Screening of novel DPP-IV inhibitory peptides derived from bovine milk proteins using a peptide array platform.

Dipeptidyl peptidase IV (DPP-IV) has become an important target in the prevention and treatment of diabetes. Although many DPP-IV inhibitory peptides have been identified by a general approach involving the repeated fractionation of food protein hydrolysates, the obtained results have been dependent on the content of each peptide and fractionation conditions. In the present study, a peptide array that provides comprehensive assays of peptide sequences was used to identify novel DPP-IV inhibitory peptides derived from bovine milk proteins; these peptides were then compared with those identified using the general approach. While the general approach identified only known peptides that were abundant in the hydrolysate, the peptide array-based approach identified 10 novel DPP-IV inhibitory peptides, all of which had proline at the second residue from the N-terminus. The proper or combined use of these two approaches, which have different advantages, will enable the efficient development of novel bioactive foods and drugs.

Erratum: Ligand-based, piggyBac-engineered CAR-T cells targeting EGFR are safe and effective against non-small cell lung cancers.

[This corrects the article DOI: 10.1016/j.omto.2023.100728.].

Ligand-based, piggyBac-engineered CAR-T cells targeting EGFR are safe and effective against non-small cell lung cancers.

Epidermal growth factor receptor (EGFR) is overexpressed in various cancers, including non-small cell lung cancer (NSCLC), and in some somatic cells at a limited level, rendering it an attractive antitumor target. In this study, we engineered chimeric antigen receptor (CAR)-T cells using the piggyBac transposon system, autologous artificial antigen-presenting cells, and natural ligands of EGFR. We showed that this approach yielded CAR-T cells with favorable phenotypes and CAR positivity. They exhibited potent antitumor activity against NSCLC both in vitro and in vivo. When administered to tumor-bearing mice and non-tumor-bearing cynomolgus macaques, they did not elicit toxicity despite their cross-reactivity to both murine and simian EGFRs. In total we tested three ligands and found that the CAR candidate with the highest affinity consistently displayed greater potency without adverse events. Taken together, our results demonstrate the feasibility and safety of targeting EGFR-expressing NSCLCs using ligand-based, piggyBac-engineered CAR-T cells. Our data also show that lowering the affinity of CAR molecules is not always beneficial.

Comprehensive genetic analyses of childhood acute leukemia in Iraq using next-generation sequencing.

Background: Molecular analyses in hematological malignancies provide insights about genetic makeup. Probable etiological factors in leukemogenesis could also be disclosed. Since genetic analyses are still primitive in Iraq, a country of repeated wars, we conceived of performing next-generation sequencing (NGS), to disclose the genomic landscape of acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) among a cohort of Iraqi children. Methods: Dried blood samples were collected from Iraqi children with ALL (n=55), or AML (n=11), and transferred to Japan where NGS was done. Whole-exome, whole-genome, and targeted gene sequencings were performed. Results: Somatic point mutations and the copy number variations among Iraqi children with acute leukemia were comparable with those in other countries, and cytosine-to-thymine nucleotide alterations were dominant. Strikingly, TCF3-PBX1 was the most recurrent fusion gene (22.4%) in B-cell precursor ALL (B-ALL), and acute promyelocytic leukemia (AML-M3) was subtyped in 5 AML cases. Additionally, a high frequency of RAS signaling pathway mutations was detected in children with B-ALL (38.8%), along with 3 AML cases that carried oncogenic RAS. Conclusions: Apart from disclosing the high frequency of TCF3-PBX1, NGS confirmed our previous finding of recurrent RAS mutations in Iraqi childhood acute leukemia. Our results suggest that the biology of Iraqi childhood acute leukemia is in part characteristic, where the war-aftermath environment or geography might play a role.

Basophil activation test for allergic and febrile non-haemolytic transfusion reactions among paediatric patients with haematological or oncological disease.

BACKGROUND AND OBJECTIVES: Allergic transfusion reactions (ATRs) and febrile non-haemolytic transfusion reactions (FNHTRs) are common, although their mechanisms remain unclear. Immunoglobulin E (IgE)-mediated type I hypersensitivity may be involved in the pathogenesis of ATR. A basophil activation test (BAT) may help elucidate this process. MATERIALS AND METHODS: The BAT was based on peripheral blood samples from paediatric patients with a haematological or oncological disease and on samples of residual blood products transfused in each case. Dasatinib was used to evaluate whether basophil activation was mediated by an IgE-dependent pathway. RESULTS: Twenty-seven patients with and 19 patients without ATR/FNHTR were included in this study, respectively. The median BAT values associated with ATR- (n = 41) and FNHTR-causing (n = 5) blood products were 22.1% (range = 6.1%-77.0%) and 27.8% (range = 15.2%-47.8%), respectively, which were higher than the median value of 8.5% (range = 1.1%-40.9%) observed in blood products without a transfusion reaction. Dasatinib suppressed basophil activity. BAT values were comparable in patients with ATR regardless of severity. Meanwhile, BAT values analysed with blood products non-causal for ATR/FNHTR were higher in patients with ATR/FNHTR than in those without. CONCLUSION: The IgE-mediated type I hypersensitivity may be involved in the pathogenesis of ATR and FNHTR. BAT analyses may help elucidate the underlying mechanisms and identify patients at risk.

Efficient differentiation of human neutrophils with recapitulation of emergency granulopoiesis in human G-CSF knockin humanized mice.

Neutrophils are critical mediators during the early stages of innate inflammation in response to bacterial or fungal infections. A human hematopoietic system reconstituted in humanized mice aids in the study of human hematology and immunology. However, the poor development of human neutrophils is a well-known limitation of humanized mice. Here, we generate a human granulocyte colony-stimulating factor (hG-CSF) knockin (KI) NOD/Shi-scid-IL2rgnull (NOG) mouse in which hG-CSF is systemically expressed while the mouse G-CSF receptor is disrupted. These mice generate high numbers of mature human neutrophils, which can be readily mobilized into the periphery, compared with conventional NOG mice. Moreover, these neutrophils exhibit infection-mediated emergency granulopoiesis and are capable of efficient phagocytosis and reactive oxygen species production. Thus, hG-CSF KI mice provide a useful model for studying the development of human neutrophils, emergency granulopoiesis, and a potential therapeutic model for sepsis.

Skin and soft tissue infections in adolescent chronic myeloid leukemia under dasatinib treatment.

Although skin complications are common adverse events from tyrosine kinase inhibitors (TKIs) for the treatment of chronic myeloid leukemia (CML), no reports have focused on skin and soft tissue infections (SSTIs) associated with TKI use. We herein present five episodes of SSTIs in three CML patients under dasatinib treatment. All patients were adolescents and had been receiving dasatinib for more than 4 years. In contrast, none of 41 adult CML patients experienced SSTIs in a retrospective analysis. Our findings suggest that long-term dasatinib treatment in adolescent patients may be associated with the increased risk of SSTIs.

Relationship between allergic transfusion reactions and allergic predisposition among pediatric patients with hematological/oncological disease.

BACKGROUND: Allergic transfusion reactions (ATRs) manifest frequently as transfusion reactions, and their onset may be related to a patient's allergic predisposition. Moreover, although pediatric patients with hematological/oncological disease are more susceptible to ATRs, the relationship between allergic predisposition and ATRs remains to be fully clarified. STUDY DESIGN AND METHODS: Patients who were diagnosed with pediatric hematological/oncological disease and received transfusion at the study institutions were included. We determined patient background information related to their allergy history, measured the levels of allergen-specific immunoglobulin E (IgE) using sera obtained on diagnosis, and analyzed their associations with ATR onset. RESULTS: Of the 363 patients analyzed, 144 developed ATRs. Multivariate analysis identified cases with high basophils in the peripheral blood, and Dermatophagoides pteronyssinus- and egg white-specific IgEs were involved in the development of ATR in all age groups. Meanwhile, a history of food allergies, and positivity for Japanese cypress- and D. pteronyssinus-specific IgEs were risk factors for developing ATRs in the <5 years age group. Moreover, patients aged 5-<10 years with a history of asthma, allergic rhinitis, pollinosis, or atopic dermatitis, and those aged ≥10 years with positivity for dog dander-specific IgE were at risk for developing ATRs. CONCLUSION: The allergic constitution of patients plays a role in ATR onset even in pediatric hematological/oncological diseases. Therefore, advance confirmation of a patient's allergic constitution may partly predict the onset of ATRs. However, since multiple allergic predispositions within complex mechanisms may be involved in the onset of ATRs, further verification is required.

PiggyBac Transposon-Mediated CD19 Chimeric Antigen Receptor-T Cells Derived From CD45RA-Positive Peripheral Blood Mononuclear Cells Possess Potent and Sustained Antileukemic Function.

The quality of chimeric antigen receptor (CAR)-T cell products, namely, memory and exhaustion markers, affects the long-term functionality of CAR-T cells. We previously reported that piggyBac (PB) transposon-mediated CD19 CAR-T cells exhibit a memory-rich phenotype that is characterized by the high proportion of CD45RA+/C-C chemokine receptor type 7 (CCR7)+ T-cell fraction. To further investigate the favorable phenotype of PB-CD19 CAR-T cells, we generated PB-CD19 CAR-T cells from CD45RA+ and CD45RA- peripheral blood mononuclear cells (PBMCs) (RA+ CAR and RA- CAR, respectively), and compared their phenotypes and antitumor activity. RA+ CAR-T cells showed better transient gene transfer efficiency 24 h after transduction and superior expansion capacity after 14 days of culture than those shown by RA- CAR-T cells. RA+ CAR-T cells exhibited dominant CD8 expression, decreased expression of the exhaustion marker programmed cell death protein-1 (PD-1) and T-cell senescence marker CD57, and enriched naïve/stem cell memory fraction, which are associated with the longevity of CAR-T cells. Transcriptome analysis showed that canonical exhaustion markers were downregulated in RA+ CAR-T, even after antigen stimulation. Although antigen stimulation could increase CAR expression, leading to tonic CAR signaling and exhaustion, the expression of CAR molecules on cell surface after antigen stimulation in RA+ CAR-T cells was controlled at a relatively lower level than that in RA- CAR-T cells. In the in vivo stress test, RA+ CAR-T cells achieved prolonged tumor control with expansion of CAR-T cells compared with RA- CAR-T cells. CAR-T cells were not detected in the control or RA- CAR-T cells but RA+ CAR-T cells were expanded even after 50 days of treatment, as confirmed by sequential bone marrow aspiration. Our results suggest that PB-mediated RA+ CAR-T cells exhibit a memory-rich phenotype and superior antitumor function, thus CD45RA+ PBMCs might be considered an efficient starting material for PB-CAR-T cell manufacturing. This novel approach will be beneficial for effective treatment of B cell malignancies.

A lymphodepleted non-human primate model for the assessment of acute on-target and off-tumor toxicity of human chimeric antigen receptor-T cells.

OBJECTIVES: Chimeric antigen receptor (CAR)-T cell therapy possesses the potential to cause unexpected on-target toxicities that may be life-threatening. Non-human primates (NHPs) share considerable structural homology and expression profiles of most proteins with humans and are therefore utilised as an animal model for non-clinical safety studies. We have developed a lymphodepleted NHP model by conditioning the animals with immunosuppressive chemotherapy designed to simulate clinical practice conditions, to induce transient mixed chimerism before the administration of human CAR-T cells redirected to target Ephrin type-B receptor 4 (EPHB4-CAR-T cells) to evaluate the toxicity of these cells. METHODS: We administered 60 mg m-2 day-1 of fludarabine for 4 days and 30 mg kg-1 day-1 of cyclophosphamide for 2 days intravenously to cynomolgus macaques for lymphodepletion; then, 3.3 × 106 kg-1 of non-transduced or EPHB4-CAR-T cells was infused into the macaques, respectively. All macaques were closely monitored and evaluated for potential toxicity for 7 days. RESULTS: Lymphodepletion was successfully achieved on day -1 before T-cell infusion and persisted over 7 days without severe organ toxicities. A single administration of human EPHB4-CAR-T cells did not induce overt organ toxicities, although EPHB4-CAR-T cells were activated in vivo as evidenced by the elevation in copy numbers of the CAR transgene 24 h after infusion. CONCLUSION: Although this NHP model is limited for the full evaluation of toxicity of human CAR-T cells and the conditioning protocol should be further optimised, this lymphodepleted NHP model could be used to assess acute on-target/off-tumor toxicities of CAR-T cells.

Mutated GM-CSF-based CAR-T cells targeting CD116/CD131 complexes exhibit enhanced anti-tumor effects against acute myeloid leukaemia.

OBJECTIVES: As the prognosis of relapsed/refractory (R/R) acute myeloid leukaemia (AML) remains poor, novel treatment strategies are urgently needed. Clinical trials have shown that chimeric antigen receptor (CAR)-T cells for AML are more challenging than those targeting CD19 in B-cell malignancies. We recently developed piggyBac-modified ligand-based CAR-T cells that target CD116/CD131 complexes, also known as the GM-CSF receptor (GMR), for the treatment of juvenile myelomonocytic leukaemia. This study therefore aimed to develop a novel therapeutic method for R/R AML using GMR CAR-T cells. METHODS: To further improve the efficacy of the original GMR CAR-T cells, we have developed novel GMR CAR vectors incorporating a mutated GM-CSF for the antigen-binding domain and G4S spacer. All GMR CAR-T cells were generated using a piggyBac-based gene transfer system. The anti-tumor effect of GMR CAR-T cells was tested in mouse AML xenograft models. RESULTS: Nearly 80% of the AML cells predominant in myelomonocytic leukaemia were found to express CD116. GMR CAR-T cells exhibited potent cytotoxic activities against CD116+ AML cells in vitro. Furthermore, GMR CAR-T cells incorporating a G4S spacer significantly improved long-term in vitro and in vivo anti-tumor effects. By employing a mutated GM-CSF at residue 21 (E21K), the anti-tumor effects of GMR CAR-T cells were also improved especially in long-term in vitro settings. Although GMR CAR-T cells exerted cytotoxic effects on normal monocytes, their lethality on normal neutrophils, T cells, B cells and NK cells was minimal. CONCLUSIONS: GMR CAR-T cell therapy represents a promising strategy for CD116+ R/R AML.

Autologous antigen-presenting cells efficiently expand piggyBac transposon CAR-T cells with predominant memory phenotype.

The quality of chimeric antigen receptor (CAR)-T cell products, including the expression of memory and exhaustion markers, has been shown to influence their long-term functionality. The manufacturing process of CAR-T cells should be optimized to prevent early T cell exhaustion during expansion. Activation of T cells by monoclonal antibodies is a critical step for T cell expansion, which may sometimes induce excess stimulation and exhaustion of T cells. Given that piggyBac transposon (PB)-based gene transfer could circumvent the conventional pre-activation of T cells, we established a manufacturing method of PB-mediated HER2-specific CAR-T cells (PB-HER2-CAR-T cells) that maintains their memory phenotype without early T cell exhaustion. Through stimulation of CAR-transduced T cells with autologous peripheral blood mononuclear cell-derived feeder cells expressing both truncated HER2, CD80, and 4-1BBL proteins, we could effectively propagate memory-rich, PD-1-negative PB-HER2-CAR-T cells. PB-HER2-CAR-T cells demonstrated sustained antitumor efficacy in vitro and debulked the HER2-positive tumors in vivo. Mice treated with PB-HER2-CAR-T cells rejected the second tumor establishment owing to the in vivo expansion of PB-HER2-CAR-T cells. Our simple and effective manufacturing process using PB system and genetically modified donor-derived feeder cells is a promising strategy for the use of PB-CAR-T cell therapy.

Investigation of risk factors associated with erythrocyte engraftment after ABO-incompatible hematopoietic stem cell transplantation.

ABO-incompatible hematopoietic stem cell transplantations (HSCTs) are widely practiced; however, the delay in erythrocyte engraftment can be problematic. While erythrocyte engraftment is usually indicated by an increase in reticulocyte levels without the need for erythrocyte transfusions, the disappearance of recipient-derived anti-A/B isoagglutinin and detection of donor-derived A/B antigens can also be used as other parameters. We conducted a retrospective analysis of 68 ABO-incompatible HSCTs, focusing on major and bidirectional mismatch. We analyzed known clinical risk factors associated with delayed erythrocyte engraftment using the three parameters (disappearance of anti-A/B isoagglutinin in recipient, detection of donor-derived A/B antigen, and reticulocyte levels >1%). Although the three parameters were well correlated, the results showed heterogeneity when analyzing the associated risk factors for delayed erythrocyte engraftment. In the analysis of all cases, the requirement for an HLA-matched platelet transfusion was a common risk factor. Furthermore, erythrocyte engraftment was slower in adults than in children. In adults, cytomegalovirus antigenemia was a risk factor for two parameters; however, in children, underlying disease was a common risk factor for all parameters. There is a complex relationship between erythrocyte engraftment and various factors related to HSCTs. Our results suggest that greater accuracy is possible by using analysis methods other than the measurement of reticulocyte levels.

A Validation Study of the Revised Diagnostic Criteria from the International Workshop on Ocular Sarcoidosis at a Single Institute in Japan.

Purpose: To validate the revised criteria of the International Workshop on Ocular Sarcoidosis (IWOS) for the diagnosis of ocular sarcoidosis (OS).Methods: A retrospective chart review study was performed on 323 patients including 51 patients with biopsy-proven sarcoidosis and 272 patients with other uveitis entities. Data on intraocular signs and systemic investigations were collected, and sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and the Youden index were calculated.Results: All intraocular signs and most systemic investigations showed high diagnostic parameters. Sensitivity, specificity, PPV, and NPV of the revised IWOS criteria were 1.000, 0.930, 0.728, and 1.000, respectively. Presumed or probable OS showed lower sensitivity and higher specificity when compared with the best Youden index.Conclusion: The revised IWOS criteria are useful in Japanese patients, but could possibly be improved by modifying the criterion of presumed or probable OS.

Reduced-intensity conditioning is effective for hematopoietic stem cell transplantation in young pediatric patients with Diamond-Blackfan anemia.

Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative therapy for the hematologic manifestations of Diamond-Blackfan anemia (DBA). However, data regarding the optimal conditioning regimen for DBA patients are limited. We retrospectively compared the outcomes of DBA patients who underwent HSCT using either myeloablative conditioning (MAC) or reduced-intensity conditioning (RIC) regimens. The patients belonged to a cohort treated at our hospitals between 2000 and 2018. HSCT was performed in 27 of 165 patients (16.4%). The median age at the time of HSCT was 3.6 years. Stem cell sources included bone marrow for 25 patients (HLA-matched sibling donors, n = 5; HLA-mismatched related donors, n = 2; HLA-matched/mismatched unrelated donors, n = 18) or cord blood for 2 patients. MAC or RIC regimens were used in 12 and 15 patients, respectively. Engraftment was successful in all 27 patients who underwent HSCT. Three patients who underwent HSCT using MAC regimens developed sinusoidal obstruction syndrome. The 3-year overall survival (OS) and failure-free survival rates (FFS) post-transplantations were 95.2% and 88.4%, respectively, with no significant differences between MAC and RIC regimens. Our data suggest that HSCTs using RIC regimens are effective and obtain engraftment with excellent OS and FFS for young DBA patients.

Autologous non-human primate model for safety assessment of piggyBac transposon-mediated chimeric antigen receptor T cells on granulocyte-macrophage colony-stimulating factor receptor.

OBJECTIVES: Chimeric antigen receptor (CAR)-T cell therapy redirected to specific antigens on tumor cells is a promising immunotherapy strategy for various cancers. Most target antigens are also expressed on normal tissues at varying levels, and therefore, a considerable challenge in the field is determining safety profiles, including life-threatening off-tumor and off-target toxicities. The granulocyte-macrophage colony-stimulating factor receptor (hGMR) is a promising target for CAR T-cell therapy for a subset of acute myelocytic leukaemia, although it is also expressed on normal cells including monocytes, macrophages, CD34-positive haematopoietic cells and vascular endothelial cells. hGMR and other immune-related proteins are highly conserved between humans and cynomolgus macaques (Macaca fascicularis). Therefore, in this study, we engineered cynomolgus T cells to express CAR molecules redirected to hGMR by piggyBac (PB) transposon-based gene transfer and adoptively transferred autologous hGMR-CAR T cells into cynomolgus macaques. METHODS: We established PB-mediated human GMR (hGMR)-specific CAR T cells using cynomolgus peripheral blood mononuclear cells and transferred them into autologous individuals, and evaluated the potential toxicity related to hGMR-CAR T cells. RESULTS: hGMR-CAR T cells did not exert overt organ toxicities such as bone marrow suppression, monocytopenia and vasculitis, although they recognised and killed cynomolgus monocytes and macrophages in vitro. CONCLUSION: Although our model did not simulate a tumor-bearing model, it supports the safety of hGMR-CAR T cells and demonstrates the usefulness of a non-human primate model to evaluate the safety of T-cell products by assessing off-tumor/off-target toxicity before clinical trials.

Direct Delivery of piggyBac CD19 CAR T Cells Has Potent Anti-tumor Activity against ALL Cells in CNS in a Xenograft Mouse Model.

The anti-CD19 chimeric antigen receptor (CAR) T cells showed excellent effect against acute lymphoblastic leukemia (ALL) in bone marrow (BM) in clinical trials. However, it remains to be elucidated whether the CD19 CAR T cell therapy is effective for ALL cells in central nervous system (CNS) because the patients with isolated or advanced CNS disease were excluded from clinical trials of systemic intravenous (i.v.) delivery of CAR T cells. Therefore, the preclinical evaluation for the efficacy of CAR T cell therapy against ALL cells in CNS is essential for clinical application. We evaluated the effect and adverse reaction of CD19 CAR T cells against ALL in CNS using a xenograft mouse model by i.v. or intra-cerebroventricular (i.c.v.) delivery of CAR T cells. Injection of piggyBac CD19 CAR T cells by i.v. had partial effects, whereas all CAR T i.c.v.-delivered mice had eliminated ALL in CNS. Although some CAR T i.c.v.-delivered mice showed transient changes of clinical symptoms during the first few days after treatment, none of CAR T i.c.v.-delivered mice displayed fatal adverse events. In this study, we demonstrated that direct delivery into CNS of CAR T cells is a possible therapeutic approach with the xenograft mouse model.

Investigating Anti-Obesity Effects by Oral Administration of Aloe vera Gel Extract (AVGE): Possible Involvement in Activation of Brown Adipose Tissue (BAT).

The aim of this study is to investigate the mechanism of anti-obesity effects of Aloe vera gel extract (AVGE) containing Aloe sterols. Previously, we reported that oral intake of Aloe vera components has an anti-diabetic and anti-obesity effect. This study was designed to assess the role of brown adipose tissue (BAT) in the anti-obesity effect of AVGE. Six-week-old male mice were divided into three groups; STD (standard diet), HFD (60% high fat diet) and AVGE (60% high fat diet with AVGE treatment). During 11 wk of AVGE administration, body weight has been monitored. Tissue samples were obtained to be measured the weight and evaluated the gene expressions. Mice treated with AVGE had suppressed body weight, and liver and fat weight gain. To investigate BAT activation, we measured the expression of mRNA related to BAT thermogenesis. Mice in the AVGE group had higher expression of Ucp1, Adrb3, and Cidea in BAT compared to HFD. Next, to investigate the possibility that AVGE induced hepatic FGF21, which is an important factor for nutrient and energy homeostasis including BAT regulation, in vitro study was conducted. HepG2 cell stimulated by AVGE were highly expressed FGF21. These results suggested that BAT activation partially contributes to mechanism of anti-obesity effect of Aloe sterols in diet-induced obesity (DIO) models. However, further study is needed to determine the predominant mechanism.