[Peripheral blood stem cell transplantation from HLA-mismatched unrelated donor or haploidentical donor for the treatment of X-linked agammaglobulinemia].

Allogeneic stem cell transplantation (allo-SCT) is currently the only curative option for patients with X-linked agammaglobulinemia (XLA). In this study, patient 1 aged 4 years who underwent allogeneic peripheral blood stem cell transplantation (allo-PBSCT) from HLA-mismatched unrelated donor; patient 2 aged 24 years (childhood onset) with primary cutaneous acral CD8+ T cell lymphoma who underwent allo-PBSCT from haploidentical relative donor. Both were treated by reduced toxicity myeloablative conditioning with post-transplantation cyclophosphamide (PTCy), anti-thymocyte globulin (ATG), methotrexate (MTX) and cyclosporine (CsA) for graft-versus-host-disease (GVHD) prophylaxis. In patient 1, neutrophil and platelet engraftment were observed on day 11 post-transplantation; the donor chimerism dropped on day 90 post-transplantation, and recovered on day 150 with donor lymphocyte infusion (DLI). In patient 2, neutrophil and platelet engraftment were observed on days 20 and 87 post-transplantation respectively, with complete donor chimerism on day 30 post-transplantation. The serum levels of IgG, IgM and IgA and the percentage of CD19+ B cells in peripheral blood of patients 1 and 2 returned to normal within 2 months and more than 1 year after transplantation respectively. There was no evidence of acute GVHD for the two patients. Patient 1 developed a limited type of skin chronic GVHD after DLI, which disappeared after anti-GVHD treatment. This is the first report of successful treatment for two XLA patients using PTCy with allo-PBSCT from HLA-mismatched unrelated donor or haploidentical donor, combining with improved conditioning, which expands the pool of eligible donors for patients with XLA.

Aptamer-Engineered Natural Killer Cells for Cell-Specific Adaptive Immunotherapy.

Natural killer (NK) cells are a key component of the innate immune system as they can attack cancer cells without prior sensitization. However, due to lack of cell-specific receptors, NK cells are not innately able to perform targeted cancer immunotherapy. Aptamers are short single-stranded oligonucleotides that specifically recognize their targets with high affinity in a similar manner to antibodies. To render NK cells with target-specificity, synthetic CD30-specific aptamers are anchored on cell surfaces to produce aptamer-engineered NK cells (ApEn-NK) without genetic alteration or cell damage. Under surface-anchored aptamer guidance, ApEn-NK specifically bind to CD30-expressing lymphoma cells but do not react to off-target cells. The resulting specific cell binding of ApEn-NK triggers higher apoptosis/death rates of lymphoma cells compared to parental NK cells. Additionally, experiments with primary human NK cells demonstrate the potential of ApEn-NK to specifically target and kill lymphoma cells, thus presenting a potential new approach for targeted immunotherapy by NK cells.

Hymeglusin Enhances the Pro-Apoptotic Effects of Venetoclax in Acute Myeloid Leukemia.

Venetoclax is used for the priority treatment of elderly patients with acute myeloid leukemia (AML). Resistance or intolerance to venetoclax offsets its clinical benefits in some patients. Combination strategies with other drugs are promising alternatives to overcome the current complications associated with venetoclax use. Hymeglusin, a specific inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A synthase 1 (HMGCS1), regulates the mevalonate pathway, which is vital for AML growth and chemosensitivity. The effects of the combination of venetoclax and hymeglusin on AML were explored in this study. The correlations between HMGCS1 and apoptosis-related genes were analyzed using the Gene Expression Profiling Interactive Analysis 2 and The Cancer Genome Atlas databases. Apoptosis and cell viability were detected in HL-60 and KG-1 cells after treatment with gradient concentrations of venetoclax or hymeglusin. The transcriptomic profiles of HL-60 and KG-1 cells were compared via RNA-Seq analysis. The effects of venetoclax and hymeglusin on apoptosis were validated in primary cells. The results showed that HMGCS1 expression was closely associated with apoptosis-related genes based on the data from large clinical databases. B cell lymphoma (BCL)-2 expression was elevated in AML and negatively associated with overall survival. Hymeglusin decreased BCL2 expression levels in HL-60 and KG-1 cells. Venetoclax and hymeglusin inhibited cell viability in both cell lines, but induced apoptosis in HL-60 cells. This discrepancy in sensitivity to hymeglusin may be attributed to the positive increase in the expression levels of HMGCS1 and multiple upregulated pro-leukemia genes in KG-1 cells. Combination treatment with venetoclax and hymeglusin significantly increased the apoptotic rates compared to single-agent treatment in both AML cell lines and primary AML cells. Furthermore, the combination strategy did not result in remarkably enhanced toxicity in normal mononuclear cells. Collectively, hymeglusin enhanced the effects of venetoclax on apoptosis. This combination strategy showed enhanced antileukemic activity with acceptable toxicity in AML.

High Expression of RhoBTB3 Predicts Favorable Chemothrapy Outcomes in non-M3 Acute Myeloid Leukemia.

Background: The expression patterns and prognostic significance of the Rho family GTPases in acute myeloid leukemia have not been systematically studied yet. Methods: In our study, we analyzed the expression patterns of 21 Rho family GTPases gene members in AML patients based on GEPIA database. 10 gene members with significant differential expression in AML tissue and healthy tissue were selected for subsequent research. Survival curve analysis in TCGA and GEO dataset preliminary showed that RhoBTB3 is related with the prognosis of non-M3 AML patients. The differential expression of RhoBTB3 on AML bone marrow and normal bone marrow was verified by RT-qPCR. We performed Kaplan-Meier survival analysis and Multivariate Cox analysis to assess the prognostic value of RhoBTB3 in non-M3 AML patients with different treatment regimens. Gene functional enrichment analysis of RhoBTB3 was performed using GO, KEGG and PPI network. Results: The AML patients from TCGA database were partitioned into 2 groups based on different treatment regimens: chemotherapy group and allo-HSCT group. In chemotherapy group, patients with higher expression level of RhoBTB3 showed relatively longer OS and EFS, multivariate Cox analysis revealed high RhoBTB3 mRNA expression as an independent favorable prognostic factor. However, in allo-HSCT group, no significant difference of OS and EFS were found between RhoBTB3 high and low subgroups. Meanwhile, allo-HSCT could circumvent the unfavorable prognosis that was associated with downregulation of RhoBTB3. Functional enrichment analysis showed the association of RhoBTB3 expression with several fundamental physiological components and pathways, including extracellular matrix components, extracellular structure organization, and cytokine-cytokine receptor interaction. Conclusions: Our study identified RhoBTB3 as a prognostic marker and may aid in the selection of the appropriate treatment options between chemotherapy and allo-HCST in non-M3 AML patients. Further researches are necessary to clarify the involvement of RhoBTB3 in the pathogenesis of AML.

Downregulation of p53 by Insufficient CTCF in CD4+ T Cells Is an Important Factor Inducing Acute Graft-Versus-Host Disease.

Recent evidence indicates that p53 plays a protective role against various systemic autoimmune diseases by suppressing pro-inflammatory cytokine production and reducing the number of pathogenic T cells. However, whether abnormal p53 expression participates in the development of acute graft-versus-host disease (aGVHD) remains unclear. In this study, we demonstrated that p53 was downregulated in CD4+ T cells from patients with aGVHD compared with the non-aGVHD group. Furthermore, we confirmed that low expression of CCCTC-binding factor (CTCF) in CD4+ T cells from aGVHD cases is an important factor affecting histone H3K9/K14 hypoacetylation in the p53 promoter and p53 downregulation. Restoring CTCF expression in CD4+ T cells from aGVHD patients increased p53 amounts and corrected the imbalance of Th17 cells/Tregs. Taken together, these results provide novel insights into p53 downregulation in CD4+ T cells from aGVHD patients.

Rapid Detection of Mycoplasma-Infected Cells by an ssDNA Aptamer Probe.

Mycoplasmas are unique cell wall-free bacteria. Because they lack a cell wall and have resistance to ß-lactam antibiotics, mycoplasma is the major pathogen that infects cultured cells in research laboratories. For rapid detection of mycoplasma-infected cells, we developed an ssDNA aptamer sequence composed of 40 nucleotides. Flow cytometry analysis showed that the synthetic aptamer probe selectively targeted mycoplasma-infected culture cells with high specificity identical to commercially available PCR-based assays. Additionally, fluorescent microscopy studies revealed that the aptamer probe rapidly stained mycoplasma-infected cells with higher sensitivity compared to Hoechst dye-mediated cellular DNA content stains. Moreover, confocal microscopy studies of trypsin-treated cells validated that the aptamer probes selectively targeted mycoplasma components on the surface of infected cells. Finally, preclinical studies of peripheral blood cells demonstrated that the aptamer probe was able to detect in vitro mycoplasma infection of primary lymphocytes. Taken together, these findings indicate that the aptamer probe will not only allow rapid detection of mycoplasma-infected culture cells for research purposes but also provide a simple method to monitor mycoplasma infection in primary cell products for clinical use.

Anterior Cruciate Ligament Reconstruction in a Rabbit Model Using a Decellularized Allogenic Semitendinous Tendon Combined with Autologous Bone Marrow-Derived Mesenchymal Stem Cells.

As a regular adoptable material for anterior cruciate ligament (ACL) reconstruction, free tendon allograft exhibits unsatisfactory outcomes, such as retarded ligamentization and tendon-bone integration. The application of bone marrow-derived mesenchymal stem cells (BMSCs), as well as a decellularized free tendon allograft developed by our group, was proven to be effective in improving ACL reconstruction results. This study aimed to investigate the efficacy and feasibility of decellularized allogenic semitendinous tendon (ST) combined with autologous BMSCs used as a substitute to free tendon allograft in a rabbit model. This study finally shows that the decellularized allogenic ST combined with autologous BMSCs could significantly improve ACL reconstruction results compared with allograft. Stem Cells Translational Medicine 2019;8:971&982.

Oligonucleotide Aptamer-Mediated Precision Therapy of Hematological Malignancies.

Precision medicine has recently emerged as a promising strategy for cancer therapy because it not only specifically targets cancer cells but it also does not have adverse effects on normal cells. Oligonucleotide aptamers are a class of small molecule ligands that can specifically bind to their targets on cell surfaces with high affinity. Aptamers have great potential in precision cancer therapy due to their unique physical, chemical, and biological properties. Therefore, aptamer technology has been widely investigated for biomedical and clinical applications. This review focuses on the potential applications of aptamer technology as a new tool for precision treatment of hematological malignancies, including leukemia, lymphoma, and multiple myeloma.

Aptamer Internalization via Endocytosis Inducing S-Phase Arrest and Priming Maver-1 Lymphoma Cells for Cytarabine Chemotherapy.

The goal of precision therapy is to efficiently treat cancer without side effects. Aptamers are a class of small ligands composed of single-stranded oligonucleotides that bind to their targets with high affinity and specificity. In this study, we identified an ssDNA aptamer specifically targeting Maver-1 lymphoma cells with high binding affinity (Kd = 70±8 pmol/L). Interestingly, cellular cycle studies revealed that exposure of Maver-1 cells to synthetic aptamers triggered S-phase arrest of 40% of the cells (vs. 18% baseline). Confocal microscopy confirmed specific cell binding of aptamers and the resultant endocytosis into Maver-1 cells. Subsequent functional assays validated the fact that aptamer internalization into targeted cells is a prerequisite for Maver-1 cell growth inhibition. Importantly, aptamer-induced S-phase arrest induced enhanced chemotherapeutic results involving cytarabine, which primarily kills lymphoma cells at S-phase. Combination treatments revealed that aptamer re-exposure considerably primed Maver-1 cells for cytarabine chemotherapy, thus achieving a synergistic killing effect by reaching cell death rates as high as 61% (vs. 13% or 14% induced by aptamer or cytarabine treatment alone). These findings demonstrated that aptamers do not only act as molecular ligands but can also function as biotherapeutic agents by inducing S-phase arrest of lymphoma cells. In addition, logical combination of aptamer and cytarabine treatments ushers the way to a unique approach in precision lymphoma chemotherapy.

ssDNA Aptamer Specifically Targets and Selectively Delivers Cytotoxic Drug Doxorubicin to HepG2 Cells.

Hepatocellular carcinoma (HCC) is the third leading cause of death due to cancer worldwide with over 500,000 people affected annually. Although chemotherapy has been widely used to treat patients with HCC, alternate modalities to specifically deliver therapeutic cargos to cancer cells have been sought in recent years due to the severe side effects of chemotherapy. In this respect, aptamer-based tumor targeted drug delivery has emerged as a promising approach to increase the efficacy of chemotherapy and reduce or eliminate drug toxicity. In this study, we developed a new HepG2-specific aptamer (HCA#3) by a procedure known as systematic evolution of ligands by exponential enrichment (SELEX) and exploited its role as a targeting ligand to deliver doxorubicin (Dox) to HepG2 cells in vitro. The selected 76-base nucleotide aptamer preferentially bound to HepG2 hepatocellular carcinoma cells but not to control cells. The aptamer HCA#3 was modified with paired CG repeats at the 5'-end to carry and deliver a high payload of intercalated Dox molecules at the CG sites. Four Dox molecules (mol/mol) were fully intercalated in each conjugate aptamer-Dox (ApDC) molecule. Biostability analysis showed that the ApDC molecules are stable in serum. Functional analysis showed that ApDC specifically targeted and released Dox within HepG2 cells but not in control cells, and treatment with HCA#3 ApDC induced HepG2 cell apoptosis but had minimal effect on control cells. Our study demonstrated that HCA#3 ApDC is a promising aptamer-targeted therapeutic that can specifically deliver and release a high doxorubicin payload in HCC cells.