM AUTHOR]

Introduction: BK polyomavirus (BKPyV)‐associated nephropathy (BKPyVAN) can cause a significant risk of allograft impairment after kidney transplantation (KT). Intact BKPyV‐specific immunity is associated with viral containment. This study investigated BKPyV‐specific immunological factors among KT recipients. Methods: This prospective study in a single transplant center from January 2019 to August 2019 assessed associations between clinical and immunological characteristics, with a focus on BKPyV‐cell‐specific immunity and BKPyVAN, among KT recipients aged ≥15 years. The numbers of interferon‐gamma (IFN‐γ)‐producing CD4+ T, CD8+ T, natural killer (NK), and natural killer T (NKT) cells were measured after stimulation with large T antigen and viral capsid protein 1 (VP1). Results: In total, 100 KT recipients were included (mean age ± SD, 42 ± 11 years); 35% of the recipients were female patients, and 70% had received induction immunosuppressive therapy. The 1‐year cumulative incidence of high‐level BKPyV DNAuria (possible BKPyVAN) and (presumptive BKPyVAN) was 18%. Among 40 patients with immunological factor data, pre‐KT %NK cells (hazard ratio [HR], 1.258; 95% confidence interval [CI], 1.077–1.469; p =.004) and %VP1‐specific NK cells (HR, 1.209; 95% CI, 1.055–1.386; p =.006) were factors independently associated with possible and presumptive BKPyVAN. KT recipients with possible and presumptive BKPyVAN were more likely to exhibit significant mean coefficients of %NK, %VP1‐specific NK, and %NKT cells at 1 month after KT than before KT (all p <.05). Conclusion: Individuals with nonspecific and VP1‐specific NK cells before KT and increasing numbers of these cells after KT may be at risk for high‐level BKPyV DNAuria and presumptive BKPyVAN. Further studies are needed to determine the utility of BKPyV‐specific innate immune surveillance in predicting the occurrence of BKPyVAN. [ABSTRACT FROM AUTHOR]

Hodgkin's lymphoma and anaplastic large cell lymphoma, especially relapsed or refractory diseases, could recently be cured by CD30-targeted immunotherapy. However, the CD30 antigen releases the soluble ectodomain of CD30, which might obscure the targeted therapy. Therefore, the membrane epitope of CD30 (mCD30), left on the cancer cells, might be a prospective target for lymphoma treatment. The discovery of novel mCD30 monoclonal antibodies (mAbs) using phage technology yielded 59 potential human single-chain variable fragments (HuscFvs). Ten candidate HuscFv clones have been selected based on various methods, i.e., direct PCR, ELISA and western blot assays, and nucleotide sequencing techniques. Fortunately, only one potential HuscFv clone, clone #A4, was determined by the prediction of HuscFv-peptide molecular docking and the binding affinity test using isothermal titration calorimetry. Finally, we proved that the HuscFv #A4, which had a binding affinity (Kd) of 421e-9 ± 2.76e-6 M, might be the novel mCD30 mAb. We generated chimeric antigen receptor-modified T lymphocytes using HuscFv #A4 as an antigen detection part (anti-mCD30-H4CART). The cytotoxicity assay of anti-mCD30-H4CART cells showed significant eradication of the CD30-expressing cell line, K562 (p = 0.0378). We found a novel mCD30 HuscFv using human phage technology. We systematically examined and proved that our HuscFv #A4 could specifically eradicate CD30-expressing cancers. [ABSTRACT FROM AUTHOR]

Objectives: Patients with high‐risk hematologic diseases require intensive modalities, including high‐dose chemotherapy and allogeneic hematopoietic stem cell transplantation (allo‐HSCT). Haploidentical T‐cell–replete transplantation is a logical choice because of the limited availability of matched sibling donors and the prolonged time needed to identify matched unrelated donors in Thailand. Methods: The clinical outcomes data of 43 patients undergoing allo‐HSCT were reviewed. All patients had high‐risk hematologic malignancies, were younger than 20 years, and were in complete cytological remission at the time of allo‐HSCT. We used two different conditioning regimens: total body irradiation (TBI) combined with cyclophosphamide, fludarabine, and melphalan (n = 23) and thiotepa combined with fludarabine and busulfan (n = 20). All patients received a graft‐versus‐host disease prophylaxis regimen consisting of cyclophosphamide, mycophenolate mofetil, and a calcineurin inhibitor or sirolimus. Results: There was no difference in engraftment between patients receiving either of the regimens. After a median follow‐up of 35.8 (range, 0.6–106.2) months, the overall survival (OS) and event‐free survival (EFS) rates were 62.4% and 54.7%, respectively. OS and EFS were comparable between the respective regimens. Conclusions: We conclude that thiotepa‐based conditioning has similar efficacy and tolerability as TBI‐based conditioning for haploidentical HSCT with post‐transplant cyclophosphamide. [ABSTRACT FROM AUTHOR]

Simple Summary: The detection of genomic aberrations in cancers has yielded a wealth of information to discover oncogenic drivers or pathogenic variants that are relevant for the development of precise treatment strategies. Recent studies have shown promising outcomes in adult cancer patients with well characterized cancer genetic biomarkers. However, the development of precise treatments for pediatric cancers is difficult due to the limited number of accessible samples and the fact that well-defined target genetic aberrations are limited. Here, we review the current landscape of pediatric precision oncology compared to adults and highlight the examples of single-arm and multiple-arm designs of pediatric precision treatments. Over the past decades, several study programs have conducted genetic testing in cancer patients to identify potential genetic targets for the development of precision therapeutic strategies. These biomarker-driven trials have demonstrated improved clinical outcomes and progression-free survival rates in various types of cancers, especially for adult malignancies. However, similar progress in pediatric cancers has been slow due to their distinguished mutation profiles compared to adults and the low frequency of recurrent genomic alterations. Recently, increased efforts to develop precision medicine for childhood malignancies have led to the identification of genomic alterations and transcriptomic profiles of pediatric patients which presents promising opportunities to study rare and difficult-to-access neoplasms. This review summarizes the current state of known and potential genetic markers for pediatric solid tumors and provides perspectives on precise therapeutic strategies that warrant further investigations. [ABSTRACT FROM AUTHOR]

T cells genetically engineered to express a chimeric antigen receptor (CAR) specifically binding to a CD19 antigen has become the frontline of hematological malignancies immunotherapy. Their remarkable antitumor effect has exerted complete remission in treating B-cell malignancies. Although successful patient treatment has been shown, improvement to the structure of CAR to enhance its safety and efficacy profile is warranted. Transduction with a lentiviral vector (LVV) leading to the expression of CARs is also a critical step in redirecting T cells to target specific tumor antigens. To improve the efficacy of CD19 CARs in this study, the transduction ability of second and third generations LVV were compared. Ex vivo expansion of CD19 CARs T cells from healthy donors' peripheral blood mononuclear cells was performed after transduction of T cells with second and third generations LVV. Transduction efficacy of transduced T cells was determined to show a higher percentage in the third generations LVV transduced cells, with no changes in viability and identity of cells characterized by immunophenotyping. Testing the cytotoxic capacity of third generations LVV-transduced T cells against target cells showed higher reactivity against control cells. Cytokine expression was detected on the CD19 CARs T cells, suggesting that these cells limit in vitro growth of B-cell leukemia via secretion of the pro-inflammatory cytokine IFN γ. To investigate whether the third generation LVV transduced T cells can limit CD19 lymphoma growth in vivo, an analysis of tumor burden in a mouse model assessed by bioluminescence imaging was performed. We found that, in the presence of CD19 CARs T cells, the level of tumor burden was markedly reduced. In addition, an increase in the length of survival in mice receiving CAR-CD19 T cells was also observed. This suggests that transduction with third generations LVV generate a functional CAR-CD19 T cells, which may provide a safer and effective therapy for B-cell malignancies. [ABSTRACT FROM AUTHOR]

Background: Recurrence of retinoblastoma (RB) following chemoreduction is common and is often managed with local (intra-arterial/intravitreal) chemotherapy. However, some tumors are resistant to even local administration of maximum feasible drug dosages, or effective tumor control and globe preservation may be achieved at the cost of vision loss due to drug-induced retinal toxicity. The aim of this study was to identify drugs with improved antitumor activity and more favorable retinal toxicity profiles via screening of potentially repurposable FDA-approved drugs in patient-derived tumor organoids. Methods: Genomic profiling of five RB organoids and the corresponding parental tissues was performed. RB organoids were screened with 133 FDA-approved drugs, and candidate drugs were selected based on cytotoxicity and potency. RNA sequencing was conducted to generate a drug signature from RB organoids, and the effects of drugs on cell cycle progression and proliferative tumor cone restriction were examined. Drug toxicity was assessed with human embryonic stem cell-derived normal retinal organoids. The efficacy/toxicity profiles of candidate drugs were compared with those of drugs in clinical use. Results: RB organoids maintained the genomic features of the parental tumors. Sunitinib was identified as highly cytotoxic against both classical RB1-deficient and novel MYCN-amplified RB organoids and inhibited proliferation while inducing differentiation in RB. Sunitinib was a more effective suppressor of proliferative tumor cones in RB organoids and had lower toxicity in normal retinal organoids than either melphalan or topotecan. Conclusion: The efficacy and retinal toxicity profiles of sunitinib suggest that it could potentially be repurposed for local chemotherapy of RB. [ABSTRACT FROM AUTHOR]

Background: Recurrence of retinoblastoma (RB) following chemoreduction is common and is often managed with local (intra-arterial/intravitreal) chemotherapy. However, some tumors are resistant to even local administration of maximum feasible drug dosages, or effective tumor control and globe preservation may be achieved at the cost of vision loss due to drug-induced retinal toxicity. The aim of this study was to identify drugs with improved antitumor activity and more favorable retinal toxicity profiles via screening of potentially repurposable FDA-approved drugs in patient-derived tumor organoids. Methods: Genomic profiling of five RB organoids and the corresponding parental tissues was performed. RB organoids were screened with 133 FDA-approved drugs, and candidate drugs were selected based on cytotoxicity and potency. RNA sequencing was conducted to generate a drug signature from RB organoids, and the effects of drugs on cell cycle progression and proliferative tumor cone restriction were examined. Drug toxicity was assessed with human embryonic stem cell-derived normal retinal organoids. The efficacy/toxicity profiles of candidate drugs were compared with those of drugs in clinical use. Results: RB organoids maintained the genomic features of the parental tumors. Sunitinib was identified as highly cytotoxic against both classical RB1-deficient and novel MYCN-amplified RB organoids and inhibited proliferation while inducing differentiation in RB. Sunitinib was a more effective suppressor of proliferative tumor cones in RB organoids and had lower toxicity in normal retinal organoids than either melphalan or topotecan. Conclusion: The efficacy and retinal toxicity profiles of sunitinib suggest that it could potentially be repurposed for local chemotherapy of RB. [ABSTRACT FROM AUTHOR]

Background: The outbreak of COVID-19 has led to the suffering of people around the world, with an inaccessibility of specific and effective medication. Fingerroot extract, which showed in vitro anti-SARS-CoV-2 activity, could alleviate the deficiency of antivirals and reduce the burden of health systems. Aim of Study: In this study, we conducted an experiment in SARS-CoV-2-infected hamsters to determine the efficacy of fingerroot extract in vivo. Materials and Methods: The infected hamsters were orally administered with vehicle control, fingerroot extract 300 or 1000 mg/kg, or favipiravir 1000 mg/kg at 48 h post-infection for 7 consecutive days. The hamsters (n = 12 each group) were sacrificed at day 2, 4 and 8 post-infection to collect the plasma and lung tissues for analyses of viral output, lung histology and lung concentration of panduratin A. Results: All animals in treatment groups reported no death, while one hamster in the control group died on day 3 post-infection. All treatments significantly reduced lung pathophysiology and inflammatory mediators, PGE2 and IL-6, compared to the control group. High levels of panduratin A were found in both the plasma and lung of infected animals. Conclusion: Fingerroot extract was shown to be a potential of reducing lung inflammation and cytokines in hamsters. Further studies of the full pharmacokinetics and toxicity are required before entering into clinical development. [ABSTRACT FROM AUTHOR]

Background: Zinc plays essential roles in immune function and epithelial integrity. Patients undergoing hematopoietic stem cell transplantation (HSCT) often have low plasma zinc levels because of poor intake and diarrhea. We hypothesized that patients with zinc deficiency before HSCT had worse infectious complications after HSCT compared with patients with normal zinc levels. Citrulline, a marker of intestinal integrity, was also hypothesized to be lower in patients with zinc deficiency. Patients and methods: Thirty patients undergoing HSCT at Ramathibodi Hospital during March 2020–September 2021 were enrolled. Blood samples for plasma zinc and citrulline were collected during the HSCT period. The 14- and 90-day outcomes after HSCT were prospectively recorded. Results: Twelve of 30 (40%) patients had zinc deficiency before HSCT. Zinc-deficient patients were younger (median (interquartile range): 6 (8.8) vs 13 (5.8) years old; p = 0.017). Zinc levels tended to increase after admission in both groups. Patients with zinc deficiency had lower citrulline levels than those with normal zinc levels. Citrulline levels decreased in both groups after stem cell infusion, and the level was not significantly different between the two groups. Zinc-deficient patients had a higher rate of bacterial infection within 90 days after HSCT than those with normal zinc levels (6 in 12 patients (50.0%) vs 1 in 18 patients (5.6%); odds ratio [OR]: 17.0; 95% confidence interval [CI]: 1.68–171.70; p = 0.016). This remained significant after adjustments for age (adjusted OR: 12.31; 95% CI: 1.084–139.92; p = 0.043). Conclusion: The prevalence of zinc deficiency in pediatric patients undergoing HSCT was high. Zinc-deficient patients had lower citrulline levels and higher incidence of bacterial infection after HSCT. However, citrulline level was not different between patients with and without bacterial infections. It is worth to investigate whether zinc supplementation before HSCT can reduce bacterial infection after HSCT. [ABSTRACT FROM AUTHOR]

The interaction of SARS‐CoV‐2 infection with extracellular vesicles (EVs) is of particular interest at the moment. Studying SARS‐CoV‐2 contaminated‐EV isolates in instruments located outside of the biosafety level‐3 (BSL‐3) environment requires knowing how viral inactivation methods affect the structure and function of extracellular vesicles (EVs). Therefore, three common viral inactivation methods, ultraviolet‐C (UVC; 1350 mJ/cm2), β‐propiolactone (BPL; 0.005%), heat (56°C, 45 min) were performed on defined EV particles and their proteins, RNAs, and function. Small EVs were isolated from the supernatant of SARS‐CoV‐2‐infected human lung epithelial Calu‐3 cells by stepwise centrifugation, ultrafiltration and qEV size‐exclusion chromatography. The EV isolates contained SARS‐CoV‐2. UVC, BPL and heat completely abolished SARS‐CoV‐2 infectivity of the contaminated EVs. Particle detection by electron microscopy and nanoparticle tracking was less affected by UVC and BPL than heat treatment. Western blot analysis of EV markers was not affected by any of these three methods. UVC reduced SARS‐CoV‐2 spike detectability by quantitative RT‐PCR and slightly altered EV‐derived β‐actin detection. Fibroblast migration‐wound healing activity of the SARS‐CoV‐2 contaminated‐EV isolate was only retained after UVC treatment. In conclusion, specific viral inactivation methods are compatible with specific measures in SARS‐CoV‐2 contaminated‐EV isolates. UVC treatment seems preferable for studying functions of EVs released from SARS‐CoV‐2 infected cells. [ABSTRACT FROM AUTHOR]

Defective hemoglobin production and ineffective erythropoiesis contribute to the pathophysiology of thalassemia syndromes. Previous studies in the field of erythropoiesis mainly focused on the severe forms of thalassemia, such as β-thalassemia major, while mechanisms underlying the pathogenesis of other thalassemia syndromes remain largely unexplored. The current study aimed to investigate the intrinsic pathophysiological properties of erythroid cells derived from the most common forms of thalassemia diseases, including α-thalassemia (hemoglobin H and hemoglobin H-Constant Spring diseases) and β-thalassemia (homozygous β0-thalassemia and β0-thalassemia/hemoglobin E diseases), under an identical in vitro erythroid culture system. Cell proliferation capacity, differentiation velocity, cell death, as well as globin synthesis and the expression levels of erythropoiesis modifying factors were determined. Accelerated expansion was found in erythroblast cells derived from all types of thalassemia, with the highest degree in β0-thalassemia/hemoglobin E. Likewise, all types of thalassemia showed limited erythroid cell differentiation, but each of them manifested varying degrees of erythroid maturation arrest corresponding with the clinical severity. Robust induction of HSP70 transcripts, an erythroid maturation-related factor, was found in both α- and β-thalassemia erythroid cells. Increased cell death was distinctly present only in homozygous β0-thalassemia erythroblasts and associated with the up-regulation of pro-apoptotic (Caspase 9, BAD, and MTCH1) genes and down-regulation of the anti-apoptotic BCL-XL gene. [ABSTRACT FROM AUTHOR]

Background: In 2013, the Thai Pediatric Oncology Group (ThaiPOG) introduced a national protocol in which highdose chemotherapy plus stem cell rescue is performed without immunotherapy. Methods: This study aimed to elucidate the outcomes of high-risk neuroblastoma (HR-NB) patients treated with the ThaiPOG protocol. This retrospective cohort review included 48 patients (30 males, 18 females) with a median age of 3 years (range, 8 months to 18 years) who were treated at 5 ThaiPOG treatment centers in Thailand in 2000-2018. Results: Eight of the 48 patients showed MYCN amplification. Twenty-three patients (48%) received 131I-meta-iodobenzylguanidine prior to high-dose chemotherapy and stem cell rescue. The majority of patients achieved a complete or very good response prior to consolidation treatment. The 5-year overall survival (OS) and event-free survival (EFS) rates were 45.1% and 40.4%, respectively. Patients aged >2 years had a nonsignificantly higher mortality risk (hazard ratio [HR], 2.66; 95% confidence interval [CI], 0.92-7.68; P=0.07). The MYCN amplification group had lower OS and EFS rates than the MYCN nonamplification group, but the difference was not statistically significant (45% OS and 37.5% EFS vs. 33.3% OS and 16.6% EFS; P=0.67 and P=0.67, respectively). Cisretinoic acid treatment for 12 months was a strong prognostic factor that could reduce mortality rates among HR-NB patients (HR, 0.27; 95% CI, 0.09-0.785; P=0.01). Conclusion: High-dose chemotherapy plus stem cell rescue followed by cis-retinoic acid for 12 months was well tolerated and could improve the survival rates of patients with HR-NB. [ABSTRACT FROM AUTHOR]

Hypoxia is associated with tumor progression and poor prognosis in several cancer types. The present study aimed to examine the contribution of hypoxia (1% O2) to cancer progression in a cholangiocarcinoma cell line, RMCCA-1. The molecular basis of the hypoxic response pathway was investigated. The results showed that hypoxia significantly accelerated cancer cell proliferation and enhanced cell invasion (P<0.05). By using receptor tyrosine kinase and intracellular signaling antibody array kits, an increased phosphorylation/activation of a number of signaling molecules, particularly hepatocyte growth factor receptor (Met) and extracellular signal-regulated kinase (ERK) 1/2, was identified. Inhibition of Met and ERK by small hairpin RNA and U0126, respectively, significantly inhibited hypoxia-induced the invasive potential of RMCCA-1 cells (P<0.05). However, according to immunohistochemical analysis, hypoxia-inducible factor-1a expression was not correlated with cancer staging or tumor differentiation in 44 samples of cholangicarcinoma cases. The findings of the present study emphasized the importance of Met/ERK pathway activation as a key molecular event that may be responsible for a more invasive phenotype in hypoxic tumors and suggest Met as a potential target for the treatment of cholangiocarcinoma. [ABSTRACT FROM AUTHOR]

The article presents a case study of a 19-year-old male adolescent with epithelioid sarcoma of lumbar vertebrae presented with fever and shock. His spinal sarcoma had been diagnosed since he was 8 years of age and was treated with surgical resection, and recurrence of a tumor with metastases had occurred since he was 12 years of age. Two weeks following the discontinuation of hydrocortisone, adrenal reserve function was evaluated, and uncommon cause of adrenal insufficiency was concluded.

Simple Summary: MYCN gene amplification, the strongest prognostic marker of aggressive neuroblastoma, is detected on invasive biopsy tissues. This study aimed to establish a less invasive method to detect MYCN status based on MYCN mRNA contents in large extracellular vesicles or microvesicles. MYCN mRNA-containing microvesicles were detectable in three distinct MYCN-amplified neuroblastoma cell lines but absent in three neuroblastoma cells with MYCN-non-amplification. The feasibility of this EV-based workflow was successfully demonstrated by using the simulated samples (prepared by pulsing neuroblastoma MVs into the normal human serum) and bone marrow plasma specimens obtained from nine patients at various disease stages. Taken together, this study established the novel EV-based method for detecting MYCN status in pediatric neuroblastoma. MYCN amplification is the strongest predictor of high-risk neuroblastoma (NB). The standard procedure to detect MYCN status requires invasive procedures. Extracellular vesicles (EVs) contain molecular signatures of originated cells, present in biofluids, and serve as an invaluable source for cancer liquid biopsies. This study aimed to establish an EV-based method to detect the MYCN status of NB. Two EV subtypes, i.e., microvesicles (MVs) and exosomes, were sequentially isolated from the culture supernatant by step-wise centrifugation, ultrafiltration, and size-exclusion chromatography. Quantitative RT-PCR was performed to detect MYCN mRNA. As a result, MYCN mRNA was detectable in the MVs, but not exosomes, of MYCN-amplified NB cells. MYCN mRNA-containing MVs (MYCN-MV) were successfully detected in three distinct MYCN-amplified NB cell lines but absent in three MYCN non-amplification cells. The simulated samples were prepared by pulsing MVs into human serum. MYCN–MV detection in the simulated samples showed a less interfering effect from the human blood matrix. Validation using clinical specimens (2 mL bone marrow plasma) obtained from patients at various disease stages showed a promising result. Five out of six specimens of MYCN-amplified patients showed positive results, while there were no false positives in four plasma samples of the MYCN non-amplification group. This study communicated a novel EV-based method for detecting the MYCN status of pediatric NB based on MYCN mRNA contents in MVs. Future studies should be pursued in a prospective cohort to determine its true diagnostic performance. [ABSTRACT FROM AUTHOR]

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most prevalent genetic diseases affecting the kidneys. A genetically specific mutation model is required to comprehend its pathophysiology and to develop a drug treatment. In this study, we successfully developed human induced pluripotent stem cells (hiPSCs) named MUi027-A from skin fibroblasts of a patient diagnosed with ADPKD and carrying the PKD1 frameshift mutation (c.7946_7947delCT). MUi027-A cells showed the same genetic fingerprints as the parental cells, including the presence of the PKD1 mutation. MUi027-A hiPSCs displayed embryonic stem cell-like characteristics with the capability of differentiating into the three germ layers. Upon directed differentiation, MUi027-A hiPSCs could be differentiated into tubular organoids with the expression of renal cell markers. Furthermore, we compared the efficiency of cyst formation in two human iPSC lines with different PKD1 mutations. When cyst formation was induced by either forskolin or blebbistatin, MUi027-A hiPSC-derived kidney organoids displayed higher frequencies of cyst formation when compared to organoids generated from an iPSC cell line with non-truncating PKD1 mutation genotype (c.5878C > T), suggesting the presence of physiological differences in the mechanism of cyst formation between different PKD1 mutants. Overall, we generated and characterized a novel human iPSC line with a specific PKD mutation and demonstrated its potential as a disease model to study the pathophysiology of genetic determinants in the development of ADPKD disease. [ABSTRACT FROM AUTHOR]

Reactivating of fetal hemoglobin (HbF; α2γ2) can ameliorate the severity of β-thalassemia disease by compensating for adult hemoglobin deficiency in patients. Previously, microarray analysis revealed that zinc finger protein (ZNF)802 (also known as Juxta-posed with another zinc finger gene-1 (JAZF1)) was upregulated in human erythroblasts derived from adult peripheral blood compared with fetal liver-derived cells, implying a potential role as a HbF repressor. However, deficiency in ZNF802 induced by lentiviral shRNA in β0-thalassemia/hemoglobinE erythroblasts had no effect on erythroblast proliferation and differentiation. Remarkably, the induction of HBG expression was observed at the transcriptional and translational levels resulting in an increase of HbF to 35.0 ± 3.5%. Interestingly, the embryonic globin transcripts were also upregulated but the translation of embryonic globin was not detected. These results suggest ZNF802 might be a transcriptional repressor of the γ-globin gene in adult erythroid cells. [ABSTRACT FROM AUTHOR]

Human pegivirus-1 (HPgV-1) is a lymphotropic human virus, typically considered nonpathogenic, but its infection can sometimes cause persistent viremia both in immunocompetent and immunosuppressed individuals. In a viral discovery research program in hematopoietic stem cell transplant (HSCT) pediatric patients, HPgV-1 was detected in 3 out of 14 patients (21.4%) using a target enrichment next-generation sequencing method, and the presence of the viruses was confirmed by agent-specific qRT-PCR assays. For the first time in this patient cohort, complete genomes of HPgV-1 were acquired and characterized. Phylogenetic analyses indicated that two patients had HPgV-1 genotype 2 and one had HPgV-1 genotype 3. Intra-host genomic variations were described and discussed. Our results highlight the necessity to screen HSCT patients and blood and stem cell donors to reduce the potential risk of HPgV-1 transmission. [ABSTRACT FROM AUTHOR]

Reactivating of fetal hemoglobin (HbF; α2γ2) can ameliorate the severity of β-thalassemia disease by compensating for adult hemoglobin deficiency in patients. Previously, microarray analysis revealed that zinc finger protein (ZNF)802 (also known as Juxta-posed with another zinc finger gene-1 (JAZF1)) was upregulated in human erythroblasts derived from adult peripheral blood compared with fetal liver-derived cells, implying a potential role as a HbF repressor. However, deficiency in ZNF802 induced by lentiviral shRNA in β0-thalassemia/hemoglobinE erythroblasts had no effect on erythroblast proliferation and differentiation. Remarkably, the induction of HBG expression was observed at the transcriptional and translational levels resulting in an increase of HbF to 35.0 ± 3.5%. Interestingly, the embryonic globin transcripts were also upregulated but the translation of embryonic globin was not detected. These results suggest ZNF802 might be a transcriptional repressor of the γ-globin gene in adult erythroid cells. [ABSTRACT FROM AUTHOR]

Anti-SARS-CoV-2 effect of extracellular vesicles released from mesenchymal stem cells As of 10 December 2021, coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 accounted for 267 million people with up to 5.3 million deaths worldwide (https://covid19.who.int). Accordingly, we applied in vitro anti-SARS-CoV-2 assays, which were previously developed by our group (Kanjanasirirat et al., 2020; Kongsomros et al., 2021; Sa-Ngiamsuntorn et al., 2021), to determine the dose dependent anti-SARS-CoV-2 effect of MSC-EVs. In this direction, MSC-EV investigations for COVID-19 treatment would be more appealing and undeniable if MSC-EVs also exhibit anti-SARS-CoV-2 effects. [Extracted from the article]

Background: Betibeglogene autotemcel (beti-cel) gene therapy for transfusion-dependent β-thalassemia contains autologous CD34+ hematopoietic stem cells and progenitor cells transduced with the BB305 lentiviral vector encoding the β-globin (βA-T87Q) gene.Methods: In this open-label, phase 3 study, we evaluated the efficacy and safety of beti-cel in adult and pediatric patients with transfusion-dependent β-thalassemia and a non-β0/β0 genotype. Patients underwent myeloablation with busulfan (with doses adjusted on the basis of pharmacokinetic analysis) and received beti-cel intravenously. The primary end point was transfusion independence (i.e., a weighted average hemoglobin level of ≥9 g per deciliter without red-cell transfusions for ≥12 months).Results: A total of 23 patients were enrolled and received treatment, with a median follow-up of 29.5 months (range, 13.0 to 48.2). Transfusion independence occurred in 20 of 22 patients who could be evaluated (91%), including 6 of 7 patients (86%) who were younger than 12 years of age. The average hemoglobin level during transfusion independence was 11.7 g per deciliter (range, 9.5 to 12.8). Twelve months after beti-cel infusion, the median level of gene therapy-derived adult hemoglobin (HbA) with a T87Q amino acid substitution (HbAT87Q) was 8.7 g per deciliter (range, 5.2 to 10.6) in patients who had transfusion independence. The safety profile of beti-cel was consistent with that of busulfan-based myeloablation. Four patients had at least one adverse event that was considered by the investigators to be related or possibly related to beti-cel; all events were nonserious except for thrombocytopenia (in 1 patient). No cases of cancer were observed.Conclusions: Treatment with beti-cel resulted in a sustained HbAT87Q level and a total hemoglobin level that was high enough to enable transfusion independence in most patients with a non-β0/β0 genotype, including those younger than 12 years of age. (Funded by Bluebird Bio; HGB-207 ClinicalTrials.gov number, NCT02906202.). [ABSTRACT FROM AUTHOR]

Aims: The purpose of this study was to design and manufacture CD19 chimeric antigen receptor (CAR)‐modified T cells for clinical use in Thailand, as a model for how this technology can be directly applied at individual institutions treating high‐risk leukemia patients. Methods: We constructed second‐generation CAR T cells expressing CD19 scFV‐CD28‐CD3ζ with different lengths of the spacer region: full, intermediate, and short length, by using a lentiviral vector. We wanted to determine whether the difference in length of the spacer would affect the cytotoxic potential of the CD19 CAR T cells against the leukemic cells. Results: We found that all constructs of CD19 CAR T cells exhibited a similar level of cytotoxicity against several human lymphoma and leukemia cell lines. For the clinical application, we chose the intermediate length spacer construct CD19 CAR T cells, hypothesizing that the highest transduction efficiency coupled with a slower initial proliferation in vitro might lead to effective leukemic cell kill, yet a lower probability for serious clinical side effects. We then tested the clinical efficacy of our CD19 CAR T cells in one patient with refractory/relapsed acute B‐cell lymphoblastic leukemia. This patient indeed had minimal clinical side effects after the CAR T‐cell infusion, and he remains in an unmaintained, ongoing complete remission 10+ months after his T‐cell treatment. Conclusion: Our CD19 CAR T cells demonstrated efficacies in acute lymphoblastic B‐cell leukemia, and will be used to establish an immunotherapeutic program for high‐risk B‐cell acute lymphoblastic leukemia in Thailand. We propose that this approach can be used as a model for how this new exciting technology can be applied directly at individual institutions that treat (a large number of) patients with high‐risk leukemia. [ABSTRACT FROM AUTHOR]

Allogeneic hematopoietic stem cell transplantation (HSCT) is the only established treatment that is potentially curative, but it is limited by the availability of donors and the medical condition of the patient. To expand the donor pool to include haploidentical related donors, we introduced a program consisting of a pharmacologic pre transplant immune suppression phase (PTIS) and two courses of dexamethasone (DXM) and fludarabine (FLU) followed by pre transplant conditioning with intravenous FLU busulfan (BU) and post transplant graft-vs.-host disease (GvHD) prophylaxis with cyclophosphamide (CPM), tacrolimus, and mycophenolate mofetil. We transplanted 83 consecutive transfusion-dependent patients with thalassemia; the 3-year projected overall and event-free survival is over 96.0%, and there have been no secondary graft failures. Of the first 31 patients, we had two graft failures, both of them occurring in patients with extremely high titers of anti-donor-specific human leukocyte antigen (HLA) antibodies [anti-donor specific antibodies (DSAs)], but after adjusting the PTIS to include bortezomib (BORT) and rituximab (RIX) for patients with high titers of anti-DSAs and using pharmacologic dose guidance for BU, we had no graft failures in the last 52 patients. Six (7.0%) of 83 patients developed severe GvHD. We conclude that this is a safe and efficacious approach to allogeneic HSCT in thalassemia. [ABSTRACT FROM AUTHOR]